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<div class="c-navbar-wrapper clearfix"> | <div class="c-navbar-wrapper clearfix"> | ||
<div class="c-brand c-pull-left"> | <div class="c-brand c-pull-left"> | ||
− | <a href="https://2016.igem.org/Team:UofC_Calgary | + | <a href="https://2016.igem.org/Team:UofC_Calgary" class="c-logo"> |
<img src="https://static.igem.org/mediawiki/2016/9/9a/Logo-1.png" alt="iGEM" class="c-desktop-logo"> | <img src="https://static.igem.org/mediawiki/2016/9/9a/Logo-1.png" alt="iGEM" class="c-desktop-logo"> | ||
<img src="https://static.igem.org/mediawiki/2016/9/9a/Logo-1.png" alt="iGEM" class="c-desktop-logo-inverse"> | <img src="https://static.igem.org/mediawiki/2016/9/9a/Logo-1.png" alt="iGEM" class="c-desktop-logo-inverse"> | ||
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<!-- BEGIN: MEGA MENU --> | <!-- BEGIN: MEGA MENU --> | ||
<ul class="nav navbar-nav c-theme-nav"> | <ul class="nav navbar-nav c-theme-nav"> | ||
− | <li class=" | + | <li class="c-menu-type-classic"> |
<a href="https://2016.igem.org/Team:UofC_Calgary" class="c-link dropdown-toggle">Home</a> | <a href="https://2016.igem.org/Team:UofC_Calgary" class="c-link dropdown-toggle">Home</a> | ||
</li> | </li> | ||
− | <li class="c-menu-type-classic"> | + | <li class="c-active c-menu-type-classic"> |
<a href="https://2016.igem.org/Team:UofC_Calgary/Description" class="c-link dropdown-toggle">Project</a> | <a href="https://2016.igem.org/Team:UofC_Calgary/Description" class="c-link dropdown-toggle">Project</a> | ||
<ul class="dropdown-menu c-menu-type-inline c-pull-left"> | <ul class="dropdown-menu c-menu-type-inline c-pull-left"> | ||
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<li> | <li> | ||
<a href="https://2016.igem.org/Team:UofC_Calgary/Model">Model</a> | <a href="https://2016.igem.org/Team:UofC_Calgary/Model">Model</a> | ||
+ | </li><li> | ||
+ | <a href="https://2016.igem.org/Team:UofC_Calgary/Design">Applied Design</a> | ||
</li> | </li> | ||
<li> | <li> | ||
<a href="https://2016.igem.org/Team:UofC_Calgary/Parts">Parts Registry</a> | <a href="https://2016.igem.org/Team:UofC_Calgary/Parts">Parts Registry</a> | ||
+ | <li> | ||
+ | <a href="https://2016.igem.org/Team:UofC_Calgary/Composite_Part">Composite Parts</a> | ||
+ | </li> | ||
</li> | </li> | ||
</ul> | </ul> | ||
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<a href="https://2016.igem.org/Team:UofC_Calgary/HP/Gold">Gold</a> | <a href="https://2016.igem.org/Team:UofC_Calgary/HP/Gold">Gold</a> | ||
</li> | </li> | ||
+ | <li> | ||
+ | <a href="https://2016.igem.org/Team:UofC_Calgary/Policy"> Policy Brief </a> | ||
+ | </li> | ||
</ul> | </ul> | ||
</li> | </li> | ||
Line 142: | Line 150: | ||
<li> | <li> | ||
− | <a href="https://2016.igem.org/Main_Page" data-toggle="modal" data-target="#login-form" class="c-btn-border-opacity-04 c-btn btn-no-focus c-btn-header btn btn-sm c-btn-border-1x c-btn-white c-btn-circle | + | <a href="https://2016.igem.org/Main_Page" data-toggle="modal" data-target="#login-form" class="c-btn-border-opacity-04 c-btn btn-no-focus c-btn-header btn btn-sm c-btn-border-1x c-btn-white c-btn-circle c-btn-sbold"><i class="icon-chemistry"></i> iGEM </a> |
</li> | </li> | ||
<li class="c-quick-sidebar-toggler-wrapper"> | <li class="c-quick-sidebar-toggler-wrapper"> | ||
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<!-- BEGIN: PAGE CONTENT --> | <!-- BEGIN: PAGE CONTENT --> | ||
<!--BEGIN: SLIDE #1 --> | <!--BEGIN: SLIDE #1 --> | ||
− | <img alt="" src="https://static.igem.org/mediawiki/2016/ | + | <img alt="" src="https://static.igem.org/mediawiki/2016/d/d2/T--UofC_Calgary--project.jpg" data-bgposition="center center" data-bgfit="cover" data-bgrepeat="no-repeat" style="width: 100%; height: 100%;"> |
<!-- END: LAYOUT/SLIDERS/REVO-SLIDER-1 --> | <!-- END: LAYOUT/SLIDERS/REVO-SLIDER-1 --> | ||
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</div> | </div> | ||
<!-- End--> | <!-- End--> | ||
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+ | content: "" counter(lst-ctn-kix_k0t5285etr0u-7, lower-latin) ". " | ||
+ | } | ||
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+ | list-style-type: none | ||
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+ | } | ||
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+ | content: "" counter(lst-ctn-kix_k0t5285etr0u-5, lower-roman) ". " | ||
+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | counter-reset: lst-ctn-kix_ghlmud29dl0e-2 0 | ||
+ | } | ||
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+ | counter-reset: lst-ctn-kix_jwmzf1bv57q2-2 0 | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | content: "" counter(lst-ctn-kix_4jptj2dntjck-6, decimal) ". " | ||
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+ | } | ||
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+ | } | ||
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+ | counter-increment: lst-ctn-kix_ghlmud29dl0e-2 | ||
+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | counter-increment: lst-ctn-kix_ugk2xbe5oobt-7 | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_ugk2xbe5oobt-7, lower-latin) ". " | ||
+ | } | ||
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+ | } | ||
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+ | counter-reset: lst-ctn-kix_ma14gllav8iv-5 0 | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_qsfm83ibxmjm-0, decimal) ". " | ||
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+ | content: "" counter(lst-ctn-kix_ugk2xbe5oobt-2, lower-roman) ". " | ||
+ | } | ||
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+ | } | ||
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+ | content: "\0025cb " | ||
+ | } | ||
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+ | counter-reset: lst-ctn-kix_i1031kzh8e9u-3 0 | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_57ctmuaxoj27-0 | ||
+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | counter-reset: lst-ctn-kix_7cr24e9okmfb-6 0 | ||
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+ | content: "" counter(lst-ctn-kix_hd8gz1vih14v-3, decimal) ". " | ||
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+ | } | ||
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+ | } | ||
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+ | counter-increment: lst-ctn-kix_pgbvm1degh2i-3 | ||
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+ | counter-reset: lst-ctn-kix_57ctmuaxoj27-6 0 | ||
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+ | counter-reset: lst-ctn-kix_ghlmud29dl0e-3 0 | ||
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+ | counter-increment: lst-ctn-kix_i1031kzh8e9u-8 | ||
+ | } | ||
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+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_hd8gz1vih14v-3 | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_pt735i4wbfer-4, lower-latin) ". " | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_k0t5285etr0u-4 | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_9z0i9ngly08f-5 | ||
+ | } | ||
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+ | counter-reset: lst-ctn-kix_dt29yuk6g8lo-1 0 | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_jwmzf1bv57q2-1 | ||
+ | } | ||
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+ | counter-reset: lst-ctn-kix_pzhk3qcax242-1 0 | ||
+ | } | ||
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+ | counter-reset: lst-ctn-kix_4jptj2dntjck-0 0 | ||
+ | } | ||
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+ | } | ||
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+ | content: "" counter(lst-ctn-kix_57ctmuaxoj27-3, decimal) ". " | ||
+ | } | ||
+ | .lst-kix_6tdibfoyikr6-1>li { | ||
+ | counter-increment: lst-ctn-kix_6tdibfoyikr6-1 | ||
+ | } | ||
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+ | counter-reset: lst-ctn-kix_57ctmuaxoj27-4 0 | ||
+ | } | ||
+ | .lst-kix_k4s75dq808da-0>li { | ||
+ | counter-increment: lst-ctn-kix_k4s75dq808da-0 | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | counter-reset: lst-ctn-kix_3v73ll15y5y-6 0 | ||
+ | } | ||
+ | .lst-kix_e6kd68i2bo5-7>li { | ||
+ | counter-increment: lst-ctn-kix_e6kd68i2bo5-7 | ||
+ | } | ||
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+ | counter-reset: lst-ctn-kix_ghlmud29dl0e-1 0 | ||
+ | } | ||
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+ | counter-reset: lst-ctn-kix_6tdibfoyikr6-2 0 | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_9z0i9ngly08f-6 | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_t27wwiwmqyyi-4, lower-latin) ". " | ||
+ | } | ||
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+ | } | ||
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+ | counter-increment: lst-ctn-kix_e6kd68i2bo5-0 | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_jwmzf1bv57q2-5, lower-roman) ". " | ||
+ | } | ||
+ | .lst-kix_t27wwiwmqyyi-6>li { | ||
+ | counter-increment: lst-ctn-kix_t27wwiwmqyyi-6 | ||
+ | } | ||
+ | .lst-kix_57ctmuaxoj27-3>li { | ||
+ | counter-increment: lst-ctn-kix_57ctmuaxoj27-3 | ||
+ | } | ||
+ | .lst-kix_t27wwiwmqyyi-6>li:before { | ||
+ | content: "" counter(lst-ctn-kix_t27wwiwmqyyi-6, decimal) ". " | ||
+ | } | ||
+ | .lst-kix_wfo24hevzk6g-3>li { | ||
+ | counter-increment: lst-ctn-kix_wfo24hevzk6g-3 | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_jwmzf1bv57q2-7, lower-latin) ". " | ||
+ | } | ||
+ | .lst-kix_pt735i4wbfer-2>li:before { | ||
+ | content: "" counter(lst-ctn-kix_pt735i4wbfer-2, lower-roman) ". " | ||
+ | } | ||
+ | ol.lst-kix_pzhk3qcax242-3.start { | ||
+ | counter-reset: lst-ctn-kix_pzhk3qcax242-3 0 | ||
+ | } | ||
+ | .lst-kix_bg6npkoi2682-8>li { | ||
+ | counter-increment: lst-ctn-kix_bg6npkoi2682-8 | ||
+ | } | ||
+ | .lst-kix_li30fqzm7zj-6>li { | ||
+ | counter-increment: lst-ctn-kix_li30fqzm7zj-6 | ||
+ | } | ||
+ | ol.lst-kix_i1031kzh8e9u-2.start { | ||
+ | counter-reset: lst-ctn-kix_i1031kzh8e9u-2 0 | ||
+ | } | ||
+ | .lst-kix_k4s75dq808da-7>li:before { | ||
+ | content: "" counter(lst-ctn-kix_k4s75dq808da-7, lower-latin) ". " | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_3v73ll15y5y-7.start { | ||
+ | counter-reset: lst-ctn-kix_3v73ll15y5y-7 0 | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_ma14gllav8iv-1 { | ||
+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
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+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | content: "" counter(lst-ctn-kix_wfo24hevzk6g-3, decimal) ". " | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_wfo24hevzk6g-7, lower-latin) ". " | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_3v73ll15y5y-4 | ||
+ | } | ||
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+ | counter-reset: lst-ctn-kix_i1031kzh8e9u-6 0 | ||
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+ | content: "" counter(lst-ctn-kix_wfo24hevzk6g-0, decimal) ". " | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_wfo24hevzk6g-8, lower-roman) ". " | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_wfo24hevzk6g-2, lower-roman) ". " | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_wfo24hevzk6g-1, lower-latin) ". " | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_k4s75dq808da-2 | ||
+ | } | ||
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+ | list-style-type: none | ||
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+ | list-style-type: none | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | } | ||
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+ | list-style-type: none | ||
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+ | list-style-type: none | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
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+ | counter-reset: lst-ctn-kix_k4s75dq808da-6 0 | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_wfo24hevzk6g-6, decimal) ". " | ||
+ | } | ||
+ | ol.lst-kix_i1031kzh8e9u-8 { | ||
+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_wfo24hevzk6g-5, lower-roman) ". " | ||
+ | } | ||
+ | ol.lst-kix_i1031kzh8e9u-7 { | ||
+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
+ | .lst-kix_7cr24e9okmfb-4>li { | ||
+ | counter-increment: lst-ctn-kix_7cr24e9okmfb-4 | ||
+ | } | ||
+ | ol.lst-kix_qsfm83ibxmjm-4 { | ||
+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | counter-reset: lst-ctn-kix_ma14gllav8iv-2 0 | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_6tdibfoyikr6-4 { | ||
+ | list-style-type: none | ||
+ | } | ||
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+ | counter-reset: lst-ctn-kix_pgbvm1degh2i-7 0 | ||
+ | } | ||
+ | ol.lst-kix_6tdibfoyikr6-8 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_57ctmuaxoj27-5.start { | ||
+ | counter-reset: lst-ctn-kix_57ctmuaxoj27-5 0 | ||
+ | } | ||
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+ | counter-reset: lst-ctn-kix_ugk2xbe5oobt-7 0 | ||
+ | } | ||
+ | .lst-kix_ma14gllav8iv-2>li { | ||
+ | counter-increment: lst-ctn-kix_ma14gllav8iv-2 | ||
+ | } | ||
+ | ol.lst-kix_jwmzf1bv57q2-0.start { | ||
+ | counter-reset: lst-ctn-kix_jwmzf1bv57q2-0 0 | ||
+ | } | ||
+ | .lst-kix_2in1h2fvxudb-5>li { | ||
+ | counter-increment: lst-ctn-kix_2in1h2fvxudb-5 | ||
+ | } | ||
+ | ol.lst-kix_yzt1xhfk9inp-7.start { | ||
+ | counter-reset: lst-ctn-kix_yzt1xhfk9inp-7 0 | ||
+ | } | ||
+ | ol.lst-kix_qsfm83ibxmjm-0 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | .lst-kix_9a5qpki6xc2a-5>li { | ||
+ | counter-increment: lst-ctn-kix_9a5qpki6xc2a-5 | ||
+ | } | ||
+ | ol.lst-kix_qsfm83ibxmjm-1 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | .lst-kix_pt735i4wbfer-0>li { | ||
+ | counter-increment: lst-ctn-kix_pt735i4wbfer-0 | ||
+ | } | ||
+ | .lst-kix_qg27jrgwyb5q-1>li:before { | ||
+ | content: "\0025cb " | ||
+ | } | ||
+ | .lst-kix_qg27jrgwyb5q-2>li:before { | ||
+ | content: "\0025a0 " | ||
+ | } | ||
+ | .lst-kix_hd8gz1vih14v-5>li { | ||
+ | counter-increment: lst-ctn-kix_hd8gz1vih14v-5 | ||
+ | } | ||
+ | .lst-kix_qg27jrgwyb5q-3>li:before { | ||
+ | content: "\0025cf " | ||
+ | } | ||
+ | .lst-kix_9a5qpki6xc2a-1>li:before { | ||
+ | content: "" counter(lst-ctn-kix_9a5qpki6xc2a-1, lower-latin) ". " | ||
+ | } | ||
+ | .lst-kix_9a5qpki6xc2a-0>li:before { | ||
+ | content: "" counter(lst-ctn-kix_9a5qpki6xc2a-0, decimal) ". " | ||
+ | } | ||
+ | .lst-kix_ghlmud29dl0e-6>li { | ||
+ | counter-increment: lst-ctn-kix_ghlmud29dl0e-6 | ||
+ | } | ||
+ | .lst-kix_qg27jrgwyb5q-0>li:before { | ||
+ | content: "\0025cf " | ||
+ | } | ||
+ | .lst-kix_qg27jrgwyb5q-8>li:before { | ||
+ | content: "\0025a0 " | ||
+ | } | ||
+ | ol.lst-kix_4jptj2dntjck-6.start { | ||
+ | counter-reset: lst-ctn-kix_4jptj2dntjck-6 0 | ||
+ | } | ||
+ | .lst-kix_yuxi9iaet81w-6>li { | ||
+ | counter-increment: lst-ctn-kix_yuxi9iaet81w-6 | ||
+ | } | ||
+ | .lst-kix_qg27jrgwyb5q-7>li:before { | ||
+ | content: "\0025cb " | ||
+ | } | ||
+ | ol.lst-kix_57ctmuaxoj27-0.start { | ||
+ | counter-reset: lst-ctn-kix_57ctmuaxoj27-0 0 | ||
+ | } | ||
+ | .lst-kix_qg27jrgwyb5q-5>li:before { | ||
+ | content: "\0025a0 " | ||
+ | } | ||
+ | .lst-kix_qg27jrgwyb5q-6>li:before { | ||
+ | content: "\0025cf " | ||
+ | } | ||
+ | .lst-kix_qg27jrgwyb5q-4>li:before { | ||
+ | content: "\0025cb " | ||
+ | } | ||
+ | ol.lst-kix_k4s75dq808da-1.start { | ||
+ | counter-reset: lst-ctn-kix_k4s75dq808da-1 0 | ||
+ | } | ||
+ | .lst-kix_9z0i9ngly08f-7>li { | ||
+ | counter-increment: lst-ctn-kix_9z0i9ngly08f-7 | ||
+ | } | ||
+ | ol.lst-kix_ugk2xbe5oobt-2.start { | ||
+ | counter-reset: lst-ctn-kix_ugk2xbe5oobt-2 0 | ||
+ | } | ||
+ | .lst-kix_4jptj2dntjck-6>li { | ||
+ | counter-increment: lst-ctn-kix_4jptj2dntjck-6 | ||
+ | } | ||
+ | .lst-kix_e6kd68i2bo5-5>li { | ||
+ | counter-increment: lst-ctn-kix_e6kd68i2bo5-5 | ||
+ | } | ||
+ | .lst-kix_9a5qpki6xc2a-8>li:before { | ||
+ | content: "" counter(lst-ctn-kix_9a5qpki6xc2a-8, lower-roman) ". " | ||
+ | } | ||
+ | .lst-kix_yzt1xhfk9inp-6>li { | ||
+ | counter-increment: lst-ctn-kix_yzt1xhfk9inp-6 | ||
+ | } | ||
+ | .lst-kix_9a5qpki6xc2a-6>li:before { | ||
+ | content: "" counter(lst-ctn-kix_9a5qpki6xc2a-6, decimal) ". " | ||
+ | } | ||
+ | .lst-kix_9a5qpki6xc2a-5>li:before { | ||
+ | content: "" counter(lst-ctn-kix_9a5qpki6xc2a-5, lower-roman) ". " | ||
+ | } | ||
+ | .lst-kix_9a5qpki6xc2a-7>li:before { | ||
+ | content: "" counter(lst-ctn-kix_9a5qpki6xc2a-7, lower-latin) ". " | ||
+ | } | ||
+ | .lst-kix_9a5qpki6xc2a-2>li:before { | ||
+ | content: "" counter(lst-ctn-kix_9a5qpki6xc2a-2, lower-roman) ". " | ||
+ | } | ||
+ | .lst-kix_pzhk3qcax242-0>li { | ||
+ | counter-increment: lst-ctn-kix_pzhk3qcax242-0 | ||
+ | } | ||
+ | .lst-kix_9a5qpki6xc2a-3>li:before { | ||
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+ | counter-reset: lst-ctn-kix_v96mlbro49yx-7 0 | ||
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+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | content: "" counter(lst-ctn-kix_li30fqzm7zj-4, lower-latin) ". " | ||
+ | } | ||
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+ | list-style-type: none | ||
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+ | counter-increment: lst-ctn-kix_yzt1xhfk9inp-8 | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_bg6npkoi2682-4 | ||
+ | } | ||
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+ | list-style-type: none | ||
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+ | } | ||
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+ | } | ||
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+ | counter-increment: lst-ctn-kix_e6kd68i2bo5-3 | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_hd8gz1vih14v-4, lower-latin) ". " | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_pt735i4wbfer-2 | ||
+ | } | ||
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+ | counter-reset: lst-ctn-kix_wfo24hevzk6g-8 0 | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_ma14gllav8iv-4 | ||
+ | } | ||
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+ | } | ||
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+ | content: "" counter(lst-ctn-kix_hd8gz1vih14v-7, lower-latin) ". " | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_7cr24e9okmfb-2 | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_ugk2xbe5oobt-6 | ||
+ | } | ||
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+ | list-style-type: none | ||
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+ | } | ||
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+ | } | ||
