Dongzhuoer (Talk | contribs) |
Dongzhuoer (Talk | contribs) |
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− | + | {{NKU_China/menubar}} | |
<html> | <html> | ||
<head> | <head> | ||
− | <!-- | + | |
− | < | + | <!--separatrix--> |
− | + | <style> | |
− | + | .main#notebook { | |
− | + | padding: 0 8vw; | |
− | + | } | |
− | + | ||
+ | #notebook .ui-accordion .ui-accordion-content { | ||
+ | height: calc(100vh - 13rem - 18px); | ||
+ | position: relative; | ||
+ | z-index: 3; | ||
+ | box-sizing: border-box; | ||
+ | background-color: transparent; | ||
+ | } | ||
− | + | #notebook .ui-tabs { | |
− | + | margin: 0.2rem; | |
− | + | background-color: transparent; | |
− | } | + | } |
+ | |||
+ | #notebook .ui-tabs .ui-tabs-nav .ui-tabs-anchor { | ||
+ | font-weight:normal; | ||
+ | } | ||
+ | |||
+ | #week16 .ui-tabs-anchor { | ||
+ | font-size:1.2rem; | ||
+ | } | ||
+ | |||
+ | #notebook .ui-tabs .ui-tabs-nav .ui-tabs-anchor.circle-number { | ||
+ | font-weight:bold; | ||
+ | } | ||
+ | |||
+ | #notebook .ui-tabs .ui-tabs-panel { | ||
+ | position: relative; | ||
+ | z-index: 0; | ||
+ | background-color: transparent; | ||
+ | height: calc(100vh - 17rem - 18px); | ||
+ | overflow: auto; | ||
+ | } | ||
− | .ui- | + | #notebook .ui-tabs-panel > .p, #notebook .ui-tabs-panel > ol, #notebook .ui-tabs-panel > ul { |
− | margin:0.8rem 1.6rem; | + | margin: 0.8rem 1.6rem; |
} | } | ||
− | .flex-container { | + | #notebook .flex-container { |
− | display:flex; | + | display: flex; |
justify-content: space-around; | justify-content: space-around; | ||
+ | align-content: space-around; | ||
flex-wrap: wrap; | flex-wrap: wrap; | ||
+ | background-color: transparent; | ||
} | } | ||
− | . | + | #notebook .table-wrapper { |
− | + | margin: auto; | |
+ | padding: 1rem; | ||
+ | position:relative; | ||
} | } | ||
− | + | .table-wrapper div.rbrace { | |
− | + | font-weight:100; | |
− | + | color:dodgerblue; | |
+ | position:absolute; | ||
+ | right:6.5rem; | ||
+ | bottom:5rem; | ||
+ | font-size:4rem; | ||
+ | z-index:4; | ||
} | } | ||
− | + | ||
− | border-top: 0. | + | #notebook table { |
− | border-bottom: 0. | + | margin: 0; |
− | margin: | + | } |
+ | |||
+ | #notebook table, #dongzhuoer td { | ||
+ | border: none; | ||
+ | } | ||
+ | |||
+ | #notebook table { | ||
+ | font-size: 1rem; | ||
+ | padding: 0; | ||
+ | } | ||
+ | |||
+ | #notebook table caption { | ||
+ | border-top: 0.1rem solid; | ||
+ | border-bottom: 0.1rem solid; | ||
+ | margin: 0; | ||
padding: 0.2rem 0; | padding: 0.2rem 0; | ||
− | + | text-align: center; | |
+ | background-color: white; | ||
} | } | ||
− | + | #notebook table tr { | |
margin: 0; | margin: 0; | ||
padding: 0; | padding: 0; | ||
+ | background-color: white; | ||
} | } | ||
− | + | #notebook table td { | |
− | border:none; | + | border: none; |
margin: 0; | margin: 0; | ||
padding: 0.2rem 1.2rem; | padding: 0.2rem 1.2rem; | ||
} | } | ||
− | + | #notebook table tr:last-child td { | |
− | border-bottom: 0. | + | border-bottom: 0.1rem solid; |
} | } | ||
− | + | figure { | |
text-align: center; | text-align: center; | ||
+ | font-size: 0.9rem; | ||
+ | margin: auto; | ||
+ | padding: 1rem; | ||
} | } | ||
− | + | figure img { | |
− | + | width: 100%; | |
− | + | margin-bottom:0.25rem; | |
} | } | ||
− | . | + | figure img.white { |
− | + | background-color:white; | |
− | + | } | |
− | + | ||
+ | figure figcaption { | ||
+ | text-align:center; | ||
+ | line-height:1.25; | ||
+ | } | ||
+ | |||
+ | #notebook figure figcaption .p { | ||
+ | text-indent:0; | ||
+ | text-align:center; | ||
+ | } | ||
+ | |||
+ | #notebook .ui-tabs-panel li { | ||
+ | margin:0.3rem; | ||
} | } | ||
</style> | </style> | ||
− | <style | + | <style title="table-theme.css"> |
+ | /**/ | ||
+ | #notebook .table-theme-0 { | ||
+ | /*color: rgb(49,132,155);*/ | ||
+ | background-color:transparent; | ||
+ | border-collapse:collapse; | ||
+ | /*color:blue;*/ | ||
+ | } | ||
+ | |||
+ | #notebook .table-theme-0 tr { | ||
+ | background-color:transparent; | ||
+ | } | ||
+ | |||
+ | #notebook .table-theme-0 tr td { | ||
+ | background-color:transparent; | ||
+ | border:0.05rem solid white; | ||
+ | border-bottom-width:0.05rem; | ||
+ | } | ||
+ | |||
+ | #notebook .table-theme-0 tr:last-child td { | ||
+ | border-bottom-width:0.05rem; | ||
+ | } | ||
+ | |||
+ | #notebook .table-theme-0 caption { | ||
+ | border:none; | ||
+ | background-color:transparent; | ||
+ | } | ||
+ | |||
/*PCR system*/ | /*PCR system*/ | ||
− | .table-theme-1 { | + | #notebook .table-theme-1 { |
color: rgb(49,132,155); | color: rgb(49,132,155); | ||
− | |||
} | } | ||
− | .table-theme-1 caption { | + | #notebook .table-theme-1 caption { |
border-top-color: rgb(75,172,198); | border-top-color: rgb(75,172,198); | ||
border-bottom-color: rgb(75,172,198); | border-bottom-color: rgb(75,172,198); | ||
} | } | ||
− | .table-theme-1 table tr:last-child td { | + | #notebook .table-theme-1 table tr:last-child td { |
border-bottom-color: rgb(75,172,198); | border-bottom-color: rgb(75,172,198); | ||
} | } | ||
− | .table-theme-1 tr:nth-child(odd) { | + | #notebook .table-theme-1 tr:nth-child(odd) { |
background-color: rgb(210,234,241); | background-color: rgb(210,234,241); | ||
− | |||
− | |||
− | |||
} | } | ||
/*PCR reaction condition*/ | /*PCR reaction condition*/ | ||
− | .table-theme-2 { | + | #notebook .table-theme-2 { |
color: rgb(118,146,60); | color: rgb(118,146,60); | ||
− | + | position:relative; | |
} | } | ||
− | .table-theme-2 caption { | + | #notebook .table-theme-2 caption { |
border-top-color: rgb(155, 187, 89); | border-top-color: rgb(155, 187, 89); | ||
border-bottom-color: rgb(155, 187, 89); | border-bottom-color: rgb(155, 187, 89); | ||
} | } | ||
− | .table-theme-2 table tr:last-child td { | + | #notebook .table-theme-2 table tr:last-child td { |
border-bottom-color: rgb(75,172,198); | border-bottom-color: rgb(75,172,198); | ||
} | } | ||
− | .table-theme-2 tr:nth-child(odd) { | + | #notebook .table-theme-2 tr:nth-child(odd) { |
background-color: rgb(230,238,213); | background-color: rgb(230,238,213); | ||
− | |||
− | |||
− | |||
} | } | ||
/*ligation system*/ | /*ligation system*/ | ||
− | .table-theme-3 { | + | #notebook .table-theme-3 { |
color: rgb(95, 73, 122); | color: rgb(95, 73, 122); | ||
− | |||
} | } | ||
− | .table-theme-3 caption { | + | #notebook .table-theme-3 caption { |
border-top-color: rgb(128, 100, 162); | border-top-color: rgb(128, 100, 162); | ||
border-bottom-color: rgb(128, 100, 162); | border-bottom-color: rgb(128, 100, 162); | ||
} | } | ||
− | .table-theme-3 table tr:last-child td { | + | #notebook .table-theme-3 table tr:last-child td { |
border-bottom-color: rgb(128, 100, 162); | border-bottom-color: rgb(128, 100, 162); | ||
} | } | ||
− | .table-theme-3 tr:nth-child(odd) { | + | #notebook .table-theme-3 tr:nth-child(odd) { |
background-color: rgb(223, 215, 232); | background-color: rgb(223, 215, 232); | ||
− | |||
− | |||
− | |||
} | } | ||
/*digestion system*/ | /*digestion system*/ | ||
− | .table-theme-4 { | + | #notebook .table-theme-4 { |
color: rgb(148, 54, 52); | color: rgb(148, 54, 52); | ||
− | |||
} | } | ||
− | .table-theme-4 caption { | + | #notebook .table-theme-4 caption { |
border-top-color: rgb(192, 80, 77); | border-top-color: rgb(192, 80, 77); | ||
border-bottom-color: rgb(192, 80, 77); | border-bottom-color: rgb(192, 80, 77); | ||
} | } | ||
− | .table-theme-4 table tr:last-child td { | + | #notebook .table-theme-4 table tr:last-child td { |
border-bottom-color: rgb(192, 80, 77); | border-bottom-color: rgb(192, 80, 77); | ||
} | } | ||
− | .table-theme-4 tr:nth-child(odd) { | + | #notebook .table-theme-4 tr:nth-child(odd) { |
background-color: rgb(239, 211, 210); | background-color: rgb(239, 211, 210); | ||
− | |||
− | |||
− | |||
} | } | ||
/*methylation system*/ | /*methylation system*/ | ||
− | .table-theme-5 { | + | #notebook .table-theme-5 { |
color: rgb(227, 108, 10); | color: rgb(227, 108, 10); | ||
− | |||
} | } | ||
− | .table-theme-5 caption { | + | #notebook .table-theme-5 caption { |
border-top-color: rgb(247, 150, 70); | border-top-color: rgb(247, 150, 70); | ||
border-bottom-color: rgb(247, 150, 70); | border-bottom-color: rgb(247, 150, 70); | ||
} | } | ||
− | .table-theme-5 table tr:last-child td { | + | #notebook .table-theme-5 table tr:last-child td { |
border-bottom-color: rgb(247, 150, 70); | border-bottom-color: rgb(247, 150, 70); | ||
} | } | ||
− | .table-theme-5 tr:nth-child(odd) { | + | #notebook .table-theme-5 tr:nth-child(odd) { |
background-color: rgb(253, 228, 208); | background-color: rgb(253, 228, 208); | ||
− | |||
− | |||
− | |||
} | } | ||
/*Groups divided*/ | /*Groups divided*/ | ||
− | .table-theme-6 { | + | #notebook .table-theme-6 { |
color: rgb(0, 0, 0); | color: rgb(0, 0, 0); | ||
− | |||
} | } | ||
− | .table-theme-6 caption { | + | #notebook .table-theme-6 caption { |
border-top-color: rgb(0, 0, 0); | border-top-color: rgb(0, 0, 0); | ||
border-bottom-color: rgb(0, 0, 0); | border-bottom-color: rgb(0, 0, 0); | ||
} | } | ||
− | .table-theme-6 table tr:last-child td { | + | #notebook .table-theme-6 table tr:last-child td { |
border-bottom-color: rgb(0, 0, 0); | border-bottom-color: rgb(0, 0, 0); | ||
} | } | ||
− | .table-theme-6 tr:nth-child(odd) { | + | #notebook .table-theme-6 tr:nth-child(odd) { |
background-color: rgb(192, 192, 192); | background-color: rgb(192, 192, 192); | ||
} | } | ||
− | + | </style> | |
− | + | <script title="ready-js"> | |
− | + | ||
− | </style> | + | |
− | <script | + | |
$(function () { | $(function () { | ||
+ | adjust_figure(1.2); | ||
+ | |||
$("#accordion").accordion({ | $("#accordion").accordion({ | ||
header: ".accordion-header", | header: ".accordion-header", | ||
Line 213: | Line 285: | ||
heightStyle: 'content', | heightStyle: 'content', | ||
collapsible: true, | collapsible: true, | ||
− | active: | + | active: 18, |
icons: false, | icons: false, | ||
}); | }); | ||
− | + | ||
$('.tabs').each( | $('.tabs').each( | ||
function (index, value) { | function (index, value) { | ||
$(value).tabs({ | $(value).tabs({ | ||
event: "mouseover", | event: "mouseover", | ||
− | heightStyle: 'content' | + | heightStyle: 'content', |
+ | active:2, | ||
}); | }); | ||
} | } | ||
− | ) | + | ) |
+ | |||
+ | |||
+ | $('#particles-js').hide(); | ||
}); | }); | ||
− | </script> | + | |
+ | </script> | ||
</head> | </head> | ||
− | <body> | + | <body id="dongzhuoer"> |
<div style="display:none"> | <div style="display:none"> | ||
− | <table class="table-theme-1" id=" | + | <table class="table-theme-1" id="50μL PCR system ×2"> |
<caption>50μL PCR system ×2</caption> | <caption>50μL PCR system ×2</caption> | ||
<tr> | <tr> | ||
Line 258: | Line 335: | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | <table class="table-theme-2" id=" | + | <table class="table-theme-2" id="PCR reaction condition"> |
<caption>PCR reaction condition</caption> | <caption>PCR reaction condition</caption> | ||
<tr> | <tr> | ||
− | <td>94 | + | <td>94<sup>o</sup>C</td> |
<td>10 min</td> | <td>10 min</td> | ||
<td></td> | <td></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>94 | + | <td>94<sup>o</sup>C</td> |
<td>30 sec</td> | <td>30 sec</td> | ||
<td></td> | <td></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>58 | + | <td>58<sup>o</sup>C</td> |
<td>15 sec</td> | <td>15 sec</td> | ||
<td>30 cycles</td> | <td>30 cycles</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>72 | + | <td>72<sup>o</sup>C</td> |
<td>30 sec</td> | <td>30 sec</td> | ||
<td></td> | <td></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>72 | + | <td>72<sup>o</sup>C</td> |
<td>10 min</td> | <td>10 min</td> | ||
<td></td> | <td></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>16 | + | <td>16<sup>o</sup>C</td> |
<td>∞</td> | <td>∞</td> | ||
<td></td> | <td></td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | <table class="table-theme-3" id=" | + | <table class="table-theme-3" id="20μL ligation system"> |
<caption>20μL ligation system</caption> | <caption>20μL ligation system</caption> | ||
<tr> | <tr> | ||
Line 318: | Line 395: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td colspan="2">Reaction condition: 16 | + | <td colspan="2">Reaction condition: 16<sup>o</sup>C overnight</td> |
</tr> | </tr> | ||
</table> | </table> | ||
− | <table class="table-theme-4" id=" | + | <table class="table-theme-4" id="20μL digestion system"> |
<caption>20μL digestion system</caption> | <caption>20μL digestion system</caption> | ||
<tr> | <tr> | ||
Line 344: | Line 421: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td colspan="2">Reaction condition: 37 | + | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> |
</tr> | </tr> | ||
</table> | </table> | ||
− | <table class="table-theme-5" id=" | + | <table class="table-theme-5" id="100μL methylation system"> |
<caption>100μL methylation system</caption> | <caption>100μL methylation system</caption> | ||
<tr> | <tr> | ||
Line 374: | Line 451: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td colspan="2">Reaction condition: 37 | + | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 1 hour</td> |
</tr> | </tr> | ||
</table> | </table> | ||
− | <table class="table-theme-6" id=" | + | <table class="table-theme-6" id="Groups divided in this experiment"> |
<caption>Groups divided in this experiment</caption> | <caption>Groups divided in this experiment</caption> | ||
<tr> | <tr> | ||
Line 392: | Line 469: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>pWH-<i>luxS</i> | + | <td>pWH-<i>luxS</i> + xyl</td> |
<td><i>luxS</i> overexpression plasmid in GR286; induced by xylose</td> | <td><i>luxS</i> overexpression plasmid in GR286; induced by xylose</td> | ||
</tr> | </tr> | ||
Line 409: | Line 486: | ||
</table> | </table> | ||
<figure> | <figure> | ||
− | <img src="image/ | + | <img src="image/Notebook/notebook-6-13-1.png" width="347" height="284"> |
<figcaption> | <figcaption> | ||
− | + | <div class="p"><span>Selection of positive clones by colony PCR</span></div> | |
(No.1 is positive control, No.2-6 are experimental groups. The result showed that we failed to transformed the plasmid pWH-<i>C2-luxS</i> into GR286) | (No.1 is positive control, No.2-6 are experimental groups. The result showed that we failed to transformed the plasmid pWH-<i>C2-luxS</i> into GR286) | ||
</figcaption> | </figcaption> | ||
− | </figure> | + | </figure> |
</div> | </div> | ||
− | <main id="notebook"> | + | <div id="particles-js"></div> |
− | <div class="h1">Laboratory Notes</div> | + | <script src="https://2016.igem.org/Template:NKU_China/js/particlesJS?action=raw&ctype=text/javascript"></script> |
+ | <div class="main" id="notebook"> | ||
+ | <div class="h1">Laboratory Notes</div> | ||
<div id="accordion"> | <div id="accordion"> | ||
