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#title{ | #title{ | ||
− | font-size: | + | font-size:260%; |
+ | } | ||
+ | .banner_link{ | ||
+ | font-size:22px; | ||
+ | } | ||
+ | .div_banner{ | ||
+ | margin-top:20px; | ||
} | } | ||
/*Makes side pictures on banner invisible on small screens*/ | /*Makes side pictures on banner invisible on small screens*/ | ||
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margin-left:0.85vw; | margin-left:0.85vw; | ||
} | } | ||
+ | } | ||
+ | #links_drop{ | ||
+ | color:#47BCC2; | ||
+ | /*font-size:20px;*/ | ||
+ | font-size:1.2vw; | ||
+ | margin:8px 0.9vw 0.5vh 0.9vw; | ||
+ | padding:0.06vw 0 0 0; | ||
+ | line-height:0px; | ||
+ | } | ||
+ | @media (max-width: 920px){ | ||
+ | #links_drop{ | ||
+ | margin-left:0; | ||
+ | padding-top:9px; | ||
+ | margin-top:-4px; | ||
+ | font-size:20px; | ||
+ | } | ||
+ | } | ||
+ | button.dropdown-toggle{ | ||
+ | padding-top:21px; !important | ||
+ | } | ||
+ | @media (max-width: 767px){ | ||
+ | button.dropdown-toggle{ | ||
+ | padding-top:5px; | ||
+ | } | ||
+ | } | ||
+ | #links_drop:hover span{ | ||
+ | color:#339499; | ||
} | } | ||
</style> | </style> | ||
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<div> | <div> | ||
<div class="collapse navbar-collapse" id="myNavbar"> | <div class="collapse navbar-collapse" id="myNavbar"> | ||
− | + | <ul class="nav navbar-nav"> | |
− | <li><div id=" | + | <li><div id="links_drop" class="dropdown"> |
<button class="dropdown-toggle" type="button" id="dropdownMenu1" data-toggle="dropdown" aria-haspopup="true" aria-expanded="true"> | <button class="dropdown-toggle" type="button" id="dropdownMenu1" data-toggle="dropdown" aria-haspopup="true" aria-expanded="true"> | ||
<span style="margin-bottom:4px;">Lab</span> | <span style="margin-bottom:4px;">Lab</span> | ||
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<li><a id="links" style="margin:10px 0 30px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Project">Lab Project</a></li> | <li><a id="links" style="margin:10px 0 30px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Project">Lab Project</a></li> | ||
<li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Labbook">Lab Book</a></li> | <li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Labbook">Lab Book</a></li> | ||
+ | <li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Safety">Safety</a></li> | ||
</ul> | </ul> | ||
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<li ><a id="links"href="https://2016.igem.org/Team:Exeter/Parts">Parts</a></li> | <li ><a id="links"href="https://2016.igem.org/Team:Exeter/Parts">Parts</a></li> | ||
<li ><a id="links"href="https://2016.igem.org/Team:Exeter/Team">Team</a></li> | <li ><a id="links"href="https://2016.igem.org/Team:Exeter/Team">Team</a></li> | ||
− | <li ><a id="links" href="https://2016.igem.org/Team:Exeter/Interlab">InterLab</a></li> | + | <li><a id="links" href="https://2016.igem.org/Team:Exeter/Interlab">InterLab</a></li> |
− | <li><div id=" | + | <li><div id="links_drop" class="dropdown"> |
<button class="dropdown-toggle" type="button" id="dropdownMenu1" data-toggle="dropdown" aria-haspopup="true" aria-expanded="true"> | <button class="dropdown-toggle" type="button" id="dropdownMenu1" data-toggle="dropdown" aria-haspopup="true" aria-expanded="true"> | ||
<span style="margin-bottom:4px;">Human Practices</span> | <span style="margin-bottom:4px;">Human Practices</span> | ||