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+ | counter-reset: lst-ctn-kix_qsfm83ibxmjm-4 0 | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_6tdibfoyikr6-4 | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | } | ||
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+ | counter-reset: lst-ctn-kix_7cr24e9okmfb-3 0 | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_k4s75dq808da-4 | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_2in1h2fvxudb-3, decimal) ". " | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_2in1h2fvxudb-5, lower-roman) ". " | ||
+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_l5833w3zpti7-8 | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_3v73ll15y5y-6 | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_2in1h2fvxudb-8, lower-roman) ". " | ||
+ | } | ||
+ | .lst-kix_4jptj2dntjck-8>li { | ||
+ | counter-increment: lst-ctn-kix_4jptj2dntjck-8 | ||
+ | } | ||
+ | .lst-kix_2in1h2fvxudb-6>li:before { | ||
+ | content: "" counter(lst-ctn-kix_2in1h2fvxudb-6, decimal) ". " | ||
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+ | } | ||
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+ | counter-reset: lst-ctn-kix_hd8gz1vih14v-6 0 | ||
+ | } | ||
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+ | } | ||
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+ | counter-reset: lst-ctn-kix_ma14gllav8iv-3 0 | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
+ | ul.lst-kix_lt1drns4mzkx-5 { | ||
+ | list-style-type: none | ||
+ | } | ||
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+ | counter-reset: lst-ctn-kix_k0t5285etr0u-7 0 | ||
+ | } | ||
+ | ul.lst-kix_lt1drns4mzkx-4 { | ||
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+ | } | ||
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+ | } | ||
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+ | content: "" counter(lst-ctn-kix_k4s75dq808da-3, decimal) ". " | ||
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+ | } | ||
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+ | } | ||
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+ | content: "" counter(lst-ctn-kix_yuxi9iaet81w-3, decimal) ". " | ||
+ | } | ||
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+ | list-style-type: none | ||
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+ | counter-increment: lst-ctn-kix_v96mlbro49yx-7 | ||
+ | } | ||
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+ | } | ||
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+ | counter-reset: lst-ctn-kix_taz15jv9qurq-7 0 | ||
+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | counter-increment: lst-ctn-kix_2in1h2fvxudb-4 | ||
+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | } | ||
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+ | counter-increment: lst-ctn-kix_3v73ll15y5y-3 | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_ugk2xbe5oobt-4, lower-latin) ". " | ||
+ | } | ||
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+ | list-style-type: none | ||
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+ | content: "" counter(lst-ctn-kix_ugk2xbe5oobt-1, lower-latin) ". " | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
+ | .lst-kix_pgbvm1degh2i-8>li { | ||
+ | counter-increment: lst-ctn-kix_pgbvm1degh2i-8 | ||
+ | } | ||
+ | ol.lst-kix_3v73ll15y5y-3 { | ||
+ | list-style-type: none | ||
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+ | counter-reset: lst-ctn-kix_li30fqzm7zj-1 0 | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_piaujb4qp22k-3 | ||
+ | } | ||
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+ | list-style-type: none | ||
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+ | } | ||
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+ | counter-increment: lst-ctn-kix_bg6npkoi2682-3 | ||
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+ | content: "" counter(lst-ctn-kix_qsfm83ibxmjm-1, lower-latin) ". " | ||
+ | } | ||
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+ | list-style-type: none | ||
+ | } | ||
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+ | content: "\0025cb " | ||
+ | } | ||
+ | .lst-kix_dt29yuk6g8lo-6>li:before { | ||
+ | content: "" counter(lst-ctn-kix_dt29yuk6g8lo-6, decimal) ". " | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_6tdibfoyikr6-7 | ||
+ | } | ||
+ | .lst-kix_3v73ll15y5y-0>li:before { | ||
+ | content: "" counter(lst-ctn-kix_3v73ll15y5y-0, decimal) ". " | ||
+ | } | ||
+ | ol.lst-kix_hd8gz1vih14v-3.start { | ||
+ | counter-reset: lst-ctn-kix_hd8gz1vih14v-3 0 | ||
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+ | content: "" counter(lst-ctn-kix_dt29yuk6g8lo-3, decimal) ". " | ||
+ | } | ||
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+ | counter-reset: lst-ctn-kix_jwmzf1bv57q2-3 0 | ||
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+ | } | ||
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+ | counter-reset: lst-ctn-kix_qsfm83ibxmjm-1 0 | ||
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+ | content: "" counter(lst-ctn-kix_3v73ll15y5y-8, lower-roman) ". " | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_wfo24hevzk6g-2 | ||
+ | } | ||
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+ | } | ||
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+ | content: "" counter(lst-ctn-kix_3v73ll15y5y-5, lower-roman) ". " | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_li30fqzm7zj-8 | ||
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+ | } | ||
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+ | content: "" counter(lst-ctn-kix_7cr24e9okmfb-6, decimal) ". " | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_li30fqzm7zj-1 | ||
+ | } | ||
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+ | .lst-kix_648mo3l1les-1>li:before { | ||
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+ | .lst-kix_bg6npkoi2682-7>li { | ||
+ | counter-increment: lst-ctn-kix_bg6npkoi2682-7 | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_e6kd68i2bo5-2, lower-roman) ". " | ||
+ | } | ||
+ | .lst-kix_57ctmuaxoj27-7>li:before { | ||
+ | content: "" counter(lst-ctn-kix_57ctmuaxoj27-7, lower-latin) ". " | ||
+ | } | ||
+ | ol.lst-kix_t27wwiwmqyyi-8.start { | ||
+ | counter-reset: lst-ctn-kix_t27wwiwmqyyi-8 0 | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_l5833w3zpti7-5, lower-roman) ". " | ||
+ | } | ||
+ | .lst-kix_k4s75dq808da-5>li { | ||
+ | counter-increment: lst-ctn-kix_k4s75dq808da-5 | ||
+ | } | ||
+ | .lst-kix_k4s75dq808da-1>li { | ||
+ | counter-increment: lst-ctn-kix_k4s75dq808da-1 | ||
+ | } | ||
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+ | counter-increment: lst-ctn-kix_7cr24e9okmfb-5 | ||
+ | } | ||
+ | .lst-kix_6tdibfoyikr6-3>li { | ||
+ | counter-increment: lst-ctn-kix_6tdibfoyikr6-3 | ||
+ | } | ||
+ | .lst-kix_ma14gllav8iv-1>li { | ||
+ | counter-increment: lst-ctn-kix_ma14gllav8iv-1 | ||
+ | } | ||
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+ | counter-reset: lst-ctn-kix_qsfm83ibxmjm-2 0 | ||
+ | } | ||
+ | .lst-kix_pzhk3qcax242-8>li:before { | ||
+ | content: "" counter(lst-ctn-kix_pzhk3qcax242-8, lower-roman) ". " | ||
+ | } | ||
+ | .lst-kix_pzhk3qcax242-3>li:before { | ||
+ | content: "" counter(lst-ctn-kix_pzhk3qcax242-3, decimal) ". " | ||
+ | } | ||
+ | ol.lst-kix_k0t5285etr0u-8.start { | ||
+ | counter-reset: lst-ctn-kix_k0t5285etr0u-8 0 | ||
+ | } | ||
+ | .lst-kix_taz15jv9qurq-5>li:before { | ||
+ | content: "" counter(lst-ctn-kix_taz15jv9qurq-5, lower-roman) ". " | ||
+ | } | ||
+ | .lst-kix_e6kd68i2bo5-2>li { | ||
+ | counter-increment: lst-ctn-kix_e6kd68i2bo5-2 | ||
+ | } | ||
+ | .lst-kix_taz15jv9qurq-0>li:before { | ||
+ | content: "" counter(lst-ctn-kix_taz15jv9qurq-0, decimal) ". " | ||
+ | } | ||
+ | .lst-kix_taz15jv9qurq-8>li:before { | ||
+ | content: "" counter(lst-ctn-kix_taz15jv9qurq-8, lower-roman) ". " | ||
+ | } | ||
+ | .lst-kix_l5833w3zpti7-2>li:before { | ||
+ | content: "" counter(lst-ctn-kix_l5833w3zpti7-2, lower-roman) ". " | ||
+ | } | ||
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+ | content: "\0025cf " | ||
+ | } | ||
+ | .lst-kix_57ctmuaxoj27-4>li { | ||
+ | counter-increment: lst-ctn-kix_57ctmuaxoj27-4 | ||
+ | } | ||
+ | .lst-kix_pzhk3qcax242-3>li { | ||
+ | counter-increment: lst-ctn-kix_pzhk3qcax242-3 | ||
+ | } | ||
+ | .lst-kix_7cr24e9okmfb-3>li:before { | ||
+ | content: "" counter(lst-ctn-kix_7cr24e9okmfb-3, decimal) ". " | ||
+ | } | ||
+ | .lst-kix_eohmc5xvvgx3-8>li:before { | ||
+ | content: "\0025a0 " | ||
+ | } | ||
+ | .lst-kix_e6kd68i2bo5-5>li:before { | ||
+ | content: "" counter(lst-ctn-kix_e6kd68i2bo5-5, lower-roman) ". " | ||
+ | } | ||
+ | .lst-kix_pzhk3qcax242-0>li:before { | ||
+ | content: "" counter(lst-ctn-kix_pzhk3qcax242-0, decimal) ". " | ||
+ | } | ||
+ | .lst-kix_ykbu90jvmajk-7>li:before { | ||
+ | content: "\0025cb " | ||
+ | } | ||
+ | ol.lst-kix_e6kd68i2bo5-8 { | ||
+ | list-style-type: none | ||
+ | } | ||
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+ | content: "" counter(lst-ctn-kix_yzt1xhfk9inp-8, lower-roman) ". " | ||
+ | } | ||
+ | .lst-kix_t27wwiwmqyyi-8>li { | ||
+ | counter-increment: lst-ctn-kix_t27wwiwmqyyi-8 | ||
+ | } | ||
+ | .lst-kix_li30fqzm7zj-2>li:before { | ||
+ | content: "" counter(lst-ctn-kix_li30fqzm7zj-2, lower-roman) ". " | ||
+ | } | ||
+ | ol.lst-kix_e6kd68i2bo5-3 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | .lst-kix_ghlmud29dl0e-4>li { | ||
+ | counter-increment: lst-ctn-kix_ghlmud29dl0e-4 | ||
+ | } | ||
+ | ol.lst-kix_e6kd68i2bo5-2 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_e6kd68i2bo5-1 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | .lst-kix_6tdibfoyikr6-0>li { | ||
+ | counter-increment: lst-ctn-kix_6tdibfoyikr6-0 | ||
+ | } | ||
+ | ol.lst-kix_e6kd68i2bo5-0 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | .lst-kix_4jptj2dntjck-4>li { | ||
+ | counter-increment: lst-ctn-kix_4jptj2dntjck-4 | ||
+ | } | ||
+ | ol.lst-kix_e6kd68i2bo5-7 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_e6kd68i2bo5-6 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_e6kd68i2bo5-5 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_e6kd68i2bo5-4 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | .lst-kix_ma14gllav8iv-1>li:before { | ||
+ | content: "" counter(lst-ctn-kix_ma14gllav8iv-1, lower-latin) ". " | ||
+ | } | ||
+ | .lst-kix_k4s75dq808da-8>li { | ||
+ | counter-increment: lst-ctn-kix_k4s75dq808da-8 | ||
+ | } | ||
+ | .lst-kix_yzt1xhfk9inp-0>li:before { | ||
+ | content: "" counter(lst-ctn-kix_yzt1xhfk9inp-0, decimal) ". " | ||
+ | } | ||
+ | .lst-kix_v96mlbro49yx-0>li { | ||
+ | counter-increment: lst-ctn-kix_v96mlbro49yx-0 | ||
+ | } | ||
+ | .lst-kix_ibs7h5r7tw81-3>li:before { | ||
+ | content: "\0025cf " | ||
+ | } | ||
+ | .lst-kix_9z0i9ngly08f-4>li { | ||
+ | counter-increment: lst-ctn-kix_9z0i9ngly08f-4 | ||
+ | } | ||
+ | ol.lst-kix_hd8gz1vih14v-7.start { | ||
+ | counter-reset: lst-ctn-kix_hd8gz1vih14v-7 0 | ||
+ | } | ||
+ | .lst-kix_ur2od9za9cv9-8>li:before { | ||
+ | content: "\0025a0 " | ||
+ | } | ||
+ | .lst-kix_9z0i9ngly08f-2>li:before { | ||
+ | content: "" counter(lst-ctn-kix_9z0i9ngly08f-2, lower-roman) ". " | ||
+ | } | ||
+ | .lst-kix_pgbvm1degh2i-8>li:before { | ||
+ | content: "" counter(lst-ctn-kix_pgbvm1degh2i-8, lower-roman) ". " | ||
+ | } | ||
+ | ol.lst-kix_yzt1xhfk9inp-1 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_yzt1xhfk9inp-0 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_jwmzf1bv57q2-7.start { | ||
+ | counter-reset: lst-ctn-kix_jwmzf1bv57q2-7 0 | ||
+ | } | ||
+ | .lst-kix_wfo24hevzk6g-5>li { | ||
+ | counter-increment: lst-ctn-kix_wfo24hevzk6g-5 | ||
+ | } | ||
+ | .lst-kix_ur2od9za9cv9-0>li:before { | ||
+ | content: "\0025cf " | ||
+ | } | ||
+ | ol.lst-kix_yuxi9iaet81w-4.start { | ||
+ | counter-reset: lst-ctn-kix_yuxi9iaet81w-4 0 | ||
+ | } | ||
+ | ol.lst-kix_qsfm83ibxmjm-8.start { | ||
+ | counter-reset: lst-ctn-kix_qsfm83ibxmjm-8 0 | ||
+ | } | ||
+ | .lst-kix_ghlmud29dl0e-1>li:before { | ||
+ | content: "" counter(lst-ctn-kix_ghlmud29dl0e-1, lower-latin) ". " | ||
+ | } | ||
+ | .lst-kix_krimb71z9a9i-4>li:before { | ||
+ | content: "\0025cb " | ||
+ | } | ||
+ | .lst-kix_57ctmuaxoj27-4>li:before { | ||
+ | content: "" counter(lst-ctn-kix_57ctmuaxoj27-4, lower-latin) ". " | ||
+ | } | ||
+ | .lst-kix_9a5qpki6xc2a-8>li { | ||
+ | counter-increment: lst-ctn-kix_9a5qpki6xc2a-8 | ||
+ | } | ||
+ | ol.lst-kix_taz15jv9qurq-6.start { | ||
+ | counter-reset: lst-ctn-kix_taz15jv9qurq-6 0 | ||
+ | } | ||
+ | .lst-kix_k0t5285etr0u-6>li { | ||
+ | counter-increment: lst-ctn-kix_k0t5285etr0u-6 | ||
+ | } | ||
+ | .lst-kix_taz15jv9qurq-4>li { | ||
+ | counter-increment: lst-ctn-kix_taz15jv9qurq-4 | ||
+ | } | ||
+ | ol.lst-kix_yuxi9iaet81w-7.start { | ||
+ | counter-reset: lst-ctn-kix_yuxi9iaet81w-7 0 | ||
+ | } | ||
+ | .lst-kix_pt735i4wbfer-6>li { | ||
+ | counter-increment: lst-ctn-kix_pt735i4wbfer-6 | ||
+ | } | ||
+ | ol.lst-kix_ghlmud29dl0e-8 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_ghlmud29dl0e-7 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_ghlmud29dl0e-6 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_ghlmud29dl0e-5 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_qsfm83ibxmjm-5.start { | ||
+ | counter-reset: lst-ctn-kix_qsfm83ibxmjm-5 0 | ||
+ | } | ||
+ | ol.lst-kix_ghlmud29dl0e-4 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_ghlmud29dl0e-3 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_ghlmud29dl0e-2 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_ghlmud29dl0e-1 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | ol.lst-kix_ghlmud29dl0e-0 { | ||
+ | list-style-type: none | ||
+ | } | ||
+ | .lst-kix_t27wwiwmqyyi-5>li:before { | ||
+ | content: "" counter(lst-ctn-kix_t27wwiwmqyyi-5, lower-roman) ". " | ||
+ | } | ||
+ | .lst-kix_i1031kzh8e9u-4>li:before { | ||
+ | content: "" counter(lst-ctn-kix_i1031kzh8e9u-4, lower-latin) ". " | ||
+ | } | ||
+ | .lst-kix_jwmzf1bv57q2-6>li:before { | ||
+ | content: "" counter(lst-ctn-kix_jwmzf1bv57q2-6, decimal) ". " | ||
+ | } | ||
+ | .lst-kix_pgbvm1degh2i-5>li { | ||
+ | counter-increment: lst-ctn-kix_pgbvm1degh2i-5 | ||
+ | } | ||
+ | .lst-kix_yuxi9iaet81w-5>li { | ||
+ | counter-increment: lst-ctn-kix_yuxi9iaet81w-5 | ||
+ | } | ||
+ | .lst-kix_6tdibfoyikr6-7>li:before { | ||
+ | content: "" counter(lst-ctn-kix_6tdibfoyikr6-7, lower-latin) ". " | ||
+ | } | ||
+ | .lst-kix_pt735i4wbfer-3>li:before { | ||
+ | content: "" counter(lst-ctn-kix_pt735i4wbfer-3, decimal) ". " | ||
+ | } | ||
+ | ol.lst-kix_hd8gz1vih14v-8.start { | ||
+ | counter-reset: lst-ctn-kix_hd8gz1vih14v-8 0 | ||
+ | } | ||
+ | ol.lst-kix_yuxi9iaet81w-6.start { | ||
+ | counter-reset: lst-ctn-kix_yuxi9iaet81w-6 0 | ||
+ | } | ||
+ | .lst-kix_yzt1xhfk9inp-1>li { | ||
+ | counter-increment: lst-ctn-kix_yzt1xhfk9inp-1 | ||
+ | } | ||
+ | .lst-kix_9a5qpki6xc2a-1>li { | ||
+ | counter-increment: lst-ctn-kix_9a5qpki6xc2a-1 | ||
+ | } | ||
+ | ol.lst-kix_taz15jv9qurq-8.start { | ||
+ | counter-reset: lst-ctn-kix_taz15jv9qurq-8 0 | ||
+ | } | ||
+ | ol.lst-kix_jwmzf1bv57q2-8.start { | ||
+ | counter-reset: lst-ctn-kix_jwmzf1bv57q2-8 0 | ||
+ | } | ||
+ | .lst-kix_dt29yuk6g8lo-4>li { | ||
+ | counter-increment: lst-ctn-kix_dt29yuk6g8lo-4 | ||
+ | } | ||
+ | ol.lst-kix_qsfm83ibxmjm-6.start { | ||
+ | counter-reset: lst-ctn-kix_qsfm83ibxmjm-6 0 | ||
+ | } | ||
+ | .lst-kix_4jptj2dntjck-7>li:before { | ||
+ | content: "" counter(lst-ctn-kix_4jptj2dntjck-7, lower-latin) ". " | ||
+ | } | ||
+ | .lst-kix_k4s75dq808da-0>li:before { | ||
+ | content: "" counter(lst-ctn-kix_k4s75dq808da-0, decimal) ". " | ||
+ | } | ||
+ | .lst-kix_k0t5285etr0u-2>li:before { | ||
+ | content: "" counter(lst-ctn-kix_k0t5285etr0u-2, lower-roman) ". " | ||
+ | } | ||
+ | ol { | ||
+ | margin: 0; | ||
+ | padding: 0 | ||
+ | } | ||
+ | table td, | ||
+ | table th { | ||
+ | padding: 0 | ||
+ | } | ||
+ | .c40 { | ||
+ | border-right-style: solid; | ||
+ | padding: 5pt 5pt 5pt 5pt; | ||
+ | border-bottom-color: #000000; | ||
+ | border-top-width: 1pt; | ||
+ | border-right-width: 1pt; | ||
+ | border-left-color: #000000; | ||
+ | vertical-align: top; | ||
+ | border-right-color: #000000; | ||
+ | border-left-width: 1pt; | ||
+ | border-top-style: solid; | ||
+ | border-left-style: solid; | ||
+ | border-bottom-width: 1pt; | ||
+ | width: 103pt; | ||
+ | border-top-color: #000000; | ||
+ | border-bottom-style: solid | ||
+ | } | ||
+ | .c17 { | ||
+ | border-right-style: solid; | ||
+ | padding: 5pt 5pt 5pt 5pt; | ||
+ | border-bottom-color: #000000; | ||
+ | border-top-width: 1pt; | ||
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+ | </style> | ||
+ | </head> | ||
+ | |||
+ | <body class="c32 c41"> | ||
+ | <p class="c42"><span class="c4 c3">BioTarget</span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.5eec12f75dbfe32a5188a821362d110487e7d5e2"></a> | ||
+ | <a id="t.0"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c43"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c4 c3 c32">Double-Stranded Break Assay using Immunofluorescence</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c20"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c2 c28"><span class="c9 c3"></span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Exposure to ionizing radiation causes DNA double strand breaks (DSBs) in cells, which are naturally repaired by cells</span><span class="c3 c32"> over time. DSBs in nuclear DNA can be visualized in cells by immunofluorescence staining of DSB markers – namely 53BP1, a protein which binds near DSBs. As a result, nuclear foci form, each corresponding to a DSB. Over time, the number of foci are reduced due to DNA repair. To test for radioprotection by mBBI, we wanted to study the repair kinetics in cells treated with mBBI against a control.</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Cells were grown to confluency, irradiated and fixed at various time points over 24 hours. Immunofluorescence staining was performed to visualize 53BP1</span><span class="c3 c22"> </span><span class="c9 c3 c32">and the resulting foci were counted.</span> | ||
+ | </p> | ||