<div class="accordion-fragment" id="week1"> | <div class="accordion-fragment" id="week1"> | ||
Line 424: | Line 503: | ||
</div> | </div> | ||
<div class="accordion-content"> | <div class="accordion-content"> | ||
− | |||
− | |||
− | |||
<div class="tabs" id="tabs1"> | <div class="tabs" id="tabs1"> | ||
<ul> | <ul> | ||
− | <li><a href="#fragment1-1">➀</a></li> | + | <li><a href="#fragment1-1">begin</a></li> |
− | <li><a href="#fragment1- | + | <li><a class="circle-number" href="#fragment1-2">➀</a></li> |
− | <li><a href="#fragment1- | + | <li><a class="circle-number" href="#fragment1-3">➁</a></li> |
+ | <li><a class="circle-number" href="#fragment1-4">➂</a></li> | ||
</ul> | </ul> | ||
<div id="fragment1-1"> | <div id="fragment1-1"> | ||
<div class="p"> | <div class="p"> | ||
− | + | In order to make sure the efficiency of our "consumer", we should first knock out the <i>luxS</i> gene in our engineering bacteria GR286(a simplified strain of <i>Bacillus amyloliquefaciens</i> LL3). We used a markerless gene replacement method to knock out the <i>luxS</i> gene. | |
− | </div> | + | </div> |
</div> | </div> | ||
<div id="fragment1-2"> | <div id="fragment1-2"> | ||
<div class="p"> | <div class="p"> | ||
− | + | Construction of targeting vector : the upstream and downstream of <i>luxS</i> gene were combined by over-lapping PCR and ligated into plasmid pKSU. | |
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment1-3"> | ||
+ | <div class="p"> | ||
+ | pKSU-Δ<i>luxS</i> was transformed into GR286, and positive clones were selected. | ||
</div> | </div> | ||
<div class="flex-container"> | <div class="flex-container"> | ||
− | <table class="table-theme-1" id="table1-2-1"> | + | <div class="table-wrapper"> |
− | + | <table class="table-theme-1" id="table1-2-1"> | |
− | + | <caption>20μL PCR system</caption> | |
− | + | <tr> | |
− | + | <td>2× Taq Master Mix</td> | |
− | + | <td>10μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>pKSU-F</td> | |
− | + | <td>1μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>pKSU-R</td> | |
− | + | <td>1μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Bacterium solution</td> | |
− | + | <td>1μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>ddH2O</td> | |
− | + | <td>7μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Total</td> | |
− | + | <td>20μL</td> | |
− | + | </tr> | |
− | <table class="table-theme-2" id="table1-2-2"> | + | </table> |
− | + | </div> | |
− | + | <div class="table-wrapper"> | |
− | + | <div class="rbrace">}</div> | |
− | + | <table class="table-theme-2" id="table1-2-2"> | |
− | + | ||
− | + | <caption>PCR reaction condition</caption> | |
− | + | <tr> | |
− | + | <td>94<sup>o</sup>C</td> | |
− | + | <td>10 min</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>94<sup>o</sup>C</td> | |
− | + | <td>30 sec</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>58<sup>o</sup>C</td> | |
− | + | <td>30 sec</td> | |
− | + | <td>30 cycles</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>72<sup>o</sup>C</td> | |
− | + | <td>1 min 30 sec</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>72<sup>o</sup>C</td> | |
− | + | <td>10 min</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
<figure> | <figure> | ||
− | <img src="https://static.igem.org/mediawiki/2016/e/e9/T--NKU_China--notebook-1-5-1.png | + | <img src="https://static.igem.org/mediawiki/2016/e/e9/T--NKU_China--notebook-1-5-1.png"> |
− | <figcaption> | + | <figcaption> |
+ | <div class="p"><span>Selection of positive clones by PCR</span></div> | ||
+ | </figcaption> | ||
</figure> | </figure> | ||
</div> | </div> | ||
− | |||
</div> | </div> | ||
− | <div id="fragment1- | + | <div id="fragment1-4"> |
<div class="p"> | <div class="p"> | ||
− | The transformants were cultured at 42 | + | The transformants were cultured at 42<sup>o</sup>C with chloramphenicol to select single-crossover clones. |
</div> | </div> | ||
<div class="flex-container"> | <div class="flex-container"> | ||
− | <table class="table-theme-1" id="table1-3-1"> | + | <div class="table-wrapper"> |
− | + | <table class="table-theme-1" id="table1-3-1"> | |
− | + | <caption>20μL PCR system</caption> | |
− | + | <tr> | |
− | + | <td>2× Taq Master Mix</td> | |
− | + | <td>10μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><i>luxS</i>-up-F</td> | |
− | + | <td>1μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><i>luxS</i>-dn-R</td> | |
− | + | <td>1μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Bacterium solution</td> | |
− | + | <td>1μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>ddH2O</td> | |
− | + | <td>7μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Total</td> | |
− | + | <td>20μL</td> | |
− | + | </tr> | |
− | <table class="table-theme-2" id="table1-3-2"> | + | </table> |
− | + | </div> | |
− | + | <div class="table-wrapper"> | |
− | + | <div class="rbrace">}</div> | |
− | + | <table class="table-theme-2" id="table1-3-2"> | |
− | + | <caption>PCR reaction condition</caption> | |
− | + | <tr> | |
− | + | <td>94<sup>o</sup>C</td> | |
− | + | <td>10 min</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>94<sup>o</sup>C</td> | |
− | + | <td>30 sec</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>56<sup>o</sup>C</td> | |
− | + | <td>30 sec</td> | |
− | + | <td>30 cycles</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>72<sup>o</sup>C</td> | |
− | + | <td>2 min</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>72<sup>o</sup>C</td> | |
− | + | <td>10 min</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>16<sup>o</sup>C</td> | |
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
<figure> | <figure> | ||
− | <img src="https://static.igem.org/mediawiki/2016/d/de/T--NKU_China--notebook-1-5-2.jpeg | + | <img src="https://static.igem.org/mediawiki/2016/d/de/T--NKU_China--notebook-1-5-2.jpeg"> |
<figcaption> | <figcaption> | ||
− | + | <div class="p"><span>Selection of single-crossover clones by PCR</span></div> | |
− | (No.1-4 are single-crossover strains, No.5 is positive control.) | + | (No.1-4 are single-crossover strains, <br>No.5 is positive control.) |
</figcaption> | </figcaption> | ||
</figure> | </figure> | ||
− | </div> | + | </div> |
</div> | </div> | ||
</div> | </div> | ||
Line 594: | Line 688: | ||
<div class="tabs" id="tabs2"> | <div class="tabs" id="tabs2"> | ||
<ul> | <ul> | ||
− | <li><a href="#fragment2-1">➀</a></li> | + | <li><a class="circle-number" href="#fragment2-1">➀</a></li> |
− | <li><a href="#fragment2-2">➁</a></li> | + | <li><a class="circle-number" href="#fragment2-2">➁</a></li> |
</ul> | </ul> | ||
<div id="fragment2-1"> | <div id="fragment2-1"> | ||
<div class="p"> | <div class="p"> | ||
− | The single-crossover strains were then cultured | + | The single-crossover strains were then cultured in LB medium and passaged every 12 hours for 4 generations. |
− | </div> | + | </div> |
</div> | </div> | ||
<div id="fragment2-2"> | <div id="fragment2-2"> | ||
<div class="p"> | <div class="p"> | ||
− | + | The last generation was cultured in medium with 5-fluorouracil to select double-crossover clones. Regretfully, we didn't get the double-crossover clones. | |
</div> | </div> | ||
− | <table class="table-theme-1" id="table2-2-1"> | + | <div class="flex-container"> |
− | + | <div class="table-wrapper"> | |
− | + | <table class="table-theme-1" id="table2-2-1"> | |
− | + | <caption>20μL PCR system</caption> | |
− | + | <tr> | |
− | + | <td>2× Taq Master Mix</td> | |
− | + | <td>10μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><i>luxS</i>-up-F</td> | |
− | + | <td>1μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><i>luxS</i>-dn-R</td> | |
− | + | <td>1μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Bacterium solution</td> | |
− | + | <td>1μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>ddH2O</td> | |
− | + | <td>7μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Total</td> | |
− | + | <td>20μL</td> | |
− | + | </tr> | |
− | + | </table> | |
− | + | </div> | |
− | + | <div class="table-wrapper"> | |
− | + | <div class="rbrace">}</div> | |
− | + | <table class="table-theme-2" id="table2-2-2"> | |
− | + | <caption>PCR reaction condition</caption> | |
− | + | <tr> | |
− | + | <td>94<sup>o</sup>C</td> | |
− | + | <td>10 min</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>94<sup>o</sup>C</td> | |
− | + | <td>30 sec</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>56<sup>o</sup>C</td> | |
− | + | <td>30 sec</td> | |
− | + | <td>30 cycles</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>72<sup>o</sup>C</td> | |
− | + | <td>2 min</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>72<sup>o</sup>C</td> | |
− | + | <td>10 min</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>16<sup>o</sup>C</td> | |
− | <img src="https://static.igem.org/mediawiki/2016/3/3d/T--NKU_China--notebook-1-5-3.png | + | <td>∞</td> |
− | + | <td></td> | |
− | + | </tr> | |
− | + | </table> | |
− | + | </div> | |
− | + | <figure> | |
− | + | <img src="https://static.igem.org/mediawiki/2016/3/3d/T--NKU_China--notebook-1-5-3.png"> | |
− | </div> | + | <figcaption> |
+ | <div class="p"><span>Selection of double-crossover clones by PCR</span></div> | ||
+ | (No.1-5 are experimental groups, No.6 is wild GR286. The result showed that we failed to get the double-crossover clones.) | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
Line 685: | Line 785: | ||
<div class="tabs" id="tabs3"> | <div class="tabs" id="tabs3"> | ||
<ul> | <ul> | ||
− | <li><a href="#fragment3-1">➀</a></li> | + | <li><a class="circle-number" href="#fragment3-1">➀</a></li> |
− | <li><a href="#fragment3-2">➁</a></li> | + | <li><a class="circle-number" href="#fragment3-2">➁</a></li> |
− | <li><a href="#fragment3-3">➂</a></li> | + | <li><a class="circle-number" href="#fragment3-3">➂</a></li> |
</ul> | </ul> | ||
<div id="fragment3-1"> | <div id="fragment3-1"> | ||
<div class="p"> | <div class="p"> | ||
− | + | Transformants were cultured at 42<sup>o</sup>C with chloramphenicol again and the single-crossover clones were selected successfully. | |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table3-1-1"> | ||
+ | <caption>20μL PCR system</caption> | ||
+ | <tr> | ||
+ | <td>2× Taq Master Mix</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxS</i>-up-F</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxS</i>-dn-R</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bacterium solution</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table3-1-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>56<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>2 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/9/93/T--NKU_China--notebook-1-5-4.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Selection of single-crossover clones by PCR</span></div> | ||
+ | (No.1&2&4 are single-crossover strains,No.5 is positive control. ) | ||
+ | </figcaption> | ||
+ | </figure> | ||
</div> | </div> | ||
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− | |||
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− | |||
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− | |||
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− | |||
</div> | </div> | ||
<div id="fragment3-2"> | <div id="fragment3-2"> | ||
<div class="p"> | <div class="p"> | ||
− | The single-crossover strains were then cultured | + | The single-crossover strains were then cultured in LB medium and passaged every 12 hours for 4 generations. |
</div> | </div> | ||
</div> | </div> | ||
<div id="fragment3-3"> | <div id="fragment3-3"> | ||
<div class="p"> | <div class="p"> | ||
− | + | The last generation was cultured in medium with 5-fluorouracil to select double-crossover clones. We finally obtained our aimed strain—GR286Δ<i>luxS</i>. | |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table3-3-1"> | ||
+ | <caption>20μL PCR system</caption> | ||
+ | <tr> | ||
+ | <td>2× Taq Master Mix</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxS</i>-up-F</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxS</i>-dn-R</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bacterium solution</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table3-3-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>56<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>2 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/a/aa/T--NKU_China--notebook-1-5-5.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Selection of Δ<i>luxS</i> clones by PCR</span></div> | ||
+ | (The No.4 is the aimed strain―GR286Δ<i>luxS</i>) | ||
+ | </figcaption> | ||
+ | </figure> | ||
</div> | </div> | ||
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</div> | </div> | ||
</div> | </div> | ||
Line 848: | Line 962: | ||
<div class="tabs" id="tabs4"> | <div class="tabs" id="tabs4"> | ||
<ul> | <ul> | ||
− | <li><a href="#fragment4-1">➀</a></li> | + | <li><a class="circle-number" href="#fragment4-1">➀</a></li> |
− | <li><a href="#fragment4-2">➁</a></li> | + | <li><a class="circle-number" href="#fragment4-2">➁</a></li> |
− | <li><a href="#fragment4-3">➂</a></li> | + | <li><a class="circle-number" href="#fragment4-3">➂</a></li> |
− | <li><a href="#fragment4-4">➃</a></li> | + | <li><a class="circle-number" href="#fragment4-4">➃</a></li> |
− | <li><a href="#fragment4-5">➄</a></li> | + | <li><a class="circle-number" href="#fragment4-5">➄</a></li> |
− | <li><a href="#fragment4-6">➅</a></li> | + | <li><a class="circle-number" href="#fragment4-6">➅</a></li> |
</ul> | </ul> | ||
<div id="fragment4-1"> | <div id="fragment4-1"> | ||
<div class="p"> | <div class="p"> | ||
− | + | The GR286Δ<i>luxS</i> strain was cultured and made competent for future use. | |
</div> | </div> | ||
</div> | </div> | ||
<div id="fragment4-2"> | <div id="fragment4-2"> | ||
<div class="p"> | <div class="p"> | ||
− | + | The <i>lsrACDB</i> gene from <i>Bacillus thuringiensis</i> was cloned and ligated to T-vector. | |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table4-2-1"> | ||
+ | <caption>50μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>2× Taq Master Mix</td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDB</i>-F</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDB</i>-R</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bacterium solution</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>19μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>50μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table4-2-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>57<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>4 min 30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-3" id="table4-2-3"> | ||
+ | <caption>20μL ligation system</caption> | ||
+ | <tr> | ||