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</button> | </button> | ||
<ul class="dropdown-menu" style="background:#e8e8e8;margin-left:25px;" aria-labelledby="dropdownMenu1"> | <ul class="dropdown-menu" style="background:#e8e8e8;margin-left:25px;" aria-labelledby="dropdownMenu1"> | ||
− | <li><a id="links" style="margin:10px | + | <li><a id="links" style="margin:10px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Integrated_Practices">Integrated</a></li> |
− | + | <li><a id="links" style="background:none;line-height:0.7vh;margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Engagement">Public Engagement<br /><br /><br /> & Education</a></li><li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Log">Log</a></li> | |
− | <li><a id="links" style="background:none;line-height:0.7vh;margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Engagement">Public Engagement<br /><br /><br /> & Education</a></li> | + | |
− | <li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Log">Log</a></li> | + | |
</ul> | </ul> | ||
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<li ><a id="links"href="https://2016.igem.org/Team:Exeter/Attributions">Attributions</a></li> | <li ><a id="links"href="https://2016.igem.org/Team:Exeter/Attributions">Attributions</a></li> | ||
− | <li><div id=" | + | <li><div id="links_drop" class="dropdown"> |
<button class="dropdown-toggle" type="button" id="dropdownMenu1" data-toggle="dropdown" aria-haspopup="true" aria-expanded="true"> | <button class="dropdown-toggle" type="button" id="dropdownMenu1" data-toggle="dropdown" aria-haspopup="true" aria-expanded="true"> | ||
<span style="margin-bottom:4px;">Awards</span> | <span style="margin-bottom:4px;">Awards</span> | ||
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<ul class="dropdown-menu" style="background:#e8e8e8;margin-left:25px;" aria-labelledby="dropdownMenu1"> | <ul class="dropdown-menu" style="background:#e8e8e8;margin-left:25px;" aria-labelledby="dropdownMenu1"> | ||
− | <li><a id="links" style="margin:10px 0 30px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Awards"> | + | <li><a id="links" style="margin:10px 0 30px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/Awards">Awards</a></li> |
− | <li><span style="margin:10px 0 30px 2px;padding:0;">Special pages</span></li> | + | <li><span style="margin:10px 0 30px 2px;padding:0;"><u>Special pages</u></span></li> |
<li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/HP/Silver">HP Silver</a></li> | <li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/HP/Silver">HP Silver</a></li> | ||
<li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/HP/Gold">HP Gold</a></li> | <li><a id="links" style="margin:30px 0 10px 2px;padding:0;font-size:1.8vh;" href="https://2016.igem.org/Team:Exeter/HP/Gold">HP Gold</a></li> | ||
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<li ><a id="links"href="https://2016.igem.org/Team:Exeter/Model">Models</a></li> | <li ><a id="links"href="https://2016.igem.org/Team:Exeter/Model">Models</a></li> | ||
<li ><a id="links"href="https://2016.igem.org/Team:Exeter/Collaborations">Collaborations</a></li> | <li ><a id="links"href="https://2016.igem.org/Team:Exeter/Collaborations">Collaborations</a></li> | ||
+ | |||
+ | |||
+ | |||
</ul> | </ul> | ||
<ul class="nav navbar-nav navbar-right"> | <ul class="nav navbar-nav navbar-right"> | ||
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<!--Contains all information of banner in the middle--> | <!--Contains all information of banner in the middle--> | ||