+ | <p class="c2 c28"><span class="c9 c3"></span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Glass coverslips and microscopy slides</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">1BR3 primary fibroblast cells in Modified Eagle Medium (MEM) - (10^5 cells/ml)</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">30 mm Cell Culture Plates</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">1x PBS</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">1x PBS solution with 3% PFA (w/v) and 2% (w/v) sucrose</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">1x PBS solution with 2% (w/v) Bovine Serum Albumin</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">1x PBS solution with 0.2% (v/v) Triton X100</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">1x PBS solution with 0.1μg/ml 4’,6-diamidino-2-phenylindole (DAPI)</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Mouse anti-53BP1 Antibody</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Rabbit anti-H2AX Antibody</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Cy3 anti-Mouse Antibody</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">FITC anti-Rabbit Antibody</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Mounting medium for fluorescence (Flouromount G or Vectashield)</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32"></span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <ol class="c10 lst-kix_dt29yuk6g8lo-0 start" start="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Prep and Irradiation</b></span></p> | ||
+ | |||
+ | <li class="c8"><span class="c3 c32">Plate 2 mLs of 1BR3 cells at the density of 10^5 cells/ml MEM into 22 30 mm plates containing glass coverslips. Incubate cells until 100% confluency is observed on the glass coverslips using a brightfield microscope.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Treat the confluent plates with mBBI (as outlined in Table 1) and incubate at 37℃ with 5% CO<sub<2</sub> for 6 hours.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Irradiate plates which are intended for irradiation (outlined in Table 1) with 2 Gy. We used a Gammacell 1000 irradiator (MDS Nordion).</span> | ||
+ | </li> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Immunofluorescence Staining</b></span></p> | ||
+ | <li class="c8"><span class="c3 c32">When plates reach their intended time point, remove the media and wash in 1X PBS. Perform washes by pouring PBS into the dish beside the coverslip, with special care not to pour media directly onto the coverslips, which could dislodge the cells. All washes hereon are done in this manner.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Cells on glass coverslips are fixed for 10 minutes by adding 100 mL of 1X PBS solution with 3% PFA (w/v) and 2% sucrose (w/v) to the top of the coverslip, forming a dome on the coverslip.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Wash cells twice in 1X PBS and store in 1X PBS until all plates have been fixed at their corresponding time points.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Add 100 mL of 1X PBS solution with 0.2% Triton X100 (v/v) to the cells for 3 minutes to allow permeabilization.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Incubate cells in 100 mL of a 1:800 dilution of Mouse anti-53BP1 and Rabbit anti-H2AX in 2% BSA for 1 hour. Pipet the solution dropwise onto the coverslips.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Wash cells three times in 1X PBS. 100 μl of secondary antibodies Cy3 anti-Mouse and FITC anti-Rabbit pipetting dropwise onto the coverslips. Cells are incubated in this solution for 20 minutes in the dark, because FITC and Cy3 are sensitive to light and lose their fluorescence if prematurely exposed.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Wash cells 3 times in 1X PBS.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Pour 2 mls of 1X PBS containing 0.1 mg/mL DAPI into the dishes and leave on for 10 mins.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Wash cells 3 times in 1X PBS.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Microscopy slides were prepared by adding one drop of Vectashield onto the slide.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Carefully lift coverslips from the cell culture dishes using a razor blade or sharp edged inplement and plate onto microscopy slides such that the cell bed (top of the coverslip) is placed face down on the slide.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Seal the edges of the coverslip onto the slide by brushing on clear nail polish over the edges.</span> | ||
+ | </li> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Counting Foci</b></span></p> | ||
+ | <li class="c8"><span class="c3 c32">Randomize cells by using opaque tape to cover their labels and get them randomly assorted by another lab member. Number them on the tape for ease of reference when counting.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Foci can be counted on an epifluorescent microscope under red and green channels.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <p class="c34"><span style="overflow: hidden; display: inline-block; margin: 0.00px 0.00px; border: 0.00px solid #000000; transform: rotate(0.00rad) translateZ(0px); -webkit-transform: rotate(0.00rad) translateZ(0px); width: 600.00px; height: 381.33px;"><img alt="" src="https://static.igem.org/mediawiki/2016/7/77/T--UofC_Calgary--H2AX_Experiments_Table1.jpg" style="width: 600.00px; height: 381.33px;""></span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Table 1: Experimental Outline of mBBI treatment and Ionizing Radiation. mBBI treatment was administered at 30uM and cells were irradiated at 2 Gy.</span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.d9801b15d4fa36a394507d0e6746bfa2bfd57ee4"></a> | ||
+ | <a id="t.1"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c43"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c24 c21 c4 c3">Clonogenic Cell Survival Assay</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c20"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Ionizing radiation is capable of killing cells by damaging their DNA, causing single and double strand breaks, which trigger apoptosis. If the DNA damage can be repaired, the cells survive. If not, they will die. To test whether mBBI elicits radioprotection, the survival of cells can be assessed. The purpose of this assay is to assess the viability of cells when treated with ionizing radiation, with and without mBBI.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">MEM media with 15% Fetal Bovine Serum (v/v) and 10% Penicillin-Streptomycin (v/v)</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">T-75 flasks</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">60 mm Cell culture plates</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">1BR3 cells</span><p class="c38 c34"><span class="c3 c32"> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Trypan Blue Dye</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">0.25% Trypsin EDTA</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">15ml Centrifuge Tube</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Preparation of Cell Lines Prior to Setting up Clonogenic Cell Survival</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_dt29yuk6g8lo-0" start="21"> | ||
+ | <li class="c8"><span class="c3 c32">Label 6 T-75 flasks with a known number of cells and radiation in Gy (0 and 5). Add 5 ml of growth medium to the flasks and keep them in a biosafety hood.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Trypsinize the stock flask of cells to be tested for radiosensitivity.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Obtain a cell count, then add 250,000 cells to the 5ml medium in the T-75 flask.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Place T-75 flasks with the cell cultures in a 37°C incubator with 5% CO<sub>2</sub>, and keep cap to the flask loose to allow gas exchange.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Allow the cells to settle and attach as a monolayer.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Irradiation of Flasks and Performance of Plating Experiment for Clonogenic Assay</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_dt29yuk6g8lo-0" start="26"> | ||
+ | <li class="c8"><span class="c3 c32">Label all 60 mm plates according to cell line, drug and radiation exposure. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Place 5mL of complete medium in each plate</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Label all 15 ml tubes according to 1:1, 1:10, 1:100 for each Gy dose level (0 and 5).</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Put 4.5 ml of complete medium into the 1:10 and 1:100 tubes, but not the 1:1 tubes.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Ensure that the Coulter counter is set to the appropriate size parameter for the cell line.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Irradiate the flasks at the appropriate dose.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Starting with the 0 Gy flask, aspirate the medium and then rinse the cells with PBS, then trypsinize.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Place these harvested cells in the 15ml tube labelled as 0 Gy, 1:1. Take 100 ul of the 0 Gy, 1:1 solution and place in the counting vial for 0 Gy. Now aspirate, rinse cells with PBS and trypsinize the next flask, 5 Gy. While the 5 Gy flask is on the warming tray, count the 0 Gy counting tray using the Coulter counter and record this data.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">As before, place the harvested 5 Gy cells into the 15 ml tube labelled 5 Gy, 1:1. Take 100 ul of the 5 Gy 1:1 solution and put into the 5 Gy counting vial. Use a Coulter counter to count cells as before.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Serial Dilutions and Incubation</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_dt29yuk6g8lo-0" start="35"> | ||
+ | <li class="c8"><span class="c3 c32">Resuspend the cell pellet of the 15 ml tube.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Place 0.5 ml of the 1:1 dilution into the 1:10 dilution tubes of both the 0 Gy and 5 Gy tubes.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Calculate the volume of the cell solution needed to plate ~50-100 cells.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Place this volume of the appropriate dilution (probably 1:100) onto the appropriate 60 mm plate. Spread this medium drop by drop, ensuring that the plate is covered equally. You can shake the plate to distribute the culture evenly, but do not swirl. Only back-and-forth motions.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Place all plates into the incubator. Incubate for 14 days at 37C with 5% CO2.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Staining of Plates</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_dt29yuk6g8lo-0" start="40"> | ||
+ | <li class="c8"><span class="c3 c32">Take out all plates from the incubator and aspirate media with vacuum </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Add 1mL fixing solution to each of the plates (3% acetic acid, 8% methanol, 89% dH2O)</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Allow cells to sit in fix solution for 2 minutes, then aspirate fix solution</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Add 1mL stain solution to each of the plates (0.2% (w/v) Gentian violet, 10% formalin in PBS)</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Let plates sit in stain solution for 5 minutes and aspirate stain solution</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Add dH<sub>2</sub>O at room temperature to the plates to de-stain excess Gentian Violet. Repeat twice, or until only the colonies are stained as opposed to the plate.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Let the plates air-dry overnight. Count the next day.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Counting of Colonies</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_dt29yuk6g8lo-0" start="47"> | ||
+ | <li class="c8"><span class="c3 c32">Take the air-dried plates and place in ColCount machine after blanking the machine. Count using small colony reference program and record data in a table. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Calculate plating efficiency (PE) by finding the average colony counts for the 3 plates and dividing that by the number of cells plated, then multiply by 100%. ((Ave colonies counted / Number of cells plated) X 100 %).</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">To find the the surviving fraction (SF), take the PE of the treated sample and divide that by the PE of the control, then multiply by 100%. ((PE treated / PE control) X 100%)</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c42"><span class="c21 c4 c3">CHASSIS</span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.932a91edf21721ecb672f843439bce66a533bda0"></a> | ||
+ | <a id="t.2"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c43"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c24 c21 c4 c3">Rehydration of Registry DNA</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">We used standard parts from the iGEM registry to ligate our synthesized genetic constructs into the pSB1c3 backbone. We also used BBa_J04450 (pSB1c3 backbone containing RFP) as a positive control to confirm transformation of <i>E. coli</i> and <i>B. subtilis</i>. </span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">iGEM 2016 distribution kit</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">ddH<sub>2</sub>O</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Protocol</b></span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <ol class="c10 lst-kix_9a5qpki6xc2a-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Add 10 uL ddH<sub>2</sub>O to desired well (it will become red).</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Pipette up and down.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Incubate at room temperature for 10 minutes.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Transform cells with 1 </span><span class="c3 c12">μ</span><span class="c9 c3">L of DNA. Store at -20°C.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.778df1fdf79b373f8d245e12314bd76b2313f784"></a> | ||
+ | <a id="t.3"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c24 c21 c4 c3">Rehydration of IDT Synthesized DNA</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">We ordered genetic constructs from IDT to clone into the pSB1c3 backbone.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Synthesized DNA from IDT</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">ddH<sub>2</sub>O</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <ol class="c10 lst-kix_k0t5285etr0u-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Centrifuge tube containing DNA for 3-5 seconds at 3000 g, ensuring all material is at the bottom of the tube.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Add ddH</span><span class="c3 c7">2</span><span class="c3">O to reach a final concentration of 50 ng/</span><span class="c5 c3">μL.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Vortex.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Incubate at 50°C for 20 minutes.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Vortex and centrifuge. Store at -80°C.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.f41d422787b3714a36fb6297c7af5746489e5fca"></a> | ||
+ | <a id="t.4"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c21 c4 c3">Plasmid MiniPrep from </span><span class="c21 c4 c3 c15">Escherichia coli </span><span class="c21 c4 c3">and </span><span class="c36 c21 c4 c3 c15">Bacillus subtilis</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Plasmids were amplified in </span><span class="c3 c15">E. coli </span><span class="c3">cells in order to use them for confirmation of ligation and transformation of </span><span class="c3 c15">B. subtilis</span><span class="c9 c3">.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">2.5 mL overnight culture of transformed bacteria in appropriate antibiotic in 16x125 mm culture tube</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Resuspension buffer (store at 4°C)</span> | ||
+ | </p> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">50 mM Tris-HCl ,pH 8</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">10 mM EDTA</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">100 </span><span class="c12 c3">μg/mL RNase A</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p class="c38 c34"><span class="c3 c32">Lysis buffer</span> | ||
+ | </p> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">200 mM NaOH</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">1% (v/v) SDS</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p class="c38 c34"><span class="c3 c32">Precipitation buffer</span> | ||
+ | </p> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">3 M CH</span><span class="c3 c7">3</span><span class="c3">CO</span><span class="c3 c7">2</span><span class="c9 c3">K, pH 5.5</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p class="c38 c34"><span class="c3 c32">Isopropanol</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">70% ethanol</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Table-top centrifuge</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">2 mL microcentrifuge tubes</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">1.5 mL microcentrifuge tubes</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <ol class="c10 lst-kix_6tdibfoyikr6-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Grow 2.5 mL of transformed culture overnight in Luria-Bertani broth with appropriate antibiotic.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Transfer 2 mL to a 2 mL microcentrifuge tube and pellet the cells by spinning at 3500 g for 1 minute. Discard supernatant.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Resuspend pellet in 300 </span><span class="c5 c3">μL Resuspension buffer.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Add 300 μL Lysis buffer, invert gently and incubate at room temperature for 3-5 minutes.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Add 300 μL Precipitation buffer, invert gently. A white precipitate should form.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Centrifuge at 14,000 g for 10 minutes at room temperature.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Retain supernatant in a clean 1.5 mL microcentrifuge tube.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Add 650 μL isopropanol, gently invert and incubate at room temperature for 10 minutes.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Centrifuge at 14,000 g for 10 minutes at 4°C. Discard supernatant.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Wash pellet with 500 μL cold 70% ethanol. Add to microcentrifuge tube, do not resuspend.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Centrifuge at 14,000 g for 5 minutes at 4°C. Discard supernatant.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Dry pellet in speed vac for 10 minutes.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Resuspend pellet in ddH<sub>2</sub>O and store at -20°C.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.5c4ad24f1f99770a47939c9912f7ad338bb32793"></a> | ||