+ | <td>10× DNA Ligase Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T4 DNA Ligase</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pMD19 T-Simple Vector</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDB</i></td> | ||
+ | <td>3μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>13μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 16<sup>o</sup>C overnight</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
</div> | </div> | ||
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</div> | </div> | ||
<div id="fragment4-3"> | <div id="fragment4-3"> | ||
<div class="p"> | <div class="p"> | ||
− | + | The T-<i>lsrACDB</i> was transformed into DH5α and plate was coated, and then positive clones were selected. | |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table4-3-1"> | ||
+ | <caption>20μL PCR system</caption> | ||
+ | <tr> | ||
+ | <td>2× Taq Master Mix</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>M13F</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>M13R</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bacterium solution</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table4-3-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>59<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>4 min 30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/7/72/T--NKU_China--notebook-1-5-6.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Selection of positive clones by PCR</span></div> | ||
+ | (No. 3&4 are positive results) | ||
+ | </figcaption> | ||
+ | </figure> | ||
</div> | </div> | ||
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</div> | </div> | ||
<div id="fragment4-4"> | <div id="fragment4-4"> | ||
<div class="p"> | <div class="p"> | ||
− | After restriction enzyme digestion verification, | + | After restriction enzyme digestion verification, the positive clones were then sequenced. Unfortunately, the sequencing result showed some mutations inside the target gene. |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table4-4-1"> | ||
+ | <caption>20μL digestion system</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BamH Ⅰ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T-<i>lsrACDB</i></td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/9/91/T--NKU_China--notebook-1-5-7.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Restriction enzyme digestion verification</span></div> | ||
+ | (No.1 are <i>lsrACDB</i> fragement, No.2 are linearized T-vector.) | ||
+ | </figcaption> | ||
+ | </figure> | ||
</div> | </div> | ||
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</div> | </div> | ||
<div id="fragment4-5"> | <div id="fragment4-5"> | ||
<div class="p"> | <div class="p"> | ||
− | + | The gene cloning process was repeated but there were still some mutations. | |
</div> | </div> | ||
</div> | </div> | ||
Line 1,076: | Line 1,210: | ||
</div> | </div> | ||
</div> | </div> | ||
− | </div> | + | </div> |
</div> | </div> | ||
<div class="accordion-fragment" id="week5"> | <div class="accordion-fragment" id="week5"> | ||
Line 1,083: | Line 1,217: | ||
</div> | </div> | ||
<div class="accordion-content"> | <div class="accordion-content"> | ||
− | |||
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<div class="tabs" id="tabs5"> | <div class="tabs" id="tabs5"> | ||
<ul> | <ul> | ||
− | <li><a href="#fragment5-1">➀</a></li> | + | <li><a href="#fragment5-1">begin</a></li> |
− | <li><a href="#fragment5- | + | <li><a class="circle-number" href="#fragment5-2">➀</a></li> |
− | <li><a href="#fragment5- | + | <li><a class="circle-number" href="#fragment5-3">➁</a></li> |
− | <li><a href="#fragment5- | + | <li><a class="circle-number" href="#fragment5-4">➂</a></li> |
+ | <li><a class="circle-number" href="#fragment5-5">➃</a></li> | ||
</ul> | </ul> | ||
<div id="fragment5-1"> | <div id="fragment5-1"> | ||
<div class="p"> | <div class="p"> | ||
− | + | This week, we started to construct another controller―supplier. | |
</div> | </div> | ||
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</div> | </div> | ||
− | + | <div id="fragment5-2"> | |
− | + | <div class="p"> | |
− | + | A strong promoter <i>C2</i> was cloned from former kit and <i>luxS</i> gene was cloned from GR286. | |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table5-1-1"> | ||
+ | <caption>50μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>2× Taq Master Mix</td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>C2-F</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>C2-R</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>p-C2</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>19μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>50μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table5-1-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>58<sup>o</sup>C</td> | ||
+ | <td>15 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table5-1-3"> | ||
+ | <caption>50μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>2× Taq Master Mix</td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxS</i>-F</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxS</i>-R</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bacterium solution</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>GR286</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>19μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>50μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table5-1-4"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>59<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/d/dc/T--NKU_China--notebook-1-5-8.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>PCR cloning product of promoter <i>C2</i> and gene <i>luxS</i></span></div> | ||
+ | (NO.1&2 are <i>C2</i>, No.3&4 are <i>luxS</i>) | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
</div> | </div> | ||
− | <table class="table-theme-1" id="table5-2-1"> | + | <div id="fragment5-3"> |
− | + | <div class="p"> | |
− | + | Two segments were fused together by fusion PCR and ligated into T-vector. After that, the vector was transformed into DH5α. | |
− | + | </div> | |
− | + | <div class="flex-container"> | |
− | + | <div class="table-wrapper"> | |
− | + | <table class="table-theme-1" id="table5-2-1"> | |
− | + | <caption>50μL PCR system ×2</caption> | |
− | + | <tr> | |
− | + | <td>2× Taq Master Mix</td> | |
− | + | <td>25μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>C2-F</td> | |
− | + | <td>2μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><i>luxS</i>-R</td> | |
− | + | <td>2μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>C2</td> | |
− | + | <td>2μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><i>luxS</i></td> | |
− | + | <td>2μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>ddH2O</td> | |
− | + | <td>17μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Total</td> | |
− | + | <td>50μL</td> | |
− | + | </tr> | |
− | + | </table> | |
− | + | </div> | |
− | + | <div class="table-wrapper"> | |
− | + | <div class="rbrace">}</div> | |
− | + | <table class="table-theme-2" id="table5-2-2"> | |
− | + | <caption>PCR reaction condition</caption> | |
− | + | <tr> | |
− | + | <td>94<sup>o</sup>C</td> | |
− | + | <td>10 min</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>94<sup>o</sup>C</td> | |
− | + | <td>30 sec</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>59<sup>o</sup>C</td> | |
− | + | <td>30 sec</td> | |
− | + | <td>30 cycles</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>72<sup>o</sup>C</td> | |
− | + | <td>40 sec</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>72<sup>o</sup>C</td> | |
− | + | <td>10 min</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>16<sup>o</sup>C</td> | |
− | + | <td>∞</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | </table> | |
− | + | </div> | |
− | + | <div class="table-wrapper"> | |
− | + | <table class="table-theme-3" id="table5-2-3"> | |
− | + | <caption>20μL ligation system</caption> | |
− | + | <tr> | |
− | + | <td>10× DNA Ligase Buffer</td> | |
− | + | <td>2μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>T4 DNA Ligase</td> | |
− | + | <td>1μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>pMD19 T-Simple Vector</td> | |
− | + | <td>1μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td><i>C2-luxS</i></td> | |
− | + | <td>4μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>ddH2O</td> | |
− | + | <td>12μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 16<sup>o</sup>C overnight</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | </div> | ||
</div> | </div> | ||
− | <table class="table-theme-1" id="table5-3-1"> | + | <div id="fragment5-4"> |
− | + | <div class="p"> | |
− | + | Positive clones were selected by colony PCR. | |
− | + | </div> | |
− | + | <div class="flex-container"> | |
− | + | <div class="table-wrapper"> | |
− | + | <table class="table-theme-1" id="table5-3-1"> | |
− | + | <caption>20μL PCR system</caption> | |
− | + | <tr> | |
− | + | <td>2× Taq Master Mix</td> | |
− | + | <td>10μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>M13-F</td> | |
− | + | <td>1μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>M13-R</td> | |
− | + | <td>1μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Bacterium solution</td> | |
− | + | <td>1μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>ddH2O</td> | |
− | + | <td>7μL</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>Total</td> | |
− | + | <td>20μL</td> | |
− | + | </tr> | |
− | + | </table> | |
− | + | </div> | |
− | + | <div class="table-wrapper"> | |
− | + | <div class="rbrace">}</div> | |
− | + | <table class="table-theme-2" id="table5-3-2"> | |
− | + | <caption>PCR reaction condition</caption> | |
− | + | <tr> | |
− | + | <td>94<sup>o</sup>C</td> | |
− | + | <td>10 min</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>94<sup>o</sup>C</td> | |
− | + | <td>30 sec</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>59<sup>o</sup>C</td> | |
− | + | <td>30 sec</td> | |
− | + | <td>30 cycles</td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>72<sup>o</sup>C</td> | |
− | + | <td>40 sec</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>72<sup>o</sup>C</td> | |
− | + | <td>10 min</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | <tr> | |
− | + | <td>16<sup>o</sup>C</td> | |
− | + | <td>∞</td> | |
− | + | <td></td> | |
− | + | </tr> | |
− | + | </table> | |
− | + | </div> | |
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/c/c9/T--NKU_China--notebook-1-5-9.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>PCR cloning product of promoter <i>C2</i> and gene <i>luxS</i></span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment5-5"> | ||
+ | <div class="p"> | ||
+ | 4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table5-4-1"> | ||
+ | <caption>20μL digestion system</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BamH Ⅰ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T-<i>lsrACDB</i></td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/f/fb/T--NKU_China--notebook-1-5-10.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Restriction enzyme digestion verification</span></div> | ||
+ | (No.1 are linearized T-vector, No.2 are <i>C2-luxS</i> fragment.) | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
</div> | </div> | ||
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</div> | </div> | ||
− | |||
</div> | </div> | ||
</div> | </div> | ||
Line 1,447: | Line 1,616: | ||
<div class="tabs" id="tabs6"> | <div class="tabs" id="tabs6"> | ||
<ul> | <ul> | ||
− | <li><a href="#fragment6-1">➀</a></li> | + | <li><a class="circle-number" href="#fragment6-1">➀</a></li> |
− | <li><a href="#fragment6-2">➁</a></li> | + | <li><a class="circle-number" href="#fragment6-2">➁</a></li> |
− | <li><a href="#fragment6-3">➂</a></li> | + | <li><a class="circle-number" href="#fragment6-3">➂</a></li> |
− | <li><a href="#fragment6-4">➃</a></li> | + | <li><a class="circle-number" href="#fragment6-4">➃</a></li> |
− | <li><a href="#fragment6-5">➄</a></li> | + | <li><a class="circle-number" href="#fragment6-5">➄</a></li> |
</ul> | </ul> | ||
<div id="fragment6-1"> | <div id="fragment6-1"> | ||
<div class="p"> | <div class="p"> | ||
− | The sequencing result showed there | + | The sequencing result showed that there was a correct strain and thus it could be used for the following experiments. We obtained the correct plasmid T-<i>C2-luxS</i> from DH5α. Then the fragment <i>C2-luxS</i> was obtained by digestion and gel extraction. |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table6-1-1"> | ||
+ | <caption>40μL digestion system</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>4μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BamH Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T-<i>C2-luxS</i></td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>9μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
</div> | </div> | ||
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− | |||
</div> | </div> | ||
<div id="fragment6-2"> | <div id="fragment6-2"> | ||
<div class="p"> | <div class="p"> | ||
− | + | The <i>C2-luxS</i> fragment was ligated to linearized plasmid pWH1520, and then the ligation product was transformed into DH5α. | |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-3" id="table6-2-1"> | ||
+ | <caption>20μL ligation system</caption> | ||
+ | <tr> | ||
+ | <td>10× DNA Ligase Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T4 DNA Ligase</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pMD19 T-Simple Vector</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>C2-luxS</i></td> | ||
+ | <td>5μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>11μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 16<sup>o</sup>C overnight</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
</div> | </div> | ||
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− | |||
− | |||
</div> | </div> | ||
<div id="fragment6-3"> | <div id="fragment6-3"> | ||
<div class="p"> | <div class="p"> | ||
− | + | The plasmid pWH-<i>C2-luxS</i> was extracted from DH5α. To prevent the plasmid from DAM&DCM methylation, we transformed it into <i>E.coli</i> JM110. | |
</div> | </div> | ||
</div> | </div> | ||
<div id="fragment6-4"> | <div id="fragment6-4"> | ||
<div class="p"> | <div class="p"> | ||
− | + | The plasmid pWH-<i>C2-luxS</i> was extracted from JM110,and then it was treated with BamH Ⅰ methylase. | |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-5" id="table6-4-1"> | ||
+ | <caption>100μL methylation system</caption> | ||
+ | <tr> | ||
+ | <td>10× BamH Ⅰ methyltransferase Buffer</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BamH Ⅰ methyltransferase</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>S-adenosylmethionine</td> | ||
+ | <td>0.5μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pWH-<i>C2-luxS</i></td> | ||