<!--of the two outer images--> | <!--of the two outer images--> | ||
− | <div class="col-xs-12 col-sm-8 subdiv_banner middle"> | + | <div class="col-xs-12 col-sm-8 subdiv_banner middle"> |
<!--Use bootstrap to add links using all 12 columns--> | <!--Use bootstrap to add links using all 12 columns--> | ||
<!--For each link please give double the coloumns for when--> | <!--For each link please give double the coloumns for when--> | ||
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<!--Give span class "oneline" or "twoline" depending on how llong the section text is--> | <!--Give span class "oneline" or "twoline" depending on how llong the section text is--> | ||
− | <a href="#section_1" class="banner_link col-xs-6 col-sm-3"><span class="oneline"> | + | <a href="#section_1" class="banner_link col-xs-6 col-sm-3"><span class="oneline">KillerRed</span></a> |
− | <a href="#section_2" class="banner_link col-xs-6 col-sm-3"><span class="oneline"> | + | <a href="#section_2" class="banner_link col-xs-6 col-sm-3"><span class="oneline">KillerOrange</span></a> |
− | <a href="#section_3" class="banner_link col-xs-6 col-sm-3"><span class="oneline"> | + | <a href="#section_3" class="banner_link col-xs-6 col-sm-3"><span class="oneline">Lysozyme</span></a> |
− | + | <a href="#section_4" class="banner_link col-xs-6 col-sm-3"><span class="oneline">DNase</span></a> | |
− | + | </div> | |
+ | |||
<!--Left picture (the teal line on left)--> | <!--Left picture (the teal line on left)--> | ||
<div class="col-sm-2 subdiv_banner right"> | <div class="col-sm-2 subdiv_banner right"> | ||
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</div> | </div> | ||
<div> | <div> | ||
− | + | ||
</div> | </div> | ||
</div> | </div> | ||
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<div id="section_1" class="link_fix"></div> | <div id="section_1" class="link_fix"></div> | ||
<div id="contentTitle"> | <div id="contentTitle"> | ||
− | + | KillerRed | |
− | + | ||
</div> | </div> | ||
− | <p id="pp"> | + | <h6>Name</h6> |
− | <p id="pp"> | + | |
+ | <p id="pp">pT7- <i>E. coli</i> optimised - KillerRed (EOKR)</p> | ||
+ | |||
+ | <h6>Description</h6> | ||
+ | |||
+ | <p id="pp">KillerRed is a red fluorescent protein that generates reactive oxygen species after illumination with light between the wavelengths of 540-580 nm[1].</p> | ||
+ | |||
+ | <p id="pp">We further characterised this kill switch by illuminating induced cultures 24 hours after induction with IPTG as well as uninduced cultures. After 6 hours in the light box CFU’s were counted to determine if the kill switch was successful. This was also reproduced on cultures grown in a ministat for 120 and 168 hours to test how long the kill switch remains functional. The full protocol can be found <a href="https://2016.igem.org/Team:Exeter/Project#section_4">here</a> and the results can be found <a href="https://2016.igem.org/Team:Exeter/Project#section_2">here</a>. </p> | ||
+ | |||
+ | <p id="pp">The mechanism by which KillerRed kills cells isn’t fully understood yet</p> | ||
+ | |||
+ | <p id="pp">Here we are submitting KillerRed as a composite part under a T7 promoter (BBa_I712074), an Elowitz ribosome binding site (BBa_B0034) and a double terminator (BBa_B0015).</p> | ||
+ | |||
+ | <p id="pp">The sequence for KillerRed protein coding region can be found here <a href="http://parts.igem.org/Part:BBa_K1914002">(BBa_K1141002)</a></p> | ||
+ | |||