+ | <a id="t.5"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c24 c21 c4 c3">Restriction Digest</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Restriction digests were performed on plasmid backbones and synthesized DNA inserts before ligating them together, as well as to confirm ligation and transformation of cells with desired plasmids.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">DNA</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Restriction enzymes</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">100X Bovine Serum Albumin (BSA)</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">10X appropriate buffer</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">ddH<sub>2</sub>O</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">0.2 mL PCR tubes</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <ol class="c10 lst-kix_piaujb4qp22k-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Into a 0.2 mL PCR tube, add the following:</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">1 μg DNA</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">1 μL 100X BSA</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">1 μL restriction enzyme 1</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">1 μL restriction enzyme 2</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">2 μL 10X appropriate buffer</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">ddH<sub>2</sub>O to a total volume of 20 μL</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ol class="c10 lst-kix_piaujb4qp22k-0" start="2"> | ||
+ | <li class="c8"><span class="c3 c32">Incubate tube at 37°C for 1 hour.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Deactivate restriction enzymes via heat shock by incubating tube at 80°C for 20 minutes.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.89d02941cbafa0f4832690098e46fa246c40b98f"></a> | ||
+ | <a id="t.6"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c24 c21 c4 c3">Antarctic Phosphatase Treatment</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Vectors were treated with Antarctic phosphatase to remove their 5’-phosphate group and prevent them from self-ligating before addition of the digested insert.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Digested DNA vector</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Antarctic phosphatase buffer</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Antarctic phosphatase</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">ddH<sub>2</sub>O</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">0.2 mL PCR tubes</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <ol class="c10 lst-kix_li30fqzm7zj-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">To vector tube from restriction digest, add:</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">1 μL 10X Antarctic phosphatase buffer</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">1 μL Antarctic phosphatase</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">8 μL ddH<sub>2</sub>O</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ol class="c10 lst-kix_li30fqzm7zj-0" start="2"> | ||
+ | <li class="c8"><span class="c3 c32">Incubate tube at 37°C for 30 minutes.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Deactivate Antarctic phosphatase via heat shock by incubating tube at 80°C for 20 minutes.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.f0c56e57be06d9762de4cbbdec85d50c005ab072"></a> | ||
+ | <a id="t.7"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c21 c4 c3 c24">Ligation of DNA Inserts to Plasmid Backbones</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c40" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c27" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Synthesized DNA inserts were ligated into plasmid backbones for propagation and use in <i>E. coli</i> and <i>B. subtilis</i> as well as for submission to the registry.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c40" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c27" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Digested vector DNA</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Digested insert DNA</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">10X DNA ligase buffer (from New England Biolabs)</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">T4 DNA ligase (1 U/</span><span class="c5 c3">μL) (from New England Biolabs)</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">ddH<sub>2</sub>O</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">0.2 mL PCR tubes</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c40" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c27" colspan="1" rowspan="1"> | ||
+ | <ol class="c10 lst-kix_57ctmuaxoj27-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">To a 0.2 mL PCR tube, add:</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">50 ng digested vector DNA</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">appropriate amount of digested insert DNA to give a 3:1 molar ratio of insert:vector</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">1 μL T4 DNA ligase</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">2 μL 10X T4 DNA ligase buffer</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">ddH<sub>2</sub>O to a total volume of 20 μL</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ol class="c10 lst-kix_57ctmuaxoj27-0" start="2"> | ||
+ | <li class="c8"><span class="c3 c32">Incubate tube at room temperature overnight.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Use 10 μL to transform cells, store at -20°C.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.feb65ebf4bd99d60e028a6b9705de5bd82c532e9"></a> | ||
+ | <a id="t.8"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c21 c4 c3">Colony PCR for </span><span class="c21 c4 c3 c15">Escherichia coli </span><span class="c21 c4 c3">and </span><span class="c21 c4 c3 c15">Bacillus subtilis </span><span class="c24 c21 c4 c3">using the KAPA HiFi PCR Kit</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Colony PCR was used to confirm the presence of plasmids with desired ligated insert in transformed <i>E. coli </i>and <i>B. subtilis</i> >cells.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Transformed bacterial colony on agar plate</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">10X Taq polymerase buffer</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Taq DNA polymerase (% U/</span><span class="c12 c3">μL)</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">10X Buffer A</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">10 mM dNTP</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">10 </span><span class="c5 c3">μM forward primer</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">10 μM reverse primer</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">PCR-grade ddH<sub>2</sub>O</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">0.2 mL PCR tubes</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Sample Preparation</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_k4s75dq808da-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Add 4 μL of PCR-grade ddH<sub>2</sub>O to 0.2 mL PCR tube.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Using aseptic technique, pick a colony and touch it with a sterile pipette tip and place it in the PCR tube for 5-10 seconds.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">To each PCR tube, add:</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">0.2 μL Taq DNA polymerase</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">5 μL 10X Buffer A</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">1 μL 10 mM dNTP</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">2 μL 10 μM forward primer</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">2 μL 10 μM reverse primer</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">8 μL PCR-grade ddH<sub>2</sub>O</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Running PCR</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_k4s75dq808da-0" start="10"> | ||
+ | <li class="c0"><span class="c3 c32">Run PCR in a thermal cycler under the following conditions:</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">Initial denaturation: 95°C for 3 minutes</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">Denature: 95°C for 30 seconds</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">Anneal: T</span><span class="c12 c3 c7">m</span><span class="c5 c3">-5°C for 30 seconds</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">Extension: 72°C for 1 minute per kilobase</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">Repeat denature, annealing, extension steps for 30-35 cycles</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">Final extension: 72°C for 5 minutes</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.2add7753f73672ae370de1bbfaf8cb380b78078e"></a> | ||
+ | <a id="t.9"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c24 c21 c4 c3">Preparation of Agar with Antibiotics</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c40" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c27" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Various antibiotics (kanamycin, chloramphenicol, ampicillin and hygromycin) were used to select for successful <i>E. coli</i> and <i>B. subtilis</i> transformants.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c40" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c27" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Luria-Bertani broth with agar</span> | ||
+ | </p> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">10% (w/v) tryptone</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">5% (w/v) NaCl</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">10% (w/v) yeast extract</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">15% (w/v) agar</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p class="c38 c34"><span class="c3 c32">Appropriate antibiotic</span> | ||
+ | </p> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">kanamycin (final concentration of 100 μg/mL)</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">chloramphenicol (final concentration of 30 μg/mL)</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">ampicillin (final concentration of 50 μg/mL)</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">hygromycin (final concentration of 100 μg/mL)</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p class="c38 c34"><span class="c3 c32">1500 mL Erlenmeyer flask</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c40" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c27" colspan="1" rowspan="1"> | ||
+ | <ol class="c10 lst-kix_ugk2xbe5oobt-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">In a 1500 Erlenmeyer flask, add 10 g tryptone, 5 g yeast extract, 10 g NaCl and 15 g agar in 1000 mL dH<sub>2</sub>O. Dissolve solids and make sure to add a stir bar.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Cover flask loosely with aluminum foil, secure with autoclave tape, and sterilize by autoclaving for 20 minutes.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Remove agar from autoclave using oven mitts. Allow agar to cool until warm to the touch before adding appropriate antibiotic. Stir on hot plate and magnetic stirrer for 30 seconds.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Pour agar into plates using aseptic technique.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.a043272a2ece69f3cb2fadd8c3061a79a0929df4"></a> | ||
+ | <a id="t.10"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c21 c4 c3">Preparation of Chemically Competent </span><span class="c21 c4 c3 c15">Escherichia coli </span><span class="c24 c21 c4 c3">Cells</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Competent <i>E. coli</i> cells were transformed with ligated plasmid to amplify them for further use in digest confirmation and <i>B. subtilis</i> transformation.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32"><i>Escherichia coli</i> TOP10 cells</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Luria-Bertani broth</span> | ||
+ | </p> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">10% (w/v) tryptone</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">5% (w/v) NaCl</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">10% (w/v) yeast extract</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p class="c38 c34"><span class="c3 c32">16x125 mm culture tubes</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">250 mL Erlenmeyer flask</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Spectrophotometer</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Centrifuge</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">50 mL Falcon tubes</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">50 mM CaCl</span><span class="c37 c26 c3 c19 c7">2</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">50 mM CaCl</span><span class="c3 c7">2</span><span class="c9 c3">, 15% glycerol</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">1.5 mL microcentrifuge tubes</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <ol class="c10 lst-kix_yzt1xhfk9inp-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Inoculate 5-10 mL of Luria-Bertani broth with <i>E. coli</i> TOP10 cells and allow it to incubate at 37°C overnight, shaking at 200 rpm.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Subculture 1 mL of <i>E. coli</i> overnight culture in 49 mL fresh Luria-Bertani broth in a 250 Erlenmeyer flask. Incubate at 37°C, shaking at 200 rpm until it reaches an OD<sub>600</sub> of 0.4-0.6 (usually takes 2.5 hours).</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Spin down cells in 50 mL Falcon tube at 8200 g for 10 minutes at 4 °C.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Resuspend cells in 12.5 mL cold 50 mM CaCl<sub>2</sub> and place on ice for 10 minutes.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Repeat Step 3.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Resuspend cells in 2 mL cold 50 mM CaCl<sub>2</sub>, 15% glycerol and place on ice for 30 minutes.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Separate cells into aliquots of 200 μL and store at -80°C.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.1dfc9937d0669c9bfddb68d3eb84c6fb063bcf6f"></a> | ||
+ | <a id="t.11"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c21 c4 c3">Glycerol Stock Preparation of Transformed </span><span class="c21 c4 c3 c15">Escherichia coli </span><span class="c21 c4 c3">and </span><span class="c36 c21 c4 c3 c15">Bacillus subtilis</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c40" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c27" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Glycerol stocks of transformed <i>E. coli</i> and <i>B. subtilis</i> were prepared for long-term storage and future use. </span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c40" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c27" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Overnight culture of transformed bacteria</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Sterile 1.5 mL cryo-tubes</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Sterile 50% glycerol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c40" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c27" colspan="1" rowspan="1"> | ||
+ | <ol class="c10 lst-kix_9z0i9ngly08f-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Using aseptic technique, pipette 0.5 mL of 50% sterile glycerol into a 1.5 mL cryo-tube.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Using aseptic technique, add 0.5 mL of overnight culture.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Pipette up and down gently to mix.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Store at -80°C for up to 1 year.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c3 c4"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.af553801c68bf412b97a40ede6acc4949d13bf82"></a> | ||
+ | <a id="t.12"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c24 c21 c4 c3">Plating Culture Broth on Agar Plates</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Culture broth was plated on agar to isolate single colonies.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Luria-Bertani agar plate with appropriate antibiotic (if required)</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Overnight culture of desired bacteria</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">70% ethanol</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Spreading rod</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Bunsen burner</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <ol class="c10 lst-kix_t27wwiwmqyyi-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Using aseptic technique, pipette 50-100 μL of bacterial culture onto antibiotic agar plate.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Dip spreading rod in 70% ethanols, pass over flame and allow for excess liquid to burn off. Cool rod on agar, avoiding bacterial culture.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Use rod to spread bacterial culture over entire plate, spinning the plate at the same time.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Dip spreading rod in 70% ethanol, pass over flame and allow for excess liquid to burn off.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Incubate plates at 37°C overnight or until growth is observed.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.a9ec5543947cf4b5c1e809e3bd93e0a39493820e"></a> | ||