+ | <td>80μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>8.5μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>100μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 1 hour</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
</div> | </div> | ||
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</div> | </div> | ||
<div id="fragment6-5"> | <div id="fragment6-5"> | ||
<div class="p"> | <div class="p"> | ||
− | + | The plasmid was transformed into GR286 by electroporation.[Failed] | |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/e/e0/T--NKU_China--notebook-6-13-1.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Selection of positive clones by colony PCR</span></div> | ||
+ | (No.1 is positive control, No.2-6 are experimental groups. The result showed that we failed to transformed the plasmid pWH-<i>C2-luxS</i> into GR286) | ||
+ | </figcaption> | ||
+ | </figure> | ||
</div> | </div> | ||
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</div> | </div> | ||
</div> | </div> | ||
Line 1,583: | Line 1,764: | ||
<div class="tabs" id="tabs7"> | <div class="tabs" id="tabs7"> | ||
<ul> | <ul> | ||
− | <li><a href="#fragment7-1">➀</a></li> | + | <li><a class="circle-number" href="#fragment7-1">➀</a></li> |
− | <li><a href="#fragment7-2">➁</a></li> | + | <li><a class="circle-number" href="#fragment7-2">➁</a></li> |
− | <li><a href="#fragment7-3">➂</a></li> | + | <li><a class="circle-number" href="#fragment7-3">➂</a></li> |
</ul> | </ul> | ||
<div id="fragment7-1"> | <div id="fragment7-1"> | ||
<div class="p"> | <div class="p"> | ||
− | This week, we tried to use different voltages to transform the plasmid. Sadly, all of these | + | This week, we tried to use different voltages to transform the plasmid. Sadly, all of these attempts rendered negative results. |
</div> | </div> | ||
− | |||
</div> | </div> | ||
<div id="fragment7-2"> | <div id="fragment7-2"> | ||
<div class="p"> | <div class="p"> | ||
− | We considered whether the <i>luxS</i> gene is | + | We considered whether the <i>luxS</i> gene is toxic for GR286, and the bacteria tends to reject the gene when a strong promoter is inserted upstream of it. So, we planned to use inducible promoter to reconstruct our expression vector. |
</div> | </div> | ||
− | |||
</div> | </div> | ||
<div id="fragment7-3"> | <div id="fragment7-3"> | ||
<div class="p"> | <div class="p"> | ||
− | The plasmid pWH1520 contains | + | The plasmid pWH1520 contains a strong <i>xylA</i> promoter originating from <i>Bacillus megaterium</i>, and transcription initiated by this promoter is xylose-inducible. Also, the gene of interest carries its own ribosome binding sequence (RBS) and translation initiation codon. Based on these points, we redesigned primers. |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/e/eb/T--NKU_China--notebook-6-13-2.png"> | ||
+ | <figcaption> | ||
+ | </figcaption> | ||
+ | </figure> | ||
</div> | </div> | ||
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</div> | </div> | ||
</div> | </div> | ||
Line 1,619: | Line 1,800: | ||
<div class="tabs" id="tabs8"> | <div class="tabs" id="tabs8"> | ||
<ul> | <ul> | ||
− | <li><a href="#fragment8-1">➀</a></li> | + | <li><a class="circle-number" href="#fragment8-1">➀</a></li> |
− | <li><a href="#fragment8-2">➁</a></li> | + | <li><a class="circle-number" href="#fragment8-2">➁</a></li> |
− | <li><a href="#fragment8-3">➂</a></li> | + | <li><a class="circle-number" href="#fragment8-3">➂</a></li> |
− | <li><a href="#fragment8-4">➃</a></li> | + | <li><a class="circle-number" href="#fragment8-4">➃</a></li> |
</ul> | </ul> | ||
<div id="fragment8-1"> | <div id="fragment8-1"> | ||
<div class="p"> | <div class="p"> | ||
− | + | <i>luxS</i> gene was cloned from GR286 using our new primers. | |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table8-1-1"> | ||
+ | <caption>50μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>2× Taq Master Mix</td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>YD-luxS-F</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>YD-luxS-R</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bacterium solution</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>19μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>50μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table8-1-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>58<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>40 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/3/3a/T--NKU_China--notebook-6-13-3.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>PCR cloning product of gene <i>luxS</i></span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
</div> | </div> | ||
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</div> | </div> | ||
<div id="fragment8-2"> | <div id="fragment8-2"> | ||
<div class="p"> | <div class="p"> | ||
− | + | The <i>luxS</i> fragment was purified by gel extraction, and ligated into linearized pWH1520. Then the vector was transformed into DH5α. | |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table8-2-1"> | ||
+ | <caption>40μL digestion system ×2</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>4μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BamH Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pWH1520</td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>9μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>40μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-3" id="table8-2-2"> | ||
+ | <caption>20μL ligation system</caption> | ||
+ | <tr> | ||
+ | <td>10× DNA Ligase Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T4 DNA Ligase</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>linearized pWH1520</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxS</i></td> | ||
+ | <td>3μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>13μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 16<sup>o</sup>C overnight</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
</div> | </div> | ||
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</div> | </div> | ||
<div id="fragment8-3"> | <div id="fragment8-3"> | ||
<div class="p"> | <div class="p"> | ||
− | + | Positive clones were selected by colony PCR. | |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table8-3-1"> | ||
+ | <caption>20μL PCR system</caption> | ||
+ | <tr> | ||
+ | <td>2× Taq Master Mix</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pWH-F</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pWH-R</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bacterium solution</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table8-3-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>58<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>40 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/4/49/T--NKU_China--notebook-6-13-4.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Selection of positive clones by colony PCR</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
</div> | </div> | ||
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</div> | </div> | ||
<div id="fragment8-4"> | <div id="fragment8-4"> | ||
<div class="p"> | <div class="p"> | ||
− | + | 4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced. | |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table8-4-1"> | ||
+ | <caption>20μL digestion system</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BamH Ⅰ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pWH-<i>luxs</i></td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/3/35/T--NKU_China--notebook-6-13-5.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Restriction enzyme digestion verification</span></div> | ||
+ | (No.1 are linearized pWH1520, No.2 are <i>luxS</i> fragments.) | ||
+ | </figcaption> | ||
+ | </figure> | ||
</div> | </div> | ||
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</div> | </div> | ||
Line 1,875: | Line 2,081: | ||
<div class="tabs" id="tabs9"> | <div class="tabs" id="tabs9"> | ||
<ul> | <ul> | ||
− | <li><a href="#fragment9-1">➀</a></li> | + | <li><a class="circle-number" href="#fragment9-1">➀</a></li> |
− | <li><a href="#fragment9-2">➁</a></li> | + | <li><a class="circle-number" href="#fragment9-2">➁</a></li> |
− | <li><a href="#fragment9-3">➂</a></li> | + | <li><a class="circle-number" href="#fragment9-3">➂</a></li> |
+ | <li><a href="#fragment9-4">end</a></li> | ||
</ul> | </ul> | ||
<div id="fragment9-1"> | <div id="fragment9-1"> | ||
<div class="p"> | <div class="p"> | ||
− | The sequencing result showed there's three | + | The sequencing result showed there's three positive strains. So one positive strain was chosen to be used for the following experiments. The pWH-<i>luxS</i> plasmid was extracted from the chosen strain. To prevent the plasmid from DAM&DCM methylation, we transformed it into E.coli JM110. |
</div> | </div> | ||
− | |||
</div> | </div> | ||
<div id="fragment9-2"> | <div id="fragment9-2"> | ||
<div class="p"> | <div class="p"> | ||
− | + | the plasmid pWH-<i>luxS</i> was extracted from JM110,and it was treated withBamH Ⅰ methylase. | |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-5" id="table6-4-1"> | ||
+ | <caption>100μL methylation system</caption> | ||
+ | <tr> | ||
+ | <td>10× BamH Ⅰ methyltransferase Buffer</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BamH Ⅰ methyltransferase</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>S-adenosylmethionine</td> | ||
+ | <td>0.5μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pWH-<i>C2-luxS</i></td> | ||
+ | <td>80μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>8.5μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>100μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 1 hour</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
</div> | </div> | ||
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</div> | </div> | ||
<div id="fragment9-3"> | <div id="fragment9-3"> | ||
<div class="p"> | <div class="p"> | ||
− | + | The plasmid was transformed into GR286 by electroporation, and positive clones were selected. | |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/a/aa/T--NKU_China--notebook-6-13-6.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Selection of positive clones by colony PCR</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment9-4"> | ||
+ | <div class="p"> | ||
+ | The construction of supplier was accomplished! | ||
</div> | </div> | ||
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</div> | </div> | ||
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</div> | </div> | ||
</div> | </div> | ||
Line 1,944: | Line 2,158: | ||
<div class="tabs" id="tabs10"> | <div class="tabs" id="tabs10"> | ||
<ul> | <ul> | ||
− | <li><a href="#fragment10-1">➀</a></li> | + | <li><a class="circle-number" href="#fragment10-1">➀</a></li> |
− | <li><a href="#fragment10-2">➁</a></li> | + | <li><a class="circle-number" href="#fragment10-2">➁</a></li> |
</ul> | </ul> | ||
<div id="fragment10-1"> | <div id="fragment10-1"> | ||
<div class="p"> | <div class="p"> | ||
− | + | We have received the product of synthetic <i>lsrACDB</i> gene. We first used restriction-ligation method to ligate <i>lsrACDB</i> to plasmid pWH1520, but we failed to select positive after several tries. | |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-3" id="table10-1-1"> | ||
+ | <caption>20μL ligation system</caption> | ||
+ | <tr> | ||
+ | <td>10× DNA Ligase Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T4 DNA Ligase</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>linearized pWH1520</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDB</i></td> | ||
+ | <td>3μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>13μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 16<sup>o</sup>C overnight</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
</div> | </div> | ||
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</div> | </div> | ||
<div id="fragment10-2"> | <div id="fragment10-2"> | ||
<div class="p"> | <div class="p"> | ||
− | Considering the <i>lsrACDB</i> gene is a large fragment (4500bp), we used ClonExpress technique | + | Considering that the <i>lsrACDB</i> gene is a large fragment (4500bp), we used ClonExpress technique to clone the gene again to improve the efficiency of ligation. The <i>lsrACDB</i> sequence was divided into two parts and they were cloned separately. Then the two segments were ligated to the plasmid pWH1520 and the recombinant vector was transformed into DH5α. After that, verification PCR was used to select the positive clones. However, we didn't get a good result. |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/1/17/T--NKU_China--notebook-6-13-7.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Selection of positive clones by colony PCR</span></div> | ||
+ | (No.6 is positive control, No.1-5 are experimental groups) | ||
+ | </figcaption> | ||
+ | </figure> | ||
</div> | </div> | ||
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</div> | </div> | ||
</div> | </div> | ||
Line 2,002: | Line 2,222: | ||
</div> | </div> | ||
<div class="accordion-content"> | <div class="accordion-content"> | ||
− | <div class=" | + | <div class="tabs" id="tabs10"> |
− | We learnt a new method called circular polymerase extension cloning (CPEC) for high-throughput cloning of complex and combinatorial DNA libraries, and we decided to use this method to try | + | <ul> |
+ | <li><a class="circle-number" href="#fragment11-1">➀</a></li> | ||
+ | </ul> | ||
+ | <div id="fragment11-1"> | ||
+ | <div class="p"> | ||
+ | We learnt a new method called circular polymerase extension cloning (CPEC) for high-throughput cloning of complex and combinatorial DNA libraries, and we decided to use this method to try to ligate our <i>lsrACDB</i> gene. It's encouraging that we succeeded to ligate the <i>lsrACDB</i> gene to the plasmid pHT-01. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/c/c2/T--NKU_China--notebook-6-13-8.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Selection of positive clones by colony PCR</span></div> | ||
+ | (No.3-6 are positive clones) | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/7/7a/T--NKU_China--notebook-6-13-9.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Restriction enzyme digestion verification</span></div> | ||
+ | (No.2&3 are positive results.) | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
</div> | </div> | ||
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</div> | </div> | ||
</div> | </div> | ||
Line 2,028: | Line 2,257: | ||
<div class="tabs" id="tabs12"> | <div class="tabs" id="tabs12"> | ||
<ul> | <ul> | ||
− | <li><a href="#fragment12-1">➀</a></li> | + | <li><a class="circle-number" href="#fragment12-1">➀</a></li> |
− | <li><a href="#fragment12-2">➁</a></li> | + | <li><a class="circle-number" href="#fragment12-2">➁</a></li> |