+ | <h6>Biobrick Code</h6> | ||
+ | |||
+ | <p id="pp"><a href="http://parts.igem.org/Part:BBa_K1914003">BBa_K1914003</a></p> | ||
+ | |||
+ | <br> | ||
+ | <br> | ||
+ | |||
+ | <div class="col-xs-12" style="padding:0;margin:0;"> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/1/1a/T--Exeter--parts-KR_png.png" style="max-width:50%;margin:auto;display:block;"> | ||
+ | <br> | ||
+ | <br> | ||
<div> | <div> | ||
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<a id="Section_link" href="#section_2" style="display:block;margin:20px auto 0 auto;width:14px;"><span style="color:#47BCC2;font-size: 25px;" class="glyphicon glyphicon-menu-down" aria-hidden="true"></span></a> | <a id="Section_link" href="#section_2" style="display:block;margin:20px auto 0 auto;width:14px;"><span style="color:#47BCC2;font-size: 25px;" class="glyphicon glyphicon-menu-down" aria-hidden="true"></span></a> | ||
</div> | </div> | ||
− | + | ||
<div class="col-xs-12 div_content"> | <div class="col-xs-12 div_content"> | ||
<div id="section_2" class="link_fix"></div> | <div id="section_2" class="link_fix"></div> | ||
<div id="contentTitle"> | <div id="contentTitle"> | ||
− | + | KillerOrange | |
</div> | </div> | ||
+ | <h6>Name:</h6> | ||
− | < | + | <p id="pp">pT7- <i>E. coli</i> optimised - KillerOrange (EOKO)</p> |
− | < | + | <h6>Description:</h6> |
− | < | + | <p id="pp">KillerOrange is a mutant of the fluorescent protein KillerRed (BBa_K1141002, BBa_K1491015) activated by blue and green light. It carries a tryptophan-based chromophore that is novel for photosensitizers [2]. </p> |
− | <p id="pp"> | + | <p id="pp">KillerOrange has an excitation maximum of 512 nm and emission maximum at 555 nm and its absorbance spectrum has two peaks, at 455 and 514 nm. <p> |
− | <p id="pp"> | + | <p id="pp">The mechanism by which KillerOrange kills cells isn’t fully understood yet. However, it is believed that KillerOrange's ability to generate reactive oxygen species depends on a water-filler channel reaching the chromophore area from the end cap of the ß-barrel [3].</p> |
− | <p id="pp"> | + | |
+ | <p id="pp">We characterised this part in the same way as KillerRed (<a href="https://2016.igem.org/Team:Exeter/Project#section_4">protocol</a>), and the results of which can be found <a href="https://2016.igem.org/Team:Exeter/Project#section_2">here</a></p> | ||
− | <p id="pp">Here we are submitting | + | <p id="pp">Here we are submitting KillerOrange as a composite part under a T7 promoter (BBa_I712074), an Elowitz ribosome binding site (BBa_B0034) and a double terminator (BBa_B0015).</p> |
− | <p id="pp">The sequence for | + | <p id="pp">The sequence for KillerOrange protein coding region can be found here <a href="http://parts.igem.org/Part:BBa_K1914000">(BBa_K1914000)</a></p> |
− | <h6>Biobrick Code</h6> | + | <h6> Biobrick Code </h6> |
+ | <p id="pp"><a href="http://parts.igem.org/Part:BBa_K1914001">BBa_K1914001</a></p> | ||
+ | |||
+ | <br> | ||
+ | <br> | ||
+ | |||
+ | <div class="col-xs-12" style="padding:0;margin:0;"> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/2/21/T--Exeter--parts-KO_png.png" style="max-width:50%;margin:auto;display:block;"> | ||
+ | <br> | ||
+ | <br> | ||
+ | |||
+ | </div> | ||
− | |||
<div> | <div> | ||
<a id="Section_link" href="#section_3" style="display:block;margin:20px auto 0 auto;width:14px;"><span style="color:#47BCC2;font-size: 25px;" class="glyphicon glyphicon-menu-down" aria-hidden="true"></span></a> | <a id="Section_link" href="#section_3" style="display:block;margin:20px auto 0 auto;width:14px;"><span style="color:#47BCC2;font-size: 25px;" class="glyphicon glyphicon-menu-down" aria-hidden="true"></span></a> | ||