+ | <a id="t.13"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c24 c21 c4 c3">Streaking of Agar Plates</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c40" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c27" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Culture broth was streaked on agar plates to isolate single colonies.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c40" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c27" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Luria-Bertani agar plate with appropriate antibiotic (if required)</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Overnight culture of desired bacteria or single isolated colony on agar plate</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Inoculation loop</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Bunsen burner</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c40" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c27" colspan="1" rowspan="1"> | ||
+ | <ol class="c10 lst-kix_pgbvm1degh2i-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Using aseptic technique, flame inoculation loop until red hot</span><span class="c5 c3">. Allow it to cool for 10 seconds or touch it to agar.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Dip the inoculation loop in bacterial culture or touch a single colony and streak the loop on ¼ of the surface of agar in a zigzag motion.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Flame the inoculation loop until red hot. Allow it to cool for 10 seconds or touch it to agar.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Run the cooled inoculation loop through one of the previous streaks ONCE, then streak 1.4 of the surface of the agar.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Repeat Steps 3 and 4 two more times.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Flame the inoculation loop until red hot. Allow it to cool for 10 seconds.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Incubate plates at 37°C overnight or until growth is observed.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.406a87a9a667877262b3842420039a15a7070795"></a> | ||
+ | <a id="t.14"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c24 c21 c4 c3">Agarose Gel Electrophoresis</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Digested and undigested plasmids were run on agarose gels to confirm the presence and proper orientation of DNA inserts.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">TAE buffer</span> | ||
+ | </p> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">40 mM Tris, pH 7.6</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">20 mM CH<sub>3</sub>COOH</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">1 mM EDTA</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p class="c38 c34"><span class="c3 c32">Agarose</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">250 mL Erlenmeyer flask</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">RedSafe Nucleic Acid Staining Solution</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Gel casting tray and comb</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">10X loading dye</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">DNA sample</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Microwave</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <ol class="c10 lst-kix_jwmzf1bv57q2-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">For a 1% gel (standard), add 1 g agarose to 100 mL TAE buffer in a 250 mL Erlenmeyer flask and microwave until agarose is fully dissolved (avoid boiling for too long).</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Allow flask to cool in fumehood until warm to the touch before adding 5 μL RedSafe Nucleic Acid Staining Solution. Gently swirl to mix.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Pour agarose into assembled gel casting tray. Remove any bubbles with a pipette tip and place comb in gel.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Allow gel to solidify and transfer to a gel running apparatus filled with TAE buffer.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Load samples of 20 μL DNA containing 2 μL loading dye.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Run gel at 120 V for 45 minutes or until loading dye is ⅔ way down the gel.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.9a06e8c35365220cdfb76525b6f3021dd3bf8cae"></a> | ||
+ | <a id="t.15"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c21 c4 c3">Growth Curve of </span><span class="c21 c4 c3 c15">Bacillus subtilis</span><span class="c24 c21 c4 c3">: Temperature Simulation</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">To simulate the temperature conditions of <i>B. subtilis</i> in our patch, we measured bacterial growth in 10 mL of Luria-Bertani (LB) broth over the course of 24 hours at varying temperatures of 35°C (average skin temperature), 22°C (average room temperature on board the International Space Station) and 4°C (negative control). Growth was measured by spectrophotometer at a wavelength of 600 nm (OD<sub>600</sub>).</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Luria-Bertani broth medium with hygromycin</span> | ||
+ | </p> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">10% (w/v) tryptone</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">5% (w/v) NaCl</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">10% (w/v) yeast extract</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">Final concentration of 100 μg/mL hygromycin</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p class="c38 c34"><span class="c3 c32"><i>Bacillus subtilis</i> WB800 cells</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">16x125 mm culture tubes</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">50 mL Falcon tubes</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Spectrophotometer</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">1 mL cuvette</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Preparation of <i>B. subtilis</i> WB800 Cells</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_e6kd68i2bo5-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Two days prior to measuring growth, prepare an overnight culture of <i>B. subtilis</i> in 10 mL LB broth with the appropriate antibiotic (100 μg/mL final concentration of hygromycin). Incubate at 37°C, shaking at 200 rpm overnight.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Inoculation</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_e6kd68i2bo5-0" start="2"> | ||
+ | <li class="c8"><span class="c3 c32">One day prior to measuring growth, inoculate 9 mL LB broth in a 50 mL Falcon tube with 1 mL of overnight culture at 16:40 (Tube 7), 16:45 (Tube 8) and 16:50 (Tube 9). Incubate the inoculated tubes at desired temperature (35°C, 22°C or 4°C) with lids loosened, shaking at 200 rpm.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Repeat Step 2 at 00:20 (Tube 4), 00:25 (Tube 5), and 00:30 (Tube 6) on the same day of growth measurement.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Repeat Step 2 at 8:00 (Tube 1), 8:05 (Tube 2), and 8:10 (Tube 3) on the same day of growth measurement.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Measurement of Growth</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_e6kd68i2bo5-0" start="5"> | ||
+ | <li class="c8"><span class="c3 c32">Set the wavelength of the spectrophotometer to a wavelength 600 nm (OD<sub>600</sub>). Blank the spectrophotometer with 1 mL uninoculated LB broth in a cuvette, <b>making sure to only blank once!</b></span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Commence measurement of samples at 8:00 (OD<sub>600</sub>). For 8 hours, measure Tube 1 every hour at :00, Tube 2 every hour at :05, Tube 3 every hour at :10, Tube 4 every hour at :20, Tube 5 every hour at :25, Tube 6 every hour at :30, Tube 7 every hour at :40, Tube 8 every hour at :45, and Tube 9 every hour at :50.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">To measure, add 1 mL of sample to a cuvette, wipe down sides with a Kim-wipe, and insert into spectrophotometer. Record value. If samples reach an OD<sub>600</sub> >0.4, dilute the sample 10X using LB-broth with 100 μg/mL hygromycin. Account for this dilution factor when recording values (multiple reading by 10). After each measurement, return the culture to its appropriate incubation temperature with lids loosened, shaking at 200 rpm.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <a id="t.e2526e129591cd9ba602101f594f214931b56a1b"></a> | ||
+ | <a id="t.16"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c21 c4 c3">Growth Curve of </span><span class="c21 c4 c3 c15">Bacillus subtilis</span><span class="c24 c21 c4 c3">: Media Optimization</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Our patch will contain three attached packets of additional media that can be ruptured to extend the cell growth and production of mBBI. In order to determine which media would be best suited for these packets, we tested three different types of media: Luria-Bertani broth, 2X Luria-Bertani broth, and Super Rich broth. We added 1 mL of these three types of media to 9 mL cultures of <i>B. subtilis</i> WB800 in LB broth containing 100 μg/mL hygromycin every 12 hours and measured bacterial growth over the course of 72 hours. Growth was measured by spectrophotometer at a wavelength of 600 nm (OD<sub>600</sub>).</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Luria-Bertani broth medium with hygromycin</span> | ||
+ | </p> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">10% (w/v) tryptone</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">5% (w/v) NaCl</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">10% (w/v) yeast extract</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">Final concentration of 100 μg/mL hygromycin</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p class="c38 c34"><span class="c3 c32">2X Luria-Bertani broth medium with hygromycin</span> | ||
+ | </p> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">20% (w/v) tryptone</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">10% (w/v) NaCl</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">20% (w/v) yeast extract</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">Final concentration of 100 μg/mL hygromycin</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p class="c38 c34"><span class="c3 c32">Super Rich broth medium</span> | ||
+ | </p> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">2.5% (w/v) tryptose</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">2% (w/v) yeast extract</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">0.3% K<sub>2</sub>HPO<sub>4</sub> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">3% glucose (v/v)</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">Notes:</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <ul> | ||
+ | <li class="c2 c39"><span class="c3 c32">Mix all components except glucose and adjust pH to 7.5</span> | ||
+ | </li> | ||
+ | <li class="c2 c39"><span class="c3 c32">Autoclave solution except for glucose. Add 1 mL/L of anti-foam prior to autoclave.</span> | ||
+ | </li> | ||
+ | <li class="c2 c39"><span class="c3 c32">Filter-sterilize glucose (20%) and add to main solution to a final concentration of 3%.</span> | ||
+ | </li> | ||
+ | <li class="c2 c39"><span class="c3 c32">Adapted from Haling <i>et al.,</i> 1997</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p class="c38 c34"><span class="c3 c32"><i>Bacillus subtilis</i> WB800 cells</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">16x125 mm culture tubes</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">50 mL Falcon tubes</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Spectrophotometer</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">1 mL cuvette</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Preparation of <i>B. subtilis</i> WB800 Cells</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_l5833w3zpti7-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Two days prior to measuring growth, prepare an overnight culture of <i>B. subtilis</i> in 10 mL LB broth with the appropriate antibiotic (100 μg/mL final concentration of hygromycin). Incubate at 35°C, shaking at 200 rpm overnight.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Inoculation</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_l5833w3zpti7-0" start="2"> | ||
+ | <li class="c8"><span class="c3 c32">One day prior to measuring growth, inoculate 9 mL LB broth in a 50 mL Falcon tube with 1 mL of overnight culture at 16:40 (Tube 7), 16:45 (Tube 8) and 16:50 (Tube 9). Incubate the inoculated tubes at 37°C with lids loosened, shaking at 200 rpm.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Repeat Step 2 at 00:20 (Tube 4), 00:25 (Tube 5), and 00:30 (Tube 6) on the same day of growth measurement.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Repeat Step 2 at 8:00 (Tube 1), 8:05 (Tube 2), and 8:10 (Tube 3) on the same day of growth measurement.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Measurement of Growth</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_l5833w3zpti7-0" start="5"> | ||
+ | <li class="c8"><span class="c3 c32">Set the wavelength of the spectrophotometer to a wavelength 600 nm (OD<sub>600</sub>). Blank the spectrophotometer with 1 mL uninoculated LB broth in a cuvette, making sure to only blank once!</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Commence measurement of samples at 8:00 (OD<sub>600</sub>). For 8 hours, measure Tube 1 every hour at :00, Tube 2 every hour at :05, Tube 3 every hour at :10, Tube 4 every hour at :20, Tube 5 every hour at :25, Tube 6 every hour at :30, Tube 7 every hour at :40, Tube 8 every hour at :45, and Tube 9 every hour at :50.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">To measure, add 1 mL of sample to a cuvette, wipe down sides with a Kim-wipe, and insert into spectrophotometer. Record value. If samples reach an OD<sub>600</sub> >0.4, dilute the sample 10X using LB-broth with 100 μg/mL hygromycin. Account for this dilution factor when recording values (multiple reading by 10). After each measurement, return the culture to incubate at 35°C with lids loosened, shaking at 200 rpm.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Add 1 mL of appropriate media (either LB, 2XLB or Super Rich) to each 50 mL Falcon tube every 12 hours for a total of three times (at 12 hours, 24 hours and 36 hours) after inoculation.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <a id="t.2a95f6008ad31bcdebcfbcb650bfb34aa6d08d4f"></a> | ||
+ | <a id="t.17"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c21 c4 c3">Transformation of </span><span class="c21 c4 c3 c15">Escherichia coli </span><span class="c24 c21 c4 c3">TOP10</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32"><i>Escherichia coli</i> TOP10 cells were used to amplify newly-ligated plasmids and were later harvested for future use in digest confirmation and <i>Bacillus subtilis</i> transformation.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Competent <i>E. coli</i> TOP10 aliquots (200 μL)</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">DNA for transformation</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Luria-Bertani broth</span> | ||
+ | </p> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">10% (w/v) tryptone</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">5% (w/v) NaCl</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">10% (w/v) yeast extract</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p class="c38 c34"><span class="c3 c32">Agar plate with appropriate antibiotic</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <ol class="c10 lst-kix_3v73ll15y5y-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Thaw 200 μL aliquot of competent <i>E. coli</i> TOP10 cells on ice just before use.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Add 0.3-1 μg DNA to cells (in maximum 20 μL), flick gently to mix, and place on ice for 30 minutes.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Heat shock for 60-75 seconds at 42°C.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Place on ice for 5 minutes.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Add 250 μL Luria-Bertani medium to aliquot of cells.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Incubate cells for 60 minutes at 37°C, shaking at 200 rpm for 1 hour.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Pellet cells in a microcentrifuge at 3500 g for 1 min and discard supernatant.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Resuspend pellet in 250 μL Luria-Bertani broth.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Plate 50-100 μL of resuspended culture on agar plate with appropriate antibiotic and spread.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Incubate plates at 37°C overnight or until desired growth is observed.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <a id="t.f717abc4b681a592e15edcbeaba0a3adfde58572"></a> | ||
+ | <a id="t.18"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c21 c4 c3">Preparation of SP1 and SP2 Media for </span><span class="c21 c4 c3 c15">Bacillus subtilis </span><span class="c24 c21 c4 c3">WB800 Transformation (adapted from Spizizen, 1958)</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c40" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c27" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32"><i>Bacillus subtilis</i> was our final chassis for our patch system, and it was transformed with our mBBI construct for continuous mBBI production.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c40" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c27" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">SP1 Salts</span> | ||
+ | </p> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">>0.2% (w/v) (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub></span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">1.4% (w/v) K<sub>2</sub>HPO<sub>4</sub></span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">0.6% (w/v) KH<sub>2</sub>PO<sub>4</sub></span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">0.1% Na<sub>3</sub>C<sub>6</sub>H<sub>5</sub>O<sub>7</sub>⋅2H<sub>2</sub>O</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">0.02% MgSO<sub>4</sub>⋅7H<sub>2</sub>O</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | <p class="c38 c34"><span class="c3 c32">50 mM CaCl<sub>2</sub></span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">250 mM MgCl<sub>2</sub></span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">50% filter-sterilized glucose</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Casamino acids/yeast extract</span> | ||