</ul> | </ul> | ||
<div id="fragment12-1"> | <div id="fragment12-1"> | ||
<div class="p"> | <div class="p"> | ||
− | Since we have constructed "supplier" and part of "consumer", we decided to measure the growth curve to explore the function of our "controller". | + | Since we have already successfully constructed "supplier" and part of "consumer", we decided to measure the growth curve to explore the function of our "controller". |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-6" id="table12-1-1"> | ||
+ | <caption>Groups divided in this experiment</caption> | ||
+ | <tr> | ||
+ | <td>GR286</td> | ||
+ | <td>wild strain as control group</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>GR286Δ<i>luxS</i></td> | ||
+ | <td>GR286 without <i>luxS</i> gene</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pWH-<i>luxS</i></td> | ||
+ | <td><i>luxS</i> overexpression plasmid in GR286; without induced by xylose</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pWH-<i>luxS</i> + xyl</td> | ||
+ | <td><i>luxS</i> overexpression plasmid in GR286; induced by xylose</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pWH1520</td> | ||
+ | <td>empty plasmid in GR286 as control group</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pHT-<i>lsrACDB</i></td> | ||
+ | <td><i>lsrACDB</i> overexpression plasmid in GR286Δ<i>luxS</i></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pHT-01</td> | ||
+ | <td>empty plasmid in GR286Δ<i>luxS</i> as control group</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/a/ad/T--NKU_China--notebook-6-13-10.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>OD<sub>600</sub> of different groups at specific times</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/0/00/T--NKU_China--notebook-6-13-11.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Growth curve of GR286 and GR286Δ<i>luxS</i></span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/a/a9/T--NKU_China--notebook-6-13-12.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Growth curve of pWH-<i>luxS</i>, pWH-<i>luxS</i> + xyl and pWH1520</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/6/6b/T--NKU_China--notebook-6-13-13.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Growth curve of pHT-<i>lsrACDB</i> and pHT-01</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
</div> | </div> | ||
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</div> | </div> | ||
<div id="fragment12-2"> | <div id="fragment12-2"> | ||
<div class="p"> | <div class="p"> | ||
− | Cultured media of our supplier was tested for the presence of AI-2 by inducing luminescence | + | Cultured media of our supplier was tested for the presence of AI-2 by inducing luminescence of <i>Vibrio harveyi</i> reporter strain BB170. |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/a/a4/T--NKU_China--notebook-6-13-14.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Fluorescence intensity of different groups at specific times</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/c/c5/T--NKU_China--notebook-6-13-15.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Relative fluorescence intensity of different groups</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
</div> | </div> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
</div> | </div> | ||
</div> | </div> | ||
Line 2,112: | Line 2,353: | ||
<div class="tabs" id="tabs12"> | <div class="tabs" id="tabs12"> | ||
<ul> | <ul> | ||
− | <li><a href="# | + | <li><a class="circle-number" href="#fragment13-1">➀</a></li> |
− | <li><a href="# | + | <li><a class="circle-number" href="#fragment13-2">➁</a></li> |
</ul> | </ul> | ||
− | <div id=" | + | <div id="fragment13-1"> |
<div class="p"> | <div class="p"> | ||
− | For our consumer, we should also | + | For our consumer, we should also overexpress the <i>lsrK</i> and <i>lsrFG</i> gene for phosphorylating and degrading phosphorylated AI-2. We used ClonExpress technique to clone the two genes and ligate them to plasmid pHT-01 successfully. |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/c/c5/T--NKU_China--notebook-6-13-16.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Restriction enzyme digestion verification</span></div> | ||
+ | (No.1&3&4 are positive results. ) | ||
+ | </figcaption> | ||
+ | </figure> | ||
</div> | </div> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
</div> | </div> | ||
− | <div id=" | + | <div id="fragment13-2"> |
<div class="p"> | <div class="p"> | ||
− | We | + | We co-cultured the supplier with BB170 and tested the fluorescence intensity to explore the function of supplier. (negative result) |
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/f/f1/T--NKU_China--notebook-6-13-17.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Fluorescence intensity of BB170 and co-culture medium</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
</div> | </div> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
</div> | </div> | ||
</div> | </div> | ||
Line 2,145: | Line 2,391: | ||
</div> | </div> | ||
<div class="accordion-content"> | <div class="accordion-content"> | ||
− | + | <div class="tabs" id="tabs14"> | |
+ | <ul> | ||
+ | <li><a href="#fragment14">begin</a></li> | ||
+ | <li><a href="#fragment14-0">outline</a></li> | ||
+ | <li><a href="#fragment14-1-1">1.1</a></li> | ||
+ | <li><a href="#fragment14-1-2">1.2</a></li> | ||
+ | <li><a href="#fragment14-1-3">1.3</a></li> | ||
+ | </ul> | ||
+ | <div id="fragment14"> | ||
+ | <div class="p">Since the controller we constructed with GR286 did not demonstrate positive results, we decided to substitute GR286 with <i>Escherichia coli</i> whose AI-2 metabolic pathway had been elucidated as the chassis of our controller.</div> | ||
+ | <ul> | ||
+ | <li> | ||
+ | The strains to be constructed are shown in the tables below. | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-0" style="font-weight:bold"> | ||
+ | <caption>AI-2 supplier</caption> | ||
+ | <tr> | ||
+ | <td></td> | ||
+ | <td>LuxS</td> | ||
+ | <td>Mtn</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrcHisB</td> | ||
+ | <td style="font-weight:normal">Native</td> | ||
+ | <td style="font-weight:normal">Native</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pLuxS</td> | ||
+ | <td>Induced</td> | ||
+ | <td style="font-weight:normal">Native</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pLuxS-Mtn</td> | ||
+ | <td>Induced</td> | ||
+ | <td>Induced</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-0" style="font-weight:bold"> | ||
+ | <caption>AI-2 consumer</caption> | ||
+ | <tr> | ||
+ | <td></td> | ||
+ | <td>LsrACDB</td> | ||
+ | <td>LsrK</td> | ||
+ | <td>LsrFG</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrcHisB</td> | ||
+ | <td style="font-weight:normal">Native</td> | ||
+ | <td style="font-weight:normal">Native</td> | ||
+ | <td style="font-weight:normal">Native</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pLsrACDB</td> | ||
+ | <td>Induced</td> | ||
+ | <td style="font-weight:normal">Native</td> | ||
+ | <td style="font-weight:normal">Native</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pLsrFG</td> | ||
+ | <td style="font-weight:normal">Native</td> | ||
+ | <td style="font-weight:normal">Native</td> | ||
+ | <td>Induced</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pLsrK</td> | ||
+ | <td style="font-weight:normal">Native</td> | ||
+ | <td>Induced</td> | ||
+ | <td style="font-weight:normal">Native</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pLsrACDBFG</td> | ||
+ | <td>Induced</td> | ||
+ | <td style="font-weight:normal">Native</td> | ||
+ | <td>Induced</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pLsrACDBK</td> | ||
+ | <td>Induced</td> | ||
+ | <td>Constitutive</td> | ||
+ | <td style="font-weight:normal">Native</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>plsrACDBFGK</td> | ||
+ | <td>Induced</td> | ||
+ | <td>Constitutive</td> | ||
+ | <td>Induced</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | <li> | ||
+ | The sequences of <i>lsrACDB</i>, <i>lsrFG</i>, <i>lsrK</i>, <i>luxS</i>, <i>mtn</i> gene in <i>E.coli K12</i> were searched in NCBI, and primers were designed according to the gene sequences. The primers used in the construction of AI-2 controller are shown in the table below. | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-0" style="word-break:break-all"> | ||
+ | <caption>Primers used in the construction of AI-2 controller</caption> | ||
+ | <tr> | ||
+ | <td style="min-width:7.5rem">Primer name</td> | ||
+ | <td>Sequence</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDBFG</i>1-F</td> | ||
+ | <td>CGACGATGACGATAAGGATCCATGCAAACGAGTGATACCCGC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDBFG</i>1-R</td> | ||
+ | <td>TGTTTGGCGTTTCCGGCAGCGGTGCGGAGAGC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDBFG</i>2-F</td> | ||
+ | <td>TGTTTGGCGTTTCCGCGATTG</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDBFG</i>2-R</td> | ||
+ | <td>GCACTCTCACACCACGTTGCATGGCGCGTTTC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDBFG</i>3-F</td> | ||
+ | <td>GTGGTGTGAGAGTGCTGAC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDBFG</i>3-R</td> | ||
+ | <td>ACCAGCTGCAGATCTCGAGCTCGTCACGGCATCAACCCATTGAAC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDB</i>1-F</td> | ||
+ | <td>CGACGATGACGATAAGGATCCATGCAAACGAGTGATACCCGC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDB</i>1-R</td> | ||
+ | <td>ACCGGAACCGCCGAGCAAACTAATGCCGCCCAGCAC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDB</i>2-F</td> | ||
+ | <td>CTCGGCGGTTCCGGTGCGAT</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDB</i>2-R</td> | ||
+ | <td>ACCAGCTGCAGATCTCGAGCTCGTCAGAAATCGTATTTGCCG</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxS</i>-F</td> | ||
+ | <td>ATGACGATAAGGATCCGAGCTCGATGCCGTTGTTAGATAGCTTC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxS</i>-R</td> | ||
+ | <td>GGACTCCCCCGGGGGACTAAATGTGCAGTTCCTGCAACTTC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>mtn</i>-F</td> | ||
+ | <td>TCCCCCGGGGGAGTCCTCTCCCGCGTGAGAAATAC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>mtn</i>-R</td> | ||
+ | <td>TATGGTACCAGCTGCAGATCTCTTAGCCATGTGCAAGTTTCTG</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxS</i>-Fd</td> | ||
+ | <td>GGAATTCCCTAAATGTGCAGTTCCTGCAACTTC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxS</i>-Rv</td> | ||
+ | <td>GGGGTACCCCATGCCGTTGTTAGATAGCTTCACAG</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrK</i>-Fd</td> | ||
+ | <td>GGGGTACCCCATGGCTCGACTCTTTACC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrK</i>-Rv</td> | ||
+ | <td>GGAATTCCCTATAACCCAGGCGCTTTCC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrFG</i>-Fd</td> | ||
+ | <td>CGGGATCCCGATGGCAGATTTAGACGATATTAAAGATGG</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrFG</i>-Rv</td> | ||
+ | <td>GAAGATCTTCTCACGGCATCAACCCATTGAAC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrK</i>-F</td> | ||
+ | <td>AACTGCAGAACCAATGCATTGGTTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGCAAAGAGGAGAAAATGGCTCGACTCTTTACC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrK</i>-R</td> | ||
+ | <td>TTCGAACTATAACCCAGGCGCTTTCC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BK-JC-F</td> | ||
+ | <td>TTTCAGTGTATGTCGCGGATGC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>GK-JC-F</td> | ||
+ | <td>TTGCGCTTCGATGTCTTACAGG</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-JC-F</td> | ||
+ | <td>TGGGCACTCGACCGGAATTATC</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-JC-R</td> | ||
+ | <td>GCTACTGCCGCCAGGCAAATTC</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | <div id="fragment14-0"> | ||
+ | <ol> | ||
+ | <li>Construction of pLuxS-Mtn</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | <div id="fragment14-1-1"> | ||
+ | <div class="p"> | ||
+ | <i>luxS</i> gene was cloned from <i>E.coli K12</i>. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table14-1-1-1-1"> | ||
+ | <caption>50μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>PrimeSTAR Max Premix(2×)</td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxS</i>-F</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxS</i>-R</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>E.coli K12</i></td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>19μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>50μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table14-1-1-1-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>63<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/f/f7/T--NKU_China--notebook-14-17-01.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>PCR cloning product of gene <i>luxS</i></span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table14-1-1-1-3"> | ||
+ | <caption>50μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>PrimeSTAR Max Premix(2×)</td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>mtn</i>-F</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>mtn</i>-R</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>E.coli K12</i></td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>19μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>50μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table14-1-1-1-4"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>63<sup>o</sup>C</td> | ||
+ | <td>30sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>45 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/6/66/T--NKU_China--notebook-14-17-02.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>PCR cloning product of gene <i>mtn</i></span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment14-1-2"> | ||
+ | <div class="p"> | ||
+ | ClonExpress technique was used to ligate <i>luxS</i> and <i>mtn</i> genes to plasmid pTrcHisB. The recombinant vector pTrc-<i>luxS</i>-<i>mtn</i> was transformed into <i>E.coli K12</i>. After that, verification PCR was performed to select the positive clones. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table14-1-1-2-1"> | ||
+ | <caption>20μL PCR system</caption> | ||
+ | <tr> | ||
+ | <td>PrimeSTAR Max Premix(2×)</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-JC-F</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-JC-R</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bacterium solution</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table14-1-1-2-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>58<sup>o</sup>C</td> | ||
+ | <td>30sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>1 min 30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/e/e4/T--NKU_China--notebook-14-17-03.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Selection of positive clones by PCR</span></div> | ||