</div> | </div> | ||
− | + | ||
<div class="col-xs-12 div_content"> | <div class="col-xs-12 div_content"> | ||
<div id="section_3" class="link_fix"></div> | <div id="section_3" class="link_fix"></div> | ||
<div id="contentTitle"> | <div id="contentTitle"> | ||
− | + | Lysozyme | |
</div> | </div> | ||
+ | <h6>Name:</h6> | ||
− | + | <p id="pp">pT7 Lysozyme <i>E. coli</i> codon optimised, signal peptide, flag tag</p> | |
− | + | ||
− | <p id="pp">pT7 | + | |
<h6>Description:</h6> | <h6>Description:</h6> | ||
− | <p id="pp"> | + | <p id="pp">Lysozyme from chicken egg white, attacks the cell wall in bacteria by hydrolysing the ß-1,4 linkages between N-acetylmuramic acid and N-acetylglucosamine of peptidoglycan [4].</p> |
− | <p id="pp"> | + | <p id="pp"> </p> |
+ | <p id="pp">Characterisation of this part involved using the Enzcheck assay kit which detects lysozyme activity as a measure of fluorescence as well as using the same protocol we used to characterise KillerRed (<a href="https://2016.igem.org/Team:Exeter/Project#section_4">protocol</a>)and the results of which can be found <a href="https://2016.igem.org/Team:Exeter/Project#section_2">here</a></p> | ||
− | <p id="pp"> | + | <p id="pp">Here we are submitting lysozyme as a composite part under a T7 promoter (BBa_I712074), an Elowitz ribosome binding site (BBa_B0034) and a double terminator (BBa_B0015).</p> |
− | <p id="pp"> | + | <p id="pp">We have codon optimised the protein coding region, added a FLAG tag and exchanged the native signal peptide for one which is <i>E. coli</i> specific and directs Lysozyme to the periplasm [5]. The sequence for Lysozyme protein coding region can be found here <a href="http://parts.igem.org/Part:BBa_K1914004">(BBa_K1914004)</a></p> |
− | < | + | <h6> Biobrick Code </h6> |
− | <p id="pp"> | + | <p id="pp"><a href="http://parts.igem.org/Part:BBa_K1914005">BBa_K1914005</a></p> |
− | + | <br> | |
+ | <br> | ||
− | + | <div class="col-xs-12" style="padding:0;margin:0;"> | |
− | + | <img src="https://static.igem.org/mediawiki/2016/c/c5/T--Exeter--parts-LYSO_png.png" style="max-width:50%;margin:auto;display:block;"> | |
+ | |||
+ | |||
+ | <br> | ||
+ | <br> | ||
<div> | <div> | ||
+ | |||
<a id="Section_link" href="#section_4" style="display:block;margin:20px auto 0 auto;width:14px;"><span style="color:#47BCC2;font-size: 25px;" class="glyphicon glyphicon-menu-down" aria-hidden="true"></span></a> | <a id="Section_link" href="#section_4" style="display:block;margin:20px auto 0 auto;width:14px;"><span style="color:#47BCC2;font-size: 25px;" class="glyphicon glyphicon-menu-down" aria-hidden="true"></span></a> | ||
</div> | </div> | ||
− | + | ||
<div class="col-xs-12 div_content"> | <div class="col-xs-12 div_content"> | ||
<div id="section_4" class="link_fix"></div> | <div id="section_4" class="link_fix"></div> | ||
<div id="contentTitle"> | <div id="contentTitle"> | ||
− | + | DNase | |
− | </div> | + | </div> |
+ | <h6>Name:</h6> | ||
− | + | <p id="pp">DNase</p> | |
− | + | ||
− | <p id="pp"> | + | |
<h6>Description:</h6> | <h6>Description:</h6> | ||
− | <p id="pp"> | + | <p id="pp"> DNase was designed [6] as a DNA degrading kill switch aiming to prevent cross contamination of DNA</p> |
− | <p id="pp"> | + | |