+ | </p> | ||
+ | <ul> | ||
+ | <li class="c0"><span class="c3 c32">2% (w/v) casamino acids</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">10% yeast extract</span> | ||
+ | </li> | ||
+ | </ul> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c40" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c27" colspan="1" rowspan="1"> | ||
+ | <ol class="c10 lst-kix_pt735i4wbfer-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">For all stock solutions, autoclave solutions for 20 minutes and store at room temperature.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">To make SP1 media, add any volume of SP1 salts plus 1/100 volume glucose, 1/100 volume casamino acids/yeast extract, and 0.5% (w/v) tryptophan.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">To make SP2 media, add 1/100 volume of CaCl<sub>2</sub> and 1/100 volume of MgCl<sub>2</sub> to any volume of SP1 media.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <a id="t.82671a9b10721bf3f55454aa3503d1d74dac74f8"></a> | ||
+ | <a id="t.19"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c21 c4 c3">Preparation of </span><span class="c4 c3 c15 c21">Bacillus subtilis </span><span class="c24 c21 c4 c3">WB800 Competent Cells and Transformation</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32"><i>B. subtilis</i> was our final chassis for our patch system, and it was transformed with our mBBI construct for continuous mBBI production.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Luria-Bertani agar plate with appropriate antibiotic</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">16x125 mm culture tubes</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">13x100 mm culture tubes</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Sterile SP1 Medium</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Sterile SP2 Medium</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">100 mM EGTA</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">DNA for transformation</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <ol class="c10 lst-kix_pt735i4wbfer-0" start="4"> | ||
+ | <li class="c8"><span class="c3 c32">Streak cells on Luria-Bertani agar plate in the evening at 17:00. Incubate cells at 30°C overnight.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">At 9:00 the next morning, transfer cells to 2 mL SP1 medium in 16x125 mm culture tubes. The culture should be slightly turbid. Incubate at 37°C for 3 hours and 45 minutes, shaking at 300 rpm.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Transfer 0.5 mL SP1 culture to 4.5 mL SP2 medium pre-warmed to 37°C in a 16x125 mm culture tube. Incubate at 37°C for 1 hour and 30 minutes, shaking at 150 rpm.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Add 50 μL 100X EGTA to the SP2 culture and incubate at 37°C for another 10 minutes, shaking at 150 rpm.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Take 0.5 mL competent cells and transfer to a sterile 13x100 mm culture tube. Add 0.1-3 g DNA in a volume of 60 μL or less. Incubate at 37°C for 1 hour and 30 minutes, shaking at 300 rpm.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Plate 0.25 mL of sample on Luria-Bertani agar plate with appropriate antibiotic.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Incubate plate at 37°C overnight or until desired growth is observed.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c42"><span class="c21 c4 c3">DEVICE</span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.e0b82d4122171dd538b17fc893bbdb300e67f7cc"></a> | ||
+ | <a id="t.20"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c24 c21 c4 c3">Semipermeable Membrane Diffusion Assay</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">The purpose of this assay was to determine quantitatively if the membrane in our transdermal patch can act as a filter and prevent bacteria cells from diffusing through the semipermeable membrane. </span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">50 mL falcon tubes</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">15 mL falcon tubes</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Distilled/non distilled water</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">NaCl</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">20 mL overnight culture (<i>B. subtilis </i> WB800) </span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">LB media </span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">CoTran 9716 and 9728 semipermeable membrane </span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Parafilm</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Preparation</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_4jptj2dntjck-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Prepare 500 mL of 0.9% w/v saline solution using sodium chloride at a pH of 7.35. Cover using parafilm and store at room temperature.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Start 20 mL overnight culture <i>B. subtilis</i>. | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">For the first replicate of the assay, first determine the wavelength needed to detect cells in the distilled water. Start 5 mL overnight culture of <i>B. subtilis</i>. | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Using distilled water as a blank, using a spectro scan spike the blank with LB media and determine a peak from the spike.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Repeat process using cell culture in distilled water.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Using the wavelength peak from the spectro scan, use this wavelength for the remainder of the assay.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">As our spectrophotometer did not have the capabilities to do a gradient scan, we used known wavelengths to determine what wavelength best detected cells. This was done by running a preliminary diffusion assay, set up exactly like the one described in the setup. Readings at 260 nm, 300 nm, 600 nm and 700 nm were done after 24 hours. 260 nm, 300 nm and 600 nm of wavelengths were used based on the readings we got.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Measurements</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_pzhk3qcax242-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Aliquot 5 mL of each overnight culture into three 15 mL falcon tubes.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">With one of the 15 mL falcon tube wrap parafilm over the top of the tube.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">With the other, place the 9716 semipermeable membrane over the top of the tube and Parafilm around the edges to prevent it from falling off. Repeat using 9728 semipermeable membrane</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">With the 50 mL falcon tubes, add 30 mL of saline solution</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Invert and submerse the 15 mL falcon tubes into the 50 mL falcon tubes. Cover the top of the 50 mL falcon tube with Parafilm.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">After a 24 hour period take a 1 mL sample of the water in the 50 mL falcon tube.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Using saline solution as a blank, spec the sample at 260 nm, 300 nm, and 600 nm.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Repeat once daily for seven days.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.912a7fb2873054bb4926b4f077bc6a864ea18b1b"></a> | ||
+ | <a id="t.21"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c24 c21 c4 c3">Filter Sterilization Membrane Diffusion Assay </span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">In running the first diffusion assay, our data analysis showed that there was an increased amount of cell detection, meaning that the cells may or may not be diffusing through. However, we suspected that there might be contamination so we repeated the assay with a few changes. </span> | ||
+ | </p> | ||
+ | <p class="c6 c28"><span class="c9 c3"></span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">This time, we are using a filter sterilization membrane that has pore sizes of 0.2 micron. We are also adding hygromycin to the saline solution for <i>B. subtilis</i> WB800 and chloramphenicol for <i>E.coli</i> mBBI-GFP. This ensured that there would be no contamination in our samples. </span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">10 mL syringes</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Filter sterilization membrane </span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Distilled/non distilled water</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">NaCl</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Hygromycin (20 mg/mL)</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">20 mL overnight culture (<i>B. subtilis </i>WB800) </span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">LB media </span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">250 mL erlenmeyer flasks</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Chloramphenicol agar plates </span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Preparation</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_wfo24hevzk6g-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Prepare 500 mL of 0.9% W/V saline solution using sodium chloride at a pH of 7.35. Add appropriate antibiotics. Cover using parafilm and store at room temperature.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Start 20 mL overnight culture <i>B. subtilis</i>.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">For the first replicate of the assay, first determine the wavelength needed to detect cells in the distilled water. Start 5 mL overnight culture of <i>B. subtilis</i>. | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Using distilled water as a blank, using a spectro scan spike the blank with LB media and determine a peak from the spike.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Repeat process using cell culture in distilled water.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Using the wavelength peak from the spectro scan, use this wavelength for the remainder of the assay.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">As our spectrophotometer did not have the capabilities to do a gradient scan, we used known wavelengths to determine what wavelength best detected cells. This was done by running a preliminary diffusion assay, set up exactly like the one described in the setup. Readings at 260 nm, 300 nm, 600 nm and 700 nm were done after 24 hours. 260 nm, 300 nm and 600 nm of wavelengths were used based on the readings we got.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Measurements</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_i1031kzh8e9u-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Take up 5 mL of each overnight culture into two 10 mL syringes. Take up 5 mL chloramphenicol LB media into one 10 mL syringe. Attach filter sterilization membrane to one of the syringes containing the 5 mL overnight culture and to the syringe containing 5 mL chlor + LB media. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">With the 250 mL erlenmeyer flasks, add 10 mL of saline solution.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Submerse each of the 10 mL filter sterilization syringes into the 250 mL erlenmeyer flasks. Cover the top of the erlenmeyer flasks with Parafilm.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">After a 24-hour period take a 1 mL sample of the saline solution in the 250 mL erlenmeyer flasks.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Using saline solution as a blank, spec the sample at 260 nm, 300 nm, and 600 nm.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Take a 150 µL sample of the saline solution and plate it on respective antibiotic plate.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Repeat once daily for seven days.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.0e1546388a76e79955d93e6042ad389a89130e18"></a> | ||
+ | <a id="t.22"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c24 c21 c4 c3">Backing Layer Growth Curve Assay</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">The purpose of this assay was to determine quantitatively if the backing layer will allow cell growth similar to those found under optimal conditions. </span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">10 mL culture tube</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">CoTran 9722 backing layer </span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">50 mL falcon tubes</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">LB media with hygromycin</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Hygromycin agar plates</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Preparation of <i>B. subtilis</i> WB800 Cells</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_2in1h2fvxudb-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Two days prior to measuring growth, prepare an overnight culture of <i>B. subtilis<i> in 10 mL LB broth with the appropriate antibiotic (100 μg/mL final concentration of hygromycin). Incubate at 37°C, shaking at 200 rpm overnight.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Inoculation</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_7cr24e9okmfb-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">One day prior to measuring growth, inoculate 9 mL LB broth in a 50 mL Falcon tube with 1 mL of overnight culture at 16:40 (Tube 7), 16:45 (Tube 8) and 16:50 (Tube 9). Incubate the inoculated tubes at desired temperature (35°C, 22°C or 4°C) with lids loosened, shaking at 200 rpm.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <ol class="c10 lst-kix_2in1h2fvxudb-0" start="2"> | ||
+ | <li class="c8"><span class="c3 c32">Repeat Step 2 at 00:20 (Tube 4), 00:25 (Tube 5), and 00:30 (Tube 6) on the same day of growth measurement.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Repeat Step 2 at 8:00 (Tube 1), 8:05 (Tube 2), and 8:10 (Tube 3) on the same day of growth measurement.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Measurement of Growth</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_v96mlbro49yx-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Set the wavelength of the spectrophotometer to a wavelength 600 nm (OD<sub>600</sub>). Blank the spectrophotometer with 1 mL uninoculated LB broth in a cuvette, making sure to only blank once!</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Commence measurement of samples at 8:00 (OD<sub>600</sub>). For 8 hours, measure Tube 1 every hour at :00, Tube 2 every hour at :05, Tube 3 every hour at :10, Tube 4 every hour at :20, Tube 5 every hour at :25, Tube 6 every hour at :30, Tube 7 every hour at :40, Tube 8 every hour at :45, and Tube 9 every hour at :50.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">To measure, add 1 mL of sample to a cuvette, wipe down sides with a Kim-wipe, and insert into spectrophotometer. Record value. If samples reach an OD<sub>600</sub> >0.4, dilute the sample 10X using LB-broth with 100 μg/mL hygromycin. Account for this dilution factor when recording values (multiple reading by 10). After each measurement, return the culture to its appropriate incubation temperature with lids loosened, shaking at 200 rpm.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c4 c3"></span> | ||
+ | </p> | ||
+ | <a id="t.1d8bbda353485a1bdfeb5ab714ef6618eeb810bc"></a> | ||
+ | <a id="t.23"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c24 c21 c4 c3">Backing Layer Survival Assay </span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Following the first backing layer growth curve assay, we consulted our mentors and determined that the assay was not effective in determining whether our backing layer would be a limiting factor for gas exchange. This is due to the fact that there is 10x the volume of oxygen in the culture tube to begin with. Therefore, the rate of gas exchange through the backing layer will be negligible. </span> | ||
+ | </p> | ||
+ | <p class="c2 c28"><span class="c9 c3"></span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">This purpose of this assay was to determine whether the bacterial cells can be starved of oxygen with our backing layer using a different protocol. </span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">10 mL culture tube</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">2x2 well nuclon plates </span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">CoTran 9722 backing layer </span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">CoTran 9719 backing layer </span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Parafilm </span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Day Zero</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_ghlmud29dl0e-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Using sterilized 2x2 culture plates, add 1.62 mL of LB media + appropriate antibiotic.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Add 180 µL O/N culture that has already been OD<sub>600</sub>. This will be the baseline reading.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Pipette 180 µL of the mixture out, discard.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Place appropriate backing layer on top of the culture plates. Secure backing layer with Parafilm on top criss cross and gain around the edges. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">For a positive control, cover culture plate with Parafilm. Puncture a hole into each of the wells using a syringe needle. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">For a negative control, place the lid on top and Parafilm to secure.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Place in incubator at 35°C at 10 rpm.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Repeat for replicates. </span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Day One</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_taz15jv9qurq-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Begin OD<sub>600</sub> readings using LB + antibiotics. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Take the reading of the day one plates.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Repeat for all membranes and controls </span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Day Three</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_ma14gllav8iv-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Repeat day one procedure using day three plates</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Day Seven</b></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_hd8gz1vih14v-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Repeat day one procedure using day seven plates </span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <a id="t.fd3f2e5883735e08b596e22b11ff0c130c39f00e"></a> | ||