+ | (NO.3&5&8 are positive results) | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment14-1-3"> | ||
+ | <div class="p"> | ||
+ | 3 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table14-1-3-1-1"> | ||
+ | <caption>20μL digestion system</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Sac Ⅰ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bgl Ⅱ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-<i>luxS</i>-<i>mtn</i></td> | ||
+ | <td>6μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/8/83/T--NKU_China--notebook-14-17-04.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Restriction enzyme digestion verification</span></div> | ||
+ | (No.1 are linearized pTrcHisB, No.2 are <i>luxS</i>-<i>mtn</i> fragment. ) | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-fragment" id="week15"> | ||
+ | <div class="accordion-header"> | ||
+ | <span class="default">☞</span><span class="active">☟</span> Week15 (Aug 22–Aug 28) | ||
+ | </div> | ||
+ | <div class="accordion-content"> | ||
+ | <div class="tabs" id="tabs15"> | ||
+ | <ul> | ||
+ | <li><a href="#fragment15-0">outline</a></li> | ||
+ | <li><a href="#fragment15-1-1">1.1</a></li> | ||
+ | <li><a href="#fragment15-1-2">1.2</a></li> | ||
+ | <li><a href="#fragment15-1-3">1.3</a></li> | ||
+ | <li><a href="#fragment15-1-4">1.4</a></li> | ||
+ | <li><a href="#fragment15-2-1">2.1</a></li> | ||
+ | <li><a href="#fragment15-2-2">2.2</a></li> | ||
+ | <li><a href="#fragment15-2-3">2.3</a></li> | ||
+ | <li><a href="#fragment15-3-1">3.1</a></li> | ||
+ | <li><a href="#fragment15-3-2">3.2</a></li> | ||
+ | <li><a href="#fragment15-3-3">3.3</a></li> | ||
+ | </ul> | ||
+ | <div id="fragment15-0"> | ||
+ | <ol> | ||
+ | <li>Construction of pLuxS</li> | ||
+ | <li>Construction of pLsrACDB</li> | ||
+ | <li>Construction of pLsrACDBFG</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | <div id="fragment15-1-1"> | ||
+ | <div class="p"> | ||
+ | <i>luxS</i> gene was cloned from <i>E.coli K12</i>. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table15-1-1-1"> | ||
+ | <caption>50μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>PrimeSTAR Max Premix(2×)</td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxS</i>-Fd</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxS</i>-Rv</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>E.coli K12</i></td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>19μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>50μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table15-1-1-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>60<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/b/bc/T--NKU_China--notebook-14-17-05.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>PCR cloning product of gene <i>luxS</i></span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment15-1-2"> | ||
+ | <div class="p"> | ||
+ | The <i>luxS</i> gene was purified by gel extraction. Then, both the <i>luxS</i> segment and pTrcHisB vector were treated with restriction enzyme. After that, <i>luxS</i> gene was ligated to linearized pTrcHisB, and the ligation product was transformed into <i>E.coli K12</i>. </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table15-1-2-1"> | ||
+ | <caption>40μL digestion system ×2</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>4μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Kpn Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcoR Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxS</i></td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>40μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table15-1-2-2"> | ||
+ | <caption>40μL digestion system ×2</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>4μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Kpn Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcoR Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrcHisB</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>12μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>40μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <table class="table-theme-3" id="table15-1-2-3"> | ||
+ | <caption>20μL ligation system</caption> | ||
+ | <tr> | ||
+ | <td>10× DNA Ligase Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T4 DNA Ligase</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>linearized pTrcHisB</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxS</i></td> | ||
+ | <td>3μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>13μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 16<sup>o</sup>C overnight</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment15-1-3"> | ||
+ | <div class="p"> | ||
+ | Verification PCR was performed to select the positive clones. </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table15-1-3-1"> | ||
+ | <caption>20μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>2× Taq Master Mix</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-JC-F</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-JC-R</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bacterium solution</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table15-1-3-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>58<sup>o</sup>C</td> | ||
+ | <td>30sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>50 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/6/65/T--NKU_China--notebook-14-17-06.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Selection of positive clones by PCR</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment15-1-4"> | ||
+ | <div class="p"> | ||
+ | One positive strain was chosen to be cultured overnight and plasmid was extracted. After restriction enzyme digestion verification, the positive clone was sequenced. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table15-1-4-1"> | ||
+ | <caption>20μL digestion system</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Kpn Ⅰ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcoR Ⅰ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-<i>luxS</i></td> | ||
+ | <td>6μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/c/ca/T--NKU_China--notebook-14-17-07.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Restriction enzyme digestion verification</span></div> | ||
+ | (No.1 is linearized pTrcHisB + <i>luxS</i>, No.2 is plasmid pTrc-<i>luxS</i>. ) | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment15-2-1"> | ||
+ | <div class="p"> | ||
+ | The <i>lsrACDBFG</i> sequence was divided into three parts and they were cloned separately. Then the three segments were ligated to the linearized pTrcHisB by ClonExpress technique, and the recombinant vector was transformed into DH5α. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table15-2-1-1"> | ||
+ | <caption>50μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>PrimeSTAR Max Premix(2×)</td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDB</i>1-F</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDB</i>1-R</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>E.coli K12</i></td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>19μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>50μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table15-2-1-2"> | ||
+ | <caption>50μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>PrimeSTAR Max Premix(2×)</td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDB</i>2-F</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDB</i>2-R</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>E.coli K12</i></td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>19μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>50μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table15-2-1-3"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>60<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>2 min 20 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/b/b6/T--NKU_China--notebook-14-17-08.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>PCR cloning product of segment <i>lsrACDB1</i>,<i>lsrACDB1</i>2</span></div> | ||
+ | |||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment15-2-2"> | ||
+ | <div class="p"> | ||
+ | Verification PCR was performed to select the positive clones. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/e/e1/T--NKU_China--notebook-14-17-09.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Selection of positive clones by PCR</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment15-2-3"> | ||
+ | <div class="p"> | ||
+ | 4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table15-2-3-1"> | ||
+ | <caption>20μL digestion system</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BamH Ⅰ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Sac Ⅰ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-<i>lsrACDB</i></td> | ||
+ | <td>6μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/5/5d/T--NKU_China--notebook-14-17-10.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Restriction enzyme digestion verification</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment15-3-1"> | ||
+ | <div class="p"> | ||
+ | The <i>lsrACDBFG</i> sequence was divided into three parts and they were cloned separately. Then the three segments were ligated to the linearized pTrcHisB by ClonExpress technique, and the recombinant vector was transformed into DH5α. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table15-3-1-1"> | ||
+ | <caption>50μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>PrimeSTAR Max Premix(2×)</td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDBFG</i>1-F</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDBFG</i>1-R</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>E.coli K12</i></td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>19μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>50μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table15-3-1-2"> | ||
+ | <caption>50μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>PrimeSTAR Max Premix(2×)</td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDBFG</i>2-F</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDBFG</i>2-R</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>E.coli K12</i></td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>19μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>50μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table15-3-1-3"> | ||
+ | <caption>50μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>PrimeSTAR Max Premix(2×)</td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDBFG</i>3-F</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrACDBFG</i>3-R</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>E.coli K12</i></td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>19μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>50μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table15-3-1-4"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>58<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>2 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/6/69/T--NKU_China--notebook-14-17-11.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>PCR cloning product of segment <i>lsrACDBFG</i>1,<i>lsrACDBFG</i>2, <i>lsrACDBFG</i>3</span> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment15-3-2"> | ||
+ | <div class="p"> | ||
+ | Verification PCR was performed to select the positive clones. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/9/97/T--NKU_China--notebook-14-17-12.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Selection of positive clones by PCR</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment15-3-3"> | ||
+ | <div class="p"> | ||
+ | 4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table15-3-2-1"> | ||
+ | <caption>20μL digestion system</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BamH Ⅰ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Sac Ⅰ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-<i>lsrACDB</i></td> | ||
+ | <td>6μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/3/33/T--NKU_China--notebook-14-17-13.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Restriction enzyme digestion verification</span></div> | ||
+ | (No.1-4 are linearized pTrcHisB + <i>lsrACDBFG</i>, No.5 is plasmid pTrc-<i>lsrACDBFG</i>.) | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-fragment" id="week16"> | ||
+ | <div class="accordion-header"> | ||
+ | <span class="default">☞</span><span class="active">☟</span> Week16 (Aug 29–Sep 04) | ||
+ | </div> | ||
+ | <div class="accordion-content"> | ||
+ | <div class="tabs" id="tabs16"> | ||
+ | <ul> | ||
+ | <li><a href="#fragment16-0">outline</a></li> | ||
+ | <li><a href="#fragment16-1-1">1.1</a></li> | ||
+ | <li><a href="#fragment16-1-2">1.2</a></li> | ||
+ | <li><a href="#fragment16-1-3">1.3</a></li> | ||
+ | <li><a href="#fragment16-1-4">1.4</a></li> | ||
+ | <li><a href="#fragment16-2-1">2.1</a></li> | ||
+ | <li><a href="#fragment16-2-2">2.2</a></li> | ||
+ | <li><a href="#fragment16-2-3">2.3</a></li> | ||
+ | <li><a href="#fragment16-2-4">2.4</a></li> | ||
+ | <li><a href="#fragment16-3-1">3.1</a></li> | ||
+ | <li><a href="#fragment16-3-2">3.2</a></li> | ||
+ | <li><a href="#fragment16-3-3">3.3</a></li> | ||
+ | <li><a href="#fragment16-3-4">3.4</a></li> | ||
+ | </ul> | ||
+ | <div id="fragment16-0"> | ||
+ | <ol> | ||
+ | <li>Construction of pLsrFG</li> | ||
+ | <li>Construction of pLsrACDBK</li> | ||
+ | <li>Construction of pLsrACDBFGK</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | <div id="fragment16-1-1"> | ||
+ | <div class="p"> | ||
+ | <i>lsrFG</i> gene was cloned from <i>E.coli K12</i>. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table16-1-1-1"> | ||
+ | <caption>50μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>PrimeSTAR Max Premix(2×)</td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrFG</i>-Fd</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrFG</i>-Rv</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>E.coli K12</i></td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>19μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>50μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table16-1-1-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>58<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>1 min 15 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/5/5c/T--NKU_China--notebook-14-17-14.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>PCR cloning product of gene <i>lsrFG</i></span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment16-1-2"> | ||
+ | <div class="p"> | ||
+ | The <i>lsrFG</i> gene was purified by gel extraction. Then, both the <i>lsrFG</i> segment and pTrcHisB vector were treated with restriction enzyme. After that, <i>lsrFG</i> gene was ligated to linearized pTrcHisB, and the ligation product was transformed into <i>E.coli K12</i>. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table16-1-2-1"> | ||