− | + | <p id="pp"> A composite part containing DNase could not be created. This could be due to the need for a tightly regulating promoter to prevent DNase protein being produced before induction and destroying DNA.</p> | |
− | + | <br> | |
+ | <br> | ||
− | + | <div class="col-xs-12" style="padding:0;margin:0;"> | |
+ | <img src="https://static.igem.org/mediawiki/2016/0/06/T--Exeter--parts-DNase_png.png" style="max-width:50%;margin:auto;display:block;"> | ||
+ | <br> | ||
+ | <br> | ||
− | < | + | <style> |
+ | ul{ | ||
+ | list-style-image:none; | ||
+ | } | ||
+ | .wrap ul{ | ||
+ | padding-top:20px; | ||
+ | padding-right:40px; | ||
+ | padding-left:80px; | ||
+ | font-size:150%; | ||
+ | } | ||
+ | .wrap ul li { | ||
+ | /* Text color */ | ||
+ | color: #333; | ||
+ | list-style-type: none; | ||
+ | } | ||
+ | |||
+ | .wrap ul li:before { | ||
+ | /* Unicode bullet symbol */ | ||
+ | content: '\25AA'; | ||
+ | /* Bullet color */ | ||
+ | color: #47BCC2; | ||
+ | padding-right: 0.5em; | ||
+ | } | ||
+ | ol{ | ||
+ | padding-top:20px; | ||
+ | padding-right:40px; | ||
+ | padding-left:80px; | ||
+ | font-size:150%; | ||
+ | } | ||
+ | ol li { | ||
+ | /* Text color */ | ||
+ | color: #333; | ||
+ | list-style-type: none; | ||
+ | } | ||
+ | ol li { | ||
+ | list-style-type: none; | ||
+ | counter-increment: list; | ||
+ | position: relative; | ||
+ | } | ||
+ | |||
+ | ol li:before { | ||
+ | content: counter(list) "."; | ||
+ | position: absolute; | ||
+ | left: -2.5em; | ||
+ | width: 2em; | ||
+ | text-align: right; | ||
+ | color: #47BCC2; | ||
+ | } | ||
+ | </style> | ||
+ | |||
+ | <h5>References</h5> | ||
+ | |||
+ | <ol style="font-size:100%;"> | ||
+ | <li>Takemoto, K., Matsuda, T., Sakai, N., Fu, D., Noda, M., Uchiyama, S., Kotera, I., Arai, Y., Horiuchi, M., Fukui, K. and Ayabe, T., 2013. SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation. Scientific reports, 3. | ||
+ | </li> | ||
+ | <li>Sarkisyan, K.S., Zlobovskaya, O.A., Gorbachev, D.A., Bozhanova, N.G., Sharonov, G.V., Staroverov, D.B., Egorov, E.S., Ryabova, A.V., Solntsev, K.M., Mishin, A.S. and Lukyanov, K.A., 2015. KillerOrange, a Genetically Encoded Photosensitizer Activated by Blue and Green Light. PloS one,10(12), p.e0145287. | ||
+ | </li> | ||
+ | <li>Pletnev, S., Gurskaya, N.G., Pletneva, N.V., Lukyanov, K.A., Chudakov, D.M., Martynov, V.I., Popov, V.O., Kovalchuk, M.V., Wlodawer, A., Dauter, Z. and Pletnev, V., 2009. Structural basis for phototoxicity of the genetically encoded photosensitizer KillerRed. Journal of Biological Chemistry, 284(46), pp.32028-32039. | ||
+ | </li> | ||
+ | <li>Blake, C.C.F., Koenig, D.F., Mair, G.A., North, A.C.T., Phillips, D.C. and Sarma, V.R., 1965. Structure of hen egg-white lysozyme: a three-dimensional Fourier synthesis at 2 Å resolution. Nature, 206(4986), pp.757-761. | ||
+ | </li> | ||
+ | <li>Fischer, B., Perry, B., Phillips, G., Sumner, I. and Goodenough, P., 1993. Physiological consequence of expression of soluble and active hen egg white lysozyme in Escherichia coli. Applied Microbiology and Biotechnology, 39, pp.537–540. | ||
+ | </li> | ||
+ | <li>Chen, C.Y., Lu, S.C. and Liao, T.H.,1998. Cloning, sequencing and expression of a cDNA encoding bovine pancreatic deoxyribonuclease I in Escherichia coli: purification and characterization of the recombinant enzyme. Gene, 206, pp.181–184. | ||
+ | </li> | ||
+ | </ol> | ||
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Latest revision as of 20:24, 19 October 2016