+ | <a id="t.24"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c21 c4 c3 c15">in vivo</span><span class="c24 c21 c4 c3"> Mouse Testing</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Ethics </span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">With the tireless support of Dr. Craig Jenne, the team wrote and submitted an extensive ethics application to the Health Sciences Animal Care Committee (HSACC) which evaluated the application on multiple grounds such as feasibility, morality, precautions, etc. of the testing of our transdermal patch on mice models. Our protocols and the scientific significance of our transdermal patch were reviewed and approved by the HSACC. </span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">This experiment allowed us to examine the effects of the prototype version of our transdermal patch <i>in vivo</i>. This experiment addresses three main aims: </span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_bg6npkoi2682-0 start" start="1"> | ||
+ | <li class="c0"><span class="c3 c32">To determine whether patches containing TD1 tagged mBBI diffused from the patch into systemic circulation of mice. </span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">To determine whether the membrane of the patch was capable of containing <i>B. subtilis</i> without leakage. </span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c3 c32">To determine whether the adhesive, the peptide or the <i>B. subtilis</i> is immunogenic. </span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32">Three cohorts of six mice each were used for this experiment. The first cohort was given patches containing water to examine the effects of the adhesive on the skin. In the second cohort, mice were given a patch containing <i>B. subtilis</i>. The third group was used to examine the diffusion of fusion protein across the skin. In order to measure the effects of these patches, the skin samples were obtained post-euthanasia and tested for any immunological responses (ie. neutrophil deposition). The blood samples were collected to check for mBBI presence using mass spectrometry. The blood samples collected were either isolated for serum or for plasma. Since it was unknown where mBBI would be found, both procedures were conducted. </span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c4 c3 c16">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">18 BALB/c adult mice</span></p> | ||
+ | <p class="c38 c34"><span class="c3 c32">18 transdermal patches with respective contents</span></p> | ||
+ | <p class="c38 c34"><span class="c3 c32">General Anesthesia</span></p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Hair removal cream</p></span> | ||
+ | <p class="c38 c34"><span class="c3 c32">0.5 M EDTA</p></span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <p class="c2 c28"><span class="c9 c3"></span> | ||
+ | </p> | ||
+ | <ol class="c10 lst-kix_yuxi9iaet81w-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Prepare patches with their respective contents in the protocol described here. Manufacture at least 6 working patches of each type.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Use 6 mice for treatment, one for each patch type.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Anesthetize mice with 300 uL of general anesthesia. The anesthesia was administered to 6 mice at a time.</span><span class="c9 c3">e.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Remove hair using hair removal cream (Nair) underneath the shoulder of the mice.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Apply one patch to one mouse on the top of lumbar vertebrae using tissue glue to ensure proper attachment. After the application, leave mice caged.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Repeat Steps 3-5 for other mice in other patch groups.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Keep mice under constant observation to ensure that patch stays on.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">After day 2, anesthetize three mice with 600uL of general anesthesia each. (Note: This amount of anesthesia is used when the mice will be sacrificed in the immediate future.)</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Once they are unconscious, draw blood samples two of the mice via the left ventricle to obtain blood. Leave the blood sample at room temperature for half an hour, and upon coagulation, isolate serum by centrifugation.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Anesthetize the remaining mouse and collect samples using 8 uL EDTA in order to obtain blood plasma. Once the blood is mixed with the anticoagulant, the plasma is isolated by spinning down the sample.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Sacrifice the mice via cervical dislocation. Obtain skin samples from the lumbar region. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">At day 5, repeat Steps 8-11.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <a id="t.73b38a8957b3ec840cd36512ba57e99487951382"></a> | ||
+ | <a id="t.25"></a> | ||
+ | <table class="c23"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c24 c21 c4 c3">Detection of mBBI by Mass Spectrometry</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Mass spectrometry was used to determine the presence of mBBI in mouse trial blood samples.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Mouse blood sample</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Heavy isotope of mBBI peptide</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">C18 Zip-Tip column from Thermo</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">50% acetonitrile</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">0.1% formic acid</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">14 cm C18 column</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Mass spectrometer</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c33" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Protocol</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c31" colspan="1" rowspan="1"> | ||
+ | <ol class="c10 lst-kix_qsfm83ibxmjm-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Extract peptides from mouse blood plasma or serum via purification on a C18 Zip-Tip column from Thermo; elute with 50% acetonitrile, 0.1% formic acid.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Concentrate samples 10x via evaporation and resuspend in 0.1% formic acid.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Inject extracted samples onto a 10 cm C18 column and elute with a 30 minute 5-40%B gradient at 300 nL/min.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Acquire MS spectra on a Thermo LTQ Orbitrap Velos in ion trap mode or in Orbitrap mode with CID.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Analyze spectra manually for the presence of mBBI (monoisotopic mass = 2577.1 Da) at various charge states.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <p class="c38 c34"><span class="c3 c32">Note: Pure sample of mBBI, synthesized by BioBasic, was used as a reference for identification.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | |||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | |||
+ | <a id="t.971ab58709574b3dabced61ce6003ae4e49580cb"></a> | ||
+ | <a id="t.0"></a> | ||
+ | <table class="c23" style="width:625px"> | ||
+ | <tbody> | ||
+ | <tr class="c18"> | ||
+ | <td class="c25" colspan="2" rowspan="1"> | ||
+ | <p class="c2"><span class="c24 c21 c4 c3">Immunohistochemical Staining of Mouse Cryo Cross-Section</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Experimental Details<br>and Rationale</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">This technique was employed to study any possible immunological responses induced by the transdermal patch, the bacteria <i>Bacillus subtilis</i>, and mBBI. Mice skin tissue samples were obtained from 9 mice at day 2 (samples 1-3 for all treatments) and the rest of the mice samples were obtained on day 5 (samples 4-6 for all treatments) of the study. The skin tissue samples were then fixed, sectioned, and stained. Due to lack of training and materials, members of the Jenne lab had to perform most of the experiment. However, a member of the team periodically assisted or observed them.</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Materials</span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c17" colspan="1" rowspan="1"> | ||
+ | <p class="c38 c34"><span class="c3 c32">Formalin </span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Optimal Cutting Temperature (OCT)</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Cryomold</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Blade (VWR Cat #: 95057-832, coated microtone blades)</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Cryostat </span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Staining Glass Container and Acetone </span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">2% BSA+PBS</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Fluorescent mounting medium (Dako, S3023)</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Conjugated Antibody:</span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Biolegend FITC anti-mouse Ly6G (clone 1A8), cat#:127606. </span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">BD Pharmingen PE anti-mouse CD49b (clone HMalpha), cat#:558759. </span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">eBioscience APC anti-mouse CD45 (clone 30-F11), cat#:17-0451-83. </span> | ||
+ | </p> | ||
+ | <p class="c38 c34"><span class="c3 c32">Fluorescent Microscope </span> | ||
+ | </p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | <tr class="c1"> | ||
+ | <td class="c29" colspan="1" rowspan="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3"><b>Protocol</b></span> | ||
+ | </p> | ||
+ | </td> | ||
+ | <td class="c6" colspan="1" rowspan="1"> | ||
+ | <ol class="c7 lst-kix_ajye8d523edn-0 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Tissue samples were collected from the lumbar region of the mice after blood draw and euthanization.</span> | ||
+ | </li> | ||
+ | <p class="c2"><span class="c16 c4 c3"><b>Tissue Fixation </b></span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <ol class="c7 lst-kix_ajye8d523edn-1 start" start="1"> | ||
+ | <p class="c2"><span class="c16 c4 c3">Formalin:</span></p> | ||
+ | |||
+ | </ol> | ||
+ | <ol class="c7 lst-kix_ajye8d523edn-2 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Remove the wax that from the application of Nair during the application of the transdermal patch. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Treat tissue with the formalin solution to fix it the skin tissue samples. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Store fixed skin tissue samples -80°C.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <ol class="c7 lst-kix_ajye8d523edn-0" start="3"> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Cryosection</b> </span> | ||
+ | </p> | ||
+ | </ol> | ||
+ | <ol class="c7 lst-kix_ajye8d523edn-1 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Fill an ice box with dry ice, ethanol, and a piece of metal. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Place Optimal Cutting Temperature (OCT) compound and fixed tissue in a cryomold. Ensure the orientation of the tissue is such that all the layers of the tissue are obtained. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Fill cryomold with OCT compound to cover the fixed tissue (approximately 0.5 cm from top of the mold).</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">When samples are completely frozen,wrap them in individually-labelled foils, seal in plastic bags, and store at -80°C.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Put the blade (VWR Cat #: 95057-832, coated microtone blades) in the Cryostat machine and tighten. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Anchor and adjust tissue sample so it is parallel to the blade.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Prime the tissue sample and slowly start cutting . </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Allow the section(s) to dry for approximately 3 hrs.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Discard the blade. </span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <ol class="c7 lst-kix_ajye8d523edn-0" start="4"> | ||
+ | <p class="c38 c34"><span class="c3 c32"><b>Cryosection FIxation</b></span> | ||
+ | </p> | ||
+ | </ol> | ||
+ | <ol class="c7 lst-kix_ajye8d523edn-1 start" start="1"> | ||
+ | <li class="c8"><span class="c3 c32">Insert tissue slide in staining glass container that containing Acetone.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Incubate at room temperature for 10 minutes. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Fill staining glass container with the slides with prepared 2% BSA+PBS. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Incubate at room temperature for 2 hours. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Wipe the slide in such a way that a dome shape is created, and added 2% BSA+PBS to the dome. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Add 1 uL of conjugated antibody to the dome. Incubate at room temperature for 1 hr. Ensure to add water to keep the slide moist at all times. Antibodies used are listed below:</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <ol class="c7 lst-kix_ajye8d523edn-2 start" start="1"> | ||
+ | <li class="c0"><span class="c1">Biolegend FITC anti-mouse Ly6G (clone 1A8), cat#:127606 </span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c1">BD Pharmingen PE anti-mouse CD49b (clone HMalpha), cat#:558759</span> | ||
+ | </li> | ||
+ | <li class="c0"><span class="c1">eBioscience APC anti-mouse CD45 (clone 30-F11), cat#:17-0451-83 </span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <ol class="c7 lst-kix_ajye8d523edn-1" start="7"> | ||
+ | <li class="c8"><span class="c3 c32">Wash the slide three times with 2% BSA+PBS.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Add 1 drop of Fluorescent mounting medium (Dako, S3023) to slide. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Gently load the cover slip. </span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Keep the tissue slide at 4°C.</span> | ||
+ | </li> | ||
+ | <li class="c8"><span class="c3 c32">Visualize sample via fluorescent microscopy.</span> | ||
+ | </li> | ||
+ | </ol> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | </table> | ||
+ | <p class="c22 c13"><span class="c10"></span> | ||
+ | </p> | ||
+ | <p class="c13 c22"><span></span> | ||
+ | </p> | ||
+ | |||
+ | |||
+ | <p class="c14"><span class="c3"></span> | ||
+ | </p> | ||
+ | <p class="c42"><span class="c21 c4 c3">References</span> | ||
+ | </p> | ||
+ | <p><font size="2">Haling, S. (1997). Super-rich medium. <i>Biochemistry</i>(16), 2280-2884.<br><br> | ||
+ | Löbrich, M., Shibata, A., Beucher, A., Fisher, A., Ensminger, M., Goodarzi, A. a., … Jeggo, P. a. (2010). H2AX foci analysis for monitoring DNA double-strand break repair: Strengths, limitations and optimization. <i>Cell Cycle</i>, 9(4), 662–669.<br><br> | ||
+ | Spizizen, J. (1958). Transformation of biochemically deficient strains of <i>Bacillus subtilis</i> by deoxyribonucleate. <i>Proceedings of the National Academy of Sciences of the United States of America</i>(44), 1072-1078.</size> | ||
+ | </p> | ||
+ | <p class="c14"><span></span> | ||
+ | </p> | ||
+ | </body> | ||
+ | |||
+ | |||
+ | |||
</div> | </div> | ||
<!-- END: CONTENT/MISC/ABOUT-1 --> | <!-- END: CONTENT/MISC/ABOUT-1 --> | ||
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− | <h3 class=" | + | <h3 class="c-font-bold c-font-white">i<span class="c-theme-font">GEM</span></h3> |
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− | + | iGEM is an international competition promoting synthetic biology as a means to solve social, economic and humanitarian problems around the globe. The iGEM Jamboree is held in Boston annually. In 2016, over 300 teams are competing against each other. | |
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<div class="c-post-content"> | <div class="c-post-content"> | ||
<h4 class="c-post-title"><a href="#">Fully Trained!</a></h4> | <h4 class="c-post-title"><a href="#">Fully Trained!</a></h4> | ||
<p class="c-text"> | <p class="c-text"> | ||
− | + | Our entire team received a full BioSafety education from the University of Calgary! This entailed going to classes to prepare for a final quiz that tested our ability to be safe in the lab. Several of our members also had radiation training and clearance to ensure that work done with radiation was safe! | |
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Latest revision as of 01:05, 20 October 2016
![](https://static.igem.org/mediawiki/2016/d/d2/T--UofC_Calgary--project.jpg)
Experiments
BioTarget
Double-Stranded Break Assay using Immunofluorescence |
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Experimental Details |
Exposure to ionizing radiation causes DNA double strand breaks (DSBs) in cells, which are naturally repaired by cells over time. DSBs in nuclear DNA can be visualized in cells by immunofluorescence staining of DSB markers – namely 53BP1, a protein which binds near DSBs. As a result, nuclear foci form, each corresponding to a DSB. Over time, the number of foci are reduced due to DNA repair. To test for radioprotection by mBBI, we wanted to study the repair kinetics in cells treated with mBBI against a control. Cells were grown to confluency, irradiated and fixed at various time points over 24 hours. Immunofluorescence staining was performed to visualize 53BP1 and the resulting foci were counted.