+ | <caption>40μL digestion system ×2</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>4μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BamH Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bgl Ⅱ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrFG</i></td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>40μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table16-1-2-2"> | ||
+ | <caption>40μL digestion system ×2</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>4μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BamH Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bgl Ⅱ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrcHisB</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>12μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>40μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-3" id="table16-1-2-3"> | ||
+ | <caption>20μL ligation system</caption> | ||
+ | <tr> | ||
+ | <td>10× DNA Ligase Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T4 DNA Ligase</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pMD19 T-Simple Vector</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>luxSFG</i></td> | ||
+ | <td>3μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>13μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 16<sup>o</sup>C overnight</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment16-1-3"> | ||
+ | <div class="p"> | ||
+ | Verification PCR was performed to select the positive clones. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table16-1-3-1"> | ||
+ | <caption>20μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>2× Taq Master Mix</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-JC-F</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-JC-R</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bacterium solution</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table16-1-3-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>58<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>1 min 30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/5/5b/T--NKU_China--notebook-14-17-15.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Selection of positive clones by PCR</span></div> | ||
+ | (NO. 1&3 are positive results) | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment16-1-4"> | ||
+ | <div class="p"> | ||
+ | Two positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table16-1-4-1"> | ||
+ | <caption>20μL digestion system</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BamH Ⅰ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bgl Ⅱ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-<i>lsrFG</i></td> | ||
+ | <td>6μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/b/b0/T--NKU_China--notebook-14-17-16.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Restriction enzyme digestion verification</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment16-2-1"> | ||
+ | <div class="p"> | ||
+ | <i>lsrK</i> gene was cloned from <i>E.coli K12</i>. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table16-2-1-1"> | ||
+ | <caption>50μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>PrimeSTAR Max Premix(2×)</td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrK</i>-F</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrK</i>-R</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>E.coli K12</i></td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>19μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>50μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table16-2-1-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>55<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>1 min 40 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/8/85/T--NKU_China--notebook-14-17-17.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>PCR cloning product of gene <i>lsrK</i></span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment16-2-2"> | ||
+ | <div class="p"> | ||
+ | The <i>lsrK</i> gene was purified by gel extraction. Then, both the <i>lsrK</i> segment and pTrc-<i>lsrACDB</i> vector were treated with restriction enzyme. After that, <i>lsrK</i> gene was ligated to linearized pTrc-<i>lsrACDB</i>, and the ligation product was transformed into <i>E.coli K12</i>. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table16-2-2-1"> | ||
+ | <caption>40μL digestion system ×2</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>4μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Pst Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BstB Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrK</i></td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>40μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table16-2-2-2"> | ||
+ | <caption>40μL digestion system ×2</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>4μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Pst Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BstB Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-<i>lsrACDB</i></td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>40μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-3" id="table16-2-2-3"> | ||
+ | <caption>20μL ligation system</caption> | ||
+ | <tr> | ||
+ | <td>10× DNA Ligase Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T4 DNA Ligase</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>linearized pTrc-<i>lsrACDB</i></td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrK</i></td> | ||
+ | <td>3μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>13μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 16<sup>o</sup>C overnight</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment16-2-3"> | ||
+ | <div class="p"> | ||
+ | Verification PCR was performed to select the positive clones. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table16-2-3-1"> | ||
+ | <caption>20μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>2× Taq Master Mix</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BK-JC-F</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-JC-R</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bacterium solution</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table16-2-3-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>58<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>2 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/c/cb/T--NKU_China--notebook-14-17-18.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Selection of positive clones by PCR</span></div> | ||
+ | (NO. 1&3 are positive results) | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment16-2-4"> | ||
+ | <div class="p"> | ||
+ | Two positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table16-2-4-1"> | ||
+ | <caption>20μL digestion system</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Pst Ⅰ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BstB Ⅰ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-<i>lsrACDBK</i></td> | ||
+ | <td>6μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/5/56/T--NKU_China--notebook-14-17-19.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Restriction enzyme digestion verification</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment16-3-1"> | ||
+ | <div class="p"> | ||
+ | <i>lsrK</i> gene was cloned from <i>E.coli K12</i>. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table16-3-1-1"> | ||
+ | <caption>50μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>PrimeSTAR Max Premix(2×)</td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrK</i>-F</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrK</i>-R</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>E.coli K12</i></td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>19μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>50μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table16-3-1-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>55<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>1 min 40 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/8/85/T--NKU_China--notebook-14-17-17.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>PCR cloning product of gene <i>lsrK</i></span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment16-3-2"> | ||
+ | <div class="p"> | ||
+ | The <i>lsrK</i> gene was purified by gel extraction. Then, both the <i>lsrK</i> segment and pTrc-<i>lsrACDBFG</i> vector were treated with restriction enzyme. After that, <i>lsrK</i> gene was ligated to linearized pTrc-<i>lsrACDBFG</i>, and the ligation product was transformed into <i>E.coli K12</i>. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table16-3-2-1"> | ||
+ | <caption>40μL digestion system ×2</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>4μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Pst Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BstB Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrK</i></td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>40μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table16-3-2-2"> | ||
+ | <caption>40μL digestion system ×2</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>4μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Pst Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BstB Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-<i>lsrACDBFG</i></td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>12μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>40μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-3" id="table16-3-2-3"> | ||
+ | <caption>20μL ligation system</caption> | ||
+ | <tr> | ||
+ | <td>10× DNA Ligase Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T4 DNA Ligase</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>linearized pTrc-<i>lsrACDFGB</i></td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrK</i></td> | ||
+ | <td>3μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>13μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 16<sup>o</sup>C overnight</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment16-3-3"> | ||
+ | <div class="p"> | ||
+ | Verification PCR was performed to select the positive clones. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table16-3-3-1"> | ||
+ | <caption>20μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>2× Taq Master Mix</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>GK-JC-F</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-JC-R</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bacterium solution</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table16-3-3-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>58<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>2 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/7/78/T--NKU_China--notebook-14-17-20.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Selection of positive clones by PCR</span></div> | ||
+ | (NO.3 is positive result) | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment16-3-4"> | ||
+ | <div class="p"> | ||
+ | Two positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table16-3-4-1"> | ||
+ | <caption>20μL digestion system</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Pst Ⅰ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BstB Ⅰ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-<i>lsrACDBKFGK</i></td> | ||
+ | <td>6μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/e/e2/T--NKU_China--notebook-14-17-21.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Restriction enzyme digestion verification</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-fragment" id="week17"> | ||
+ | <div class="accordion-header"> | ||
+ | <span class="default">☞</span><span class="active">☟</span> Week17 (Sep 05–Sep 11) | ||
+ | </div> | ||
+ | <div class="accordion-content"> | ||
+ | <div class="tabs" id="tabs17"> | ||
+ | <ul> | ||
+ | <li><a href="#fragment17-0">outline</a></li> | ||
+ | <li><a href="#fragment17-1-1">1.1</a></li> | ||
+ | <li><a href="#fragment17-1-2">1.2</a></li> | ||
+ | <li><a href="#fragment17-1-3">1.3</a></li> | ||
+ | <li><a href="#fragment17-1-4">1.4</a></li> | ||
+ | </ul> | ||
+ | <div id="fragment17-0"> | ||
+ | <ol> | ||
+ | <li>Construction of pLsrK</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | <div id="fragment17-1-1"> | ||
+ | <div class="p"> | ||
+ | <i>lsrK</i> gene was cloned from <i>E.coli K12</i>. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table17-1-1-1"> | ||
+ | <caption>50μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>PrimeSTAR Max Premix(2×)</td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrK</i>-Fd</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrK</i>-Rv</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>E.coli K12</i></td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>19μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>50μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table17-1-2-1"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>55<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>1 min 40 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/2/24/T--NKU_China--notebook-14-17-22.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>PCR cloning product of gene <i>lsrK</i></span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment17-1-2"> | ||
+ | <div class="p"> | ||
+ | The <i>lsrK</i> gene was purified by gel extraction. Then, both the <i>lsrK</i> segment and pTrc-<i>lsrACDBFG</i> vector were treated with restriction enzyme. After that, <i>lsrK</i> gene was ligated to linearized pTrc-<i>lsrACDBFG</i>, and the ligation product was transformed into <i>E.coli K12</i>. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table17-1-2-1"> | ||
+ | <caption>40μL digestion system ×2 ×2</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>4μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Kpn Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcoR Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrK</i></td> | ||
+ | <td>25μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>40μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table17-1-2-2"> | ||
+ | <caption>40μL digestion system ×2 ×2</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>4μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Pst Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>BstB Ⅰ</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-HisB</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>12μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>40μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-3" id="table17-1-2-3"> | ||
+ | <caption>20μL ligation system</caption> | ||
+ | <tr> | ||