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Materials |
Glass coverslips and microscopy slides 1BR3 primary fibroblast cells in Modified Eagle Medium (MEM) - (10^5 cells/ml) 30 mm Cell Culture Plates 1x PBS 1x PBS solution with 3% PFA (w/v) and 2% (w/v) sucrose 1x PBS solution with 2% (w/v) Bovine Serum Albumin 1x PBS solution with 0.2% (v/v) Triton X100 1x PBS solution with 0.1μg/ml 4’,6-diamidino-2-phenylindole (DAPI) Mouse anti-53BP1 Antibody Rabbit anti-H2AX Antibody Cy3 anti-Mouse Antibody FITC anti-Rabbit Antibody Mounting medium for fluorescence (Flouromount G or Vectashield)
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Protocol |
Prep and Irradiation Immunofluorescence Staining Counting Foci |
Table 1: Experimental Outline of mBBI treatment and Ionizing Radiation. mBBI treatment was administered at 30uM and cells were irradiated at 2 Gy.
Clonogenic Cell Survival Assay |
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Experimental Details |
Ionizing radiation is capable of killing cells by damaging their DNA, causing single and double strand breaks, which trigger apoptosis. If the DNA damage can be repaired, the cells survive. If not, they will die. To test whether mBBI elicits radioprotection, the survival of cells can be assessed. The purpose of this assay is to assess the viability of cells when treated with ionizing radiation, with and without mBBI. |
Materials |
MEM media with 15% Fetal Bovine Serum (v/v) and 10% Penicillin-Streptomycin (v/v) T-75 flasks 60 mm Cell culture plates 1BR3 cells
Trypan Blue Dye 0.25% Trypsin EDTA 15ml Centrifuge Tube |
Protocol |
Preparation of Cell Lines Prior to Setting up Clonogenic Cell Survival
Irradiation of Flasks and Performance of Plating Experiment for Clonogenic Assay
Serial Dilutions and Incubation
Staining of Plates
Counting of Colonies
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CHASSIS
Rehydration of Registry DNA |
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Experimental Details |
We used standard parts from the iGEM registry to ligate our synthesized genetic constructs into the pSB1c3 backbone. We also used BBa_J04450 (pSB1c3 backbone containing RFP) as a positive control to confirm transformation of E. coli and B. subtilis. |
Materials |
iGEM 2016 distribution kit ddH2O |
Protocol |
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Rehydration of IDT Synthesized DNA |
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Experimental Details |
We ordered genetic constructs from IDT to clone into the pSB1c3 backbone. |
Materials |
Synthesized DNA from IDT ddH2O |
Protocol |
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Plasmid MiniPrep from Escherichia coli and Bacillus subtilis |
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Experimental Details |
Plasmids were amplified in E. coli cells in order to use them for confirmation of ligation and transformation of B. subtilis. |
Materials |
2.5 mL overnight culture of transformed bacteria in appropriate antibiotic in 16x125 mm culture tube Resuspension buffer (store at 4°C)
Lysis buffer
Precipitation buffer
Isopropanol 70% ethanol Table-top centrifuge 2 mL microcentrifuge tubes 1.5 mL microcentrifuge tubes |
Protocol |
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Restriction Digest |
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Experimental Details |
Restriction digests were performed on plasmid backbones and synthesized DNA inserts before ligating them together, as well as to confirm ligation and transformation of cells with desired plasmids. |
Materials |
DNA Restriction enzymes 100X Bovine Serum Albumin (BSA) 10X appropriate buffer ddH2O 0.2 mL PCR tubes |
Protocol |
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Antarctic Phosphatase Treatment |
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Experimental Details |
Vectors were treated with Antarctic phosphatase to remove their 5’-phosphate group and prevent them from self-ligating before addition of the digested insert. |
Materials |
Digested DNA vector Antarctic phosphatase buffer Antarctic phosphatase ddH2O 0.2 mL PCR tubes |
Protocol |
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Ligation of DNA Inserts to Plasmid Backbones |
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Experimental Details |
Synthesized DNA inserts were ligated into plasmid backbones for propagation and use in E. coli and B. subtilis as well as for submission to the registry. |
Materials |
Digested vector DNA Digested insert DNA 10X DNA ligase buffer (from New England Biolabs) T4 DNA ligase (1 U/μL) (from New England Biolabs) ddH2O 0.2 mL PCR tubes |
Protocol |
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Colony PCR for Escherichia coli and Bacillus subtilis using the KAPA HiFi PCR Kit |
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Experimental Details |
Colony PCR was used to confirm the presence of plasmids with desired ligated insert in transformed E. coli and B. subtilis >cells. |
Materials |
Transformed bacterial colony on agar plate 10X Taq polymerase buffer Taq DNA polymerase (% U/μL) 10X Buffer A 10 mM dNTP 10 μM forward primer 10 μM reverse primer PCR-grade ddH2O 0.2 mL PCR tubes |
Protocol |
Sample Preparation
Running PCR
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Preparation of Agar with Antibiotics |
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Experimental Details |
Various antibiotics (kanamycin, chloramphenicol, ampicillin and hygromycin) were used to select for successful E. coli and B. subtilis transformants. |
Materials |
Luria-Bertani broth with agar
Appropriate antibiotic
1500 mL Erlenmeyer flask |
Protocol |
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Preparation of Chemically Competent Escherichia coli Cells |
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Experimental Details |
Competent E. coli cells were transformed with ligated plasmid to amplify them for further use in digest confirmation and B. subtilis transformation. |
Materials |
Escherichia coli TOP10 cells Luria-Bertani broth
16x125 mm culture tubes 250 mL Erlenmeyer flask Spectrophotometer Centrifuge 50 mL Falcon tubes 50 mM CaCl2 50 mM CaCl2, 15% glycerol 1.5 mL microcentrifuge tubes |
Protocol |
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Glycerol Stock Preparation of Transformed Escherichia coli and Bacillus subtilis |
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Experimental Details |
Glycerol stocks of transformed E. coli and B. subtilis were prepared for long-term storage and future use. |
Materials |
Overnight culture of transformed bacteria Sterile 1.5 mL cryo-tubes Sterile 50% glycerol |
Protocol |
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Plating Culture Broth on Agar Plates |
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Experimental Details |
Culture broth was plated on agar to isolate single colonies. |
Materials |
Luria-Bertani agar plate with appropriate antibiotic (if required) Overnight culture of desired bacteria 70% ethanol Spreading rod Bunsen burner |
Protocol |
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Streaking of Agar Plates |
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Experimental Details |
Culture broth was streaked on agar plates to isolate single colonies. |
Materials |
Luria-Bertani agar plate with appropriate antibiotic (if required) Overnight culture of desired bacteria or single isolated colony on agar plate Inoculation loop Bunsen burner |
Protocol |
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Agarose Gel Electrophoresis |
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Experimental Details |
Digested and undigested plasmids were run on agarose gels to confirm the presence and proper orientation of DNA inserts. |
Materials |
TAE buffer
Agarose 250 mL Erlenmeyer flask RedSafe Nucleic Acid Staining Solution Gel casting tray and comb 10X loading dye DNA sample Microwave |
Protocol |
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Growth Curve of Bacillus subtilis: Temperature Simulation |
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Experimental Details |
To simulate the temperature conditions of B. subtilis in our patch, we measured bacterial growth in 10 mL of Luria-Bertani (LB) broth over the course of 24 hours at varying temperatures of 35°C (average skin temperature), 22°C (average room temperature on board the International Space Station) and 4°C (negative control). Growth was measured by spectrophotometer at a wavelength of 600 nm (OD600). |
Materials |
Luria-Bertani broth medium with hygromycin
Bacillus subtilis WB800 cells 16x125 mm culture tubes 50 mL Falcon tubes Spectrophotometer 1 mL cuvette |
Protocol |
Preparation of B. subtilis WB800 Cells
Inoculation
Measurement of Growth
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Growth Curve of Bacillus subtilis: Media Optimization |
|
Experimental Details |
Our patch will contain three attached packets of additional media that can be ruptured to extend the cell growth and production of mBBI. In order to determine which media would be best suited for these packets, we tested three different types of media: Luria-Bertani broth, 2X Luria-Bertani broth, and Super Rich broth. We added 1 mL of these three types of media to 9 mL cultures of B. subtilis WB800 in LB broth containing 100 μg/mL hygromycin every 12 hours and measured bacterial growth over the course of 72 hours. Growth was measured by spectrophotometer at a wavelength of 600 nm (OD600). |
Materials |
Luria-Bertani broth medium with hygromycin
2X Luria-Bertani broth medium with hygromycin
Super Rich broth medium
Bacillus subtilis WB800 cells 16x125 mm culture tubes 50 mL Falcon tubes Spectrophotometer 1 mL cuvette |
Protocol |
Preparation of B. subtilis WB800 Cells
Inoculation
Measurement of Growth
|
Transformation of Escherichia coli TOP10 |
|
Experimental Details |
Escherichia coli TOP10 cells were used to amplify newly-ligated plasmids and were later harvested for future use in digest confirmation and Bacillus subtilis transformation. |
Materials |
Competent E. coli TOP10 aliquots (200 μL) DNA for transformation Luria-Bertani broth
Agar plate with appropriate antibiotic |
Protocol |
|
Preparation of SP1 and SP2 Media for Bacillus subtilis WB800 Transformation (adapted from Spizizen, 1958) |
|
Experimental Details |
Bacillus subtilis was our final chassis for our patch system, and it was transformed with our mBBI construct for continuous mBBI production. |
Materials |
SP1 Salts
50 mM CaCl2 250 mM MgCl2 50% filter-sterilized glucose Casamino acids/yeast extract
|
Protocol |
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Preparation of Bacillus subtilis WB800 Competent Cells and Transformation |
|
Experimental Details |
B. subtilis was our final chassis for our patch system, and it was transformed with our mBBI construct for continuous mBBI production. |
Materials |
Luria-Bertani agar plate with appropriate antibiotic 16x125 mm culture tubes 13x100 mm culture tubes Sterile SP1 Medium Sterile SP2 Medium 100 mM EGTA DNA for transformation |
Protocol |
|
DEVICE
Semipermeable Membrane Diffusion Assay |
|
Experimental Details |
The purpose of this assay was to determine quantitatively if the membrane in our transdermal patch can act as a filter and prevent bacteria cells from diffusing through the semipermeable membrane. |
Materials |
50 mL falcon tubes 15 mL falcon tubes Distilled/non distilled water NaCl 20 mL overnight culture (B. subtilis WB800) LB media CoTran 9716 and 9728 semipermeable membrane Parafilm |
Protocol |
Preparation
Measurements
|
Filter Sterilization Membrane Diffusion Assay |
|
Experimental Details |
In running the first diffusion assay, our data analysis showed that there was an increased amount of cell detection, meaning that the cells may or may not be diffusing through. However, we suspected that there might be contamination so we repeated the assay with a few changes.
This time, we are using a filter sterilization membrane that has pore sizes of 0.2 micron. We are also adding hygromycin to the saline solution for B. subtilis WB800 and chloramphenicol for E.coli mBBI-GFP. This ensured that there would be no contamination in our samples. |
Materials |
10 mL syringes Filter sterilization membrane Distilled/non distilled water NaCl Hygromycin (20 mg/mL) 20 mL overnight culture (B. subtilis WB800) LB media 250 mL erlenmeyer flasks Chloramphenicol agar plates |
Protocol |
Preparation
Measurements
|
Backing Layer Growth Curve Assay |
|
Experimental Details |
The purpose of this assay was to determine quantitatively if the backing layer will allow cell growth similar to those found under optimal conditions. |
Materials |
10 mL culture tube CoTran 9722 backing layer 50 mL falcon tubes LB media with hygromycin Hygromycin agar plates |
Protocol |
Preparation of B. subtilis WB800 Cells
Inoculation
Measurement of Growth
|
Backing Layer Survival Assay |
|
Experimental Details |
Following the first backing layer growth curve assay, we consulted our mentors and determined that the assay was not effective in determining whether our backing layer would be a limiting factor for gas exchange. This is due to the fact that there is 10x the volume of oxygen in the culture tube to begin with. Therefore, the rate of gas exchange through the backing layer will be negligible.
This purpose of this assay was to determine whether the bacterial cells can be starved of oxygen with our backing layer using a different protocol. |
Materials |
10 mL culture tube 2x2 well nuclon plates CoTran 9722 backing layer CoTran 9719 backing layer Parafilm |
Protocol |
Day Zero
Day One
Day Three
Day Seven
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in vivo Mouse Testing |
|
Ethics |
With the tireless support of Dr. Craig Jenne, the team wrote and submitted an extensive ethics application to the Health Sciences Animal Care Committee (HSACC) which evaluated the application on multiple grounds such as feasibility, morality, precautions, etc. of the testing of our transdermal patch on mice models. Our protocols and the scientific significance of our transdermal patch were reviewed and approved by the HSACC. |
Experimental Details |
This experiment allowed us to examine the effects of the prototype version of our transdermal patch in vivo. This experiment addresses three main aims:
Three cohorts of six mice each were used for this experiment. The first cohort was given patches containing water to examine the effects of the adhesive on the skin. In the second cohort, mice were given a patch containing B. subtilis. The third group was used to examine the diffusion of fusion protein across the skin. In order to measure the effects of these patches, the skin samples were obtained post-euthanasia and tested for any immunological responses (ie. neutrophil deposition). The blood samples were collected to check for mBBI presence using mass spectrometry. The blood samples collected were either isolated for serum or for plasma. Since it was unknown where mBBI would be found, both procedures were conducted. |
Materials |
18 BALB/c adult mice 18 transdermal patches with respective contents General Anesthesia Hair removal cream 0.5 M EDTA |
Protocol |
|
Detection of mBBI by Mass Spectrometry |
|
Experimental Details |
Mass spectrometry was used to determine the presence of mBBI in mouse trial blood samples. |
Materials |
Mouse blood sample Heavy isotope of mBBI peptide C18 Zip-Tip column from Thermo 50% acetonitrile 0.1% formic acid 14 cm C18 column Mass spectrometer |
Protocol |
Note: Pure sample of mBBI, synthesized by BioBasic, was used as a reference for identification. |
Immunohistochemical Staining of Mouse Cryo Cross-Section |
|
Experimental Details |
This technique was employed to study any possible immunological responses induced by the transdermal patch, the bacteria Bacillus subtilis, and mBBI. Mice skin tissue samples were obtained from 9 mice at day 2 (samples 1-3 for all treatments) and the rest of the mice samples were obtained on day 5 (samples 4-6 for all treatments) of the study. The skin tissue samples were then fixed, sectioned, and stained. Due to lack of training and materials, members of the Jenne lab had to perform most of the experiment. However, a member of the team periodically assisted or observed them. |
Materials |
Formalin Optimal Cutting Temperature (OCT) Cryomold Blade (VWR Cat #: 95057-832, coated microtone blades) Cryostat Staining Glass Container and Acetone 2% BSA+PBS Fluorescent mounting medium (Dako, S3023) Conjugated Antibody: Biolegend FITC anti-mouse Ly6G (clone 1A8), cat#:127606. BD Pharmingen PE anti-mouse CD49b (clone HMalpha), cat#:558759. eBioscience APC anti-mouse CD45 (clone 30-F11), cat#:17-0451-83. Fluorescent Microscope |
Protocol |
Tissue Fixation Formalin:
Cryosection
Cryosection FIxation
|
References
Haling, S. (1997). Super-rich medium. Biochemistry(16), 2280-2884.
Löbrich, M., Shibata, A., Beucher, A., Fisher, A., Ensminger, M., Goodarzi, A. a., … Jeggo, P. a. (2010). H2AX foci analysis for monitoring DNA double-strand break repair: Strengths, limitations and optimization. Cell Cycle, 9(4), 662–669.
Spizizen, J. (1958). Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proceedings of the National Academy of Sciences of the United States of America(44), 1072-1078.