+ | <td>10× DNA Ligase Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T4 DNA Ligase</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>linearized pTrcHisB</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><i>lsrK</i></td> | ||
+ | <td>3μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>13μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 16<sup>o</sup>C overnight</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment17-1-3"> | ||
+ | <div class="p"> | ||
+ | Verification PCR was performed to select the positive clones. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-1" id="table17-1-3-1"> | ||
+ | <caption>20μL PCR system ×2</caption> | ||
+ | <tr> | ||
+ | <td>2× Taq Master Mix</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-JC-F</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-JC-R</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Bacterium solution</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>7μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <div class="table-wrapper"> | ||
+ | <div class="rbrace">}</div> | ||
+ | <table class="table-theme-2" id="table17-1-3-2"> | ||
+ | <caption>PCR reaction condition</caption> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>94<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>58<sup>o</sup>C</td> | ||
+ | <td>30 sec</td> | ||
+ | <td>30 cycles</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>2 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>72<sup>o</sup>C</td> | ||
+ | <td>10 min</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16<sup>o</sup>C</td> | ||
+ | <td>∞</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/3/30/T--NKU_China--notebook-14-17-23.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Selection of positive clones by PCR</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment17-1-4"> | ||
+ | <div class="p"> | ||
+ | 4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <div class="table-wrapper"> | ||
+ | <table class="table-theme-4" id="table17-1-4-1"> | ||
+ | <caption>20μL digestion system</caption> | ||
+ | <tr> | ||
+ | <td>10× FastDigest Buffer</td> | ||
+ | <td>2μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Kpn Ⅰ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcoR Ⅰ</td> | ||
+ | <td>1μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pTrc-<i>lsrK</i></td> | ||
+ | <td>6μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ddH2O</td> | ||
+ | <td>10μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td colspan="2">Reaction condition: 37<sup>o</sup>C for 40 min</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </div> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/2/22/T--NKU_China--notebook-14-17-24.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Restriction enzyme digestion verification</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-fragment" id="week18"> | ||
+ | <div class="accordion-header"> | ||
+ | <span class="default">☞</span><span class="active">☟</span> Week18 (Seq 12–Seq 18) | ||
+ | </div> | ||
+ | <div class="accordion-content"> | ||
+ | <div class="tabs" id="tabs7"> | ||
+ | <ul> | ||
+ | <li><a class="circle-number" href="#fragment18-1">➀</a></li> | ||
+ | <li><a class="circle-number" href="#fragment18-2">➁</a></li> | ||
+ | </ul> | ||
+ | <div id="fragment18-1"> | ||
+ | <div class="p"> | ||
+ | We detected the expression of several overexpressed genes at the mRNA level by RT-PCR, and obtained positive results. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/f/f8/T--NKU_China--notebook-18-20-01.png" class="white"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Relative expression of several overexpressed genes at the mRNA level</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment18-2"> | ||
+ | <div class="p"> | ||
+ | SDS-PAGE was performed to detect the expression of these genes at the protein level. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/d/d2/T--NKU_China--notebook-18-20-02.png" class="white"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>pTrcHisB, pLuxS, pLuxS-Mtn, pLsrACDB, pLsrACDBFG SDS-PAGE</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-fragment" id="week19"> | ||
+ | <div class="accordion-header"> | ||
+ | <span class="default">☞</span><span class="active">☟</span> Week19 (Seq 19–Seq 25) | ||
+ | </div> | ||
+ | <div class="accordion-content"> | ||
+ | <div class="tabs" id="tabs7"> | ||
+ | <ul> | ||
+ | <li><a class="circle-number" href="#fragment19-1">➀</a></li> | ||
+ | <li><a class="circle-number" href="#fragment19-2">➁</a></li> | ||
+ | </ul> | ||
+ | <div id="fragment19-1"> | ||
+ | <div class="p"> | ||
+ | Cultured media of controllers were tested for the presence of AI-2 by inducing luminescence of <i>Vibrio harveyi</i> reporter strain BB170. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/4/4c/T--NKU_China--notebook-18-20-03.jpg"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Relative fluorescence intensity of suppliers at specific times</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/f/f7/T--NKU_China--notebook-18-20-04.jpg"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Relative fluorescence intensity of consumers at specific times</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment19-2"> | ||
+ | <div class="p"> | ||
+ | HPLC was performed to detect the production of AI-2 of the strain pLuxS. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/d/d9/T--NKU_China--notebook-18-20-05.png"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>HPLC result of AI-2 production of luxS overexpression strain and negative control</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="accordion-fragment" id="week20"> | ||
+ | <div class="accordion-header"> | ||
+ | <span class="default">☞</span><span class="active">☟</span> Week20 (Seq 26–Oct 02) | ||
+ | </div> | ||
+ | <div class="accordion-content"> | ||
+ | <div class="tabs" id="tabs7"> | ||
+ | <ul> | ||
+ | <li><a class="circle-number" href="#fragment20-1">➀</a></li> | ||
+ | <li><a class="circle-number" href="#fragment20-2">➁</a></li> | ||
+ | </ul> | ||
+ | <div id="fragment20-1"> | ||
+ | <div class="p"> | ||
+ | Biofilm studies and evaluation:<br> | ||
+ | pTrcHisB, pLuxS, pLuxSMtn, pLsrACDB, pLsrACDBFG, pLsrACDBK, pLsrACDBFGK were diluted to OD~0.05 and reinoculated at a total volume of 200 μL at a 1:1 (v/v) ratio. IPTG (1 mM) was added at OD ~ 0.4, and biofilms were cultured for ~24 h (+/−30 min) at 30 <sup>o</sup>C in static conditions. After incubation, optical density was read on a plate reader at 600 nm. The supernatant was gently decanted, and each well was washed 3 times with 300 μL of sterile PBS to detach loosely adhered cells. The plate was then incubated at 60 <sup>o</sup>C with the lid off for 60 min, and afterwards, 250 μL of 0.1% crystal violet was added to each well and incubated for 15 min at room temperature. Crystal violet stain was aspirated with a pipette and excess stain was washed off by gently submerging and mixing in a tray filled with distilled water until washings were free of the stain. After the microplate was airdried, the dye was resolubilized by adding 250 μL of 95% ethanol, and incubated at room temperature with shaking for 30 min. The optical density of each well stained with crystal violet was measured at 540 nm. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/3/33/T--NKU_China--notebook-18-20-06.jpg"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Relative biofilm production of suppliers</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/8/8b/T--NKU_China--notebook-18-20-07.jpg"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>Relative biofilm production of consumers</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div id="fragment20-2"> | ||
+ | <div class="p"> | ||
+ | Cultured media of controllers were tested for the presence of AI-2 by HPLC. AI-2 output and uptake profiles of controllers were drawn according to the HPLC results. | ||
+ | </div> | ||
+ | <div class="flex-container"> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/e/ed/T--NKU_China--notebook-18-20-08.jpg"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>AI-2 output profiles of suppliers</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | <figure> | ||
+ | <img src="https://static.igem.org/mediawiki/2016/0/04/T--NKU_China--notebook-18-20-09.jpg"> | ||
+ | <figcaption> | ||
+ | <div class="p"><span>AI-2 uptake profiles of consumers</span></div> | ||
+ | </figcaption> | ||
+ | </figure> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
− | </ | + | </div> |
</body> | </body> | ||
</html> | </html> | ||
+ | {{NKU_China/footer}} |
Latest revision as of 16:17, 19 October 2016
Laboratory Notes
☞☟ Week1 (May 16–May 22)
In order to make sure the efficiency of our "consumer", we should first knock out the luxS gene in our engineering bacteria GR286(a simplified strain of Bacillus amyloliquefaciens LL3). We used a markerless gene replacement method to knock out the luxS gene.
Construction of targeting vector : the upstream and downstream of luxS gene were combined by over-lapping PCR and ligated into plasmid pKSU.
pKSU-ΔluxS was transformed into GR286, and positive clones were selected.
2× Taq Master Mix | 10μL |
pKSU-F | 1μL |
pKSU-R | 1μL |
Bacterium solution | 1μL |
ddH2O | 7μL |
Total | 20μL |
}
94oC | 10 min | |
94oC | 30 sec | |
58oC | 30 sec | 30 cycles |
72oC | 1 min 30 sec | |
72oC | 10 min | |
16oC | ∞ |
The transformants were cultured at 42oC with chloramphenicol to select single-crossover clones.
2× Taq Master Mix | 10μL |
luxS-up-F | 1μL |
luxS-dn-R | 1μL |
Bacterium solution | 1μL |
ddH2O | 7μL |
Total | 20μL |
}
94oC | 10 min | |
94oC | 30 sec | |
56oC | 30 sec | 30 cycles |
72oC | 2 min | |
72oC | 10 min | |
16oC | ∞ |
☞☟ Week2 (May 23–May 29)
The single-crossover strains were then cultured in LB medium and passaged every 12 hours for 4 generations.
The last generation was cultured in medium with 5-fluorouracil to select double-crossover clones. Regretfully, we didn't get the double-crossover clones.
2× Taq Master Mix | 10μL |
luxS-up-F | 1μL |
luxS-dn-R | 1μL |
Bacterium solution | 1μL |
ddH2O | 7μL |
Total | 20μL |
}
94oC | 10 min | |
94oC | 30 sec | |
56oC | 30 sec | 30 cycles |
72oC | 2 min | |
72oC | 10 min | |
16oC | ∞ |
☞☟ Week3 (May 30–Jun 05)
Transformants were cultured at 42oC with chloramphenicol again and the single-crossover clones were selected successfully.
2× Taq Master Mix | 10μL |
luxS-up-F | 1μL |
luxS-dn-R | 1μL |
Bacterium solution | 1μL |
ddH2O | 7μL |
Total | 20μL |
}
94oC | 10 min | |
94oC | 30 sec | |
56oC | 30 sec | 30 cycles |
72oC | 2 min | |
72oC | 10 min | |
16oC | ∞ |
The single-crossover strains were then cultured in LB medium and passaged every 12 hours for 4 generations.
The last generation was cultured in medium with 5-fluorouracil to select double-crossover clones. We finally obtained our aimed strain—GR286ΔluxS.
2× Taq Master Mix | 10μL |
luxS-up-F | 1μL |
luxS-dn-R | 1μL |
Bacterium solution | 1μL |
ddH2O | 7μL |
Total | 20μL |
}
94oC | 10 min | |
94oC | 30 sec | |
56oC | 30 sec | 30 cycles |
72oC | 2 min | |
72oC | 10 min | |
16oC | ∞ |
☞☟ Week4 (Jun 06–Jun 12)
The GR286ΔluxS strain was cultured and made competent for future use.
The lsrACDB gene from Bacillus thuringiensis was cloned and ligated to T-vector.
2× Taq Master Mix | 25μL |
lsrACDB-F | 2μL |
lsrACDB-R | 2μL |
Bacterium solution | 2μL |
ddH2O | 19μL |
Total | 50μL |
}
94oC | 10 min | |
94oC | 30 sec | |
57oC | 30 sec | 30 cycles |
72oC | 4 min 30 sec | |
72oC | 10 min | |
16oC | ∞ |
10× DNA Ligase Buffer | 2μL |
T4 DNA Ligase | 1μL |
pMD19 T-Simple Vector | 1μL |
lsrACDB | 3μL |
ddH2O | 13μL |
Total | 20μL |
Reaction condition: 16oC overnight |
The T-lsrACDB was transformed into DH5α and plate was coated, and then positive clones were selected.
2× Taq Master Mix | 10μL |
M13F | 1μL |
M13R | 1μL |
Bacterium solution | 1μL |
ddH2O | 7μL |
Total | 20μL |
}
94oC | 10 min | |
94oC | 30 sec | |
59oC | 30 sec | 30 cycles |
72oC | 4 min 30 sec | |
72oC | 10 min | |
16oC | ∞ |
After restriction enzyme digestion verification, the positive clones were then sequenced. Unfortunately, the sequencing result showed some mutations inside the target gene.
10× FastDigest Buffer | 2μL |
BamH Ⅰ | 1μL |
T-lsrACDB | 1μL |
ddH2O | 7μL |
Total | 20μL |
Reaction condition: 37oC for 40 min |
The gene cloning process was repeated but there were still some mutations.
We finally decided to request the gene company to synthesize the lsrACDB gene.
☞☟ Week5 (Jun 13–Jun 19)
This week, we started to construct another controller―supplier.
A strong promoter C2 was cloned from former kit and luxS gene was cloned from GR286.
2× Taq Master Mix | 25μL |
C2-F | 2μL |
C2-R | 2μL |
p-C2 | 2μL |
ddH2O | 19μL |
Total | 50μL |
}
94oC | 10 min | |
94oC | 30 sec | |
58oC | 15 sec | 30 cycles |
72oC | 30 sec | |
72oC | 10 min | |
16oC | ∞ |
2× Taq Master Mix | 25μL |
luxS-F | 2μL |
luxS-R | 2μL |
Bacterium solution | 1μL |
GR286 | 2μL |
ddH2O | 19μL |
Total | 50μL |
}
94oC | 10 min | |
94oC | 30 sec | |
59oC | 30 sec | 30 cycles |
72oC | 30 sec | |
72oC | 10 min | |
16oC | ∞ |
Two segments were fused together by fusion PCR and ligated into T-vector. After that, the vector was transformed into DH5α.
2× Taq Master Mix | 25μL |
C2-F | 2μL |
luxS-R | 2μL |
C2 | 2μL |
luxS | 2μL |
ddH2O | 17μL |
Total | 50μL |
}
94oC | 10 min | |
94oC | 30 sec | |
59oC | 30 sec | 30 cycles |
72oC | 40 sec | |
72oC | 10 min | |
16oC | ∞ |
10× DNA Ligase Buffer | 2μL |
T4 DNA Ligase | 1μL |
pMD19 T-Simple Vector | 1μL |
C2-luxS | 4μL |
ddH2O | 12μL |
Total | 20μL |
Reaction condition: 16oC overnight |
Positive clones were selected by colony PCR.
2× Taq Master Mix | 10μL |
M13-F | 1μL |
M13-R | 1μL |
Bacterium solution | 1μL |
ddH2O | 7μL |
Total | 20μL |
}
94oC | 10 min | |
94oC | 30 sec | |
59oC | 30 sec | 30 cycles |
72oC | 40 sec | |
72oC | 10 min | |
16oC | ∞ |
4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.
10× FastDigest Buffer | 2μL |
BamH Ⅰ | 1μL |
T-lsrACDB | 1μL |
ddH2O | 7μL |
Total | 20μL |
Reaction condition: 37oC for 40 min |
☞☟ Week6 (Jun 20–Jun 26)
The sequencing result showed that there was a correct strain and thus it could be used for the following experiments. We obtained the correct plasmid T-C2-luxS from DH5α. Then the fragment C2-luxS was obtained by digestion and gel extraction.
10× FastDigest Buffer | 4μL |
BamH Ⅰ | 2μL |
T-C2-luxS | 25μL |
ddH2O | 9μL |
Total | 20μL |
Reaction condition: 37oC for 40 min |
The C2-luxS fragment was ligated to linearized plasmid pWH1520, and then the ligation product was transformed into DH5α.
10× DNA Ligase Buffer | 2μL |
T4 DNA Ligase | 1μL |
pMD19 T-Simple Vector | 1μL |
C2-luxS | 5μL |
ddH2O | 11μL |
Total | 20μL |
Reaction condition: 16oC overnight |
The plasmid pWH-C2-luxS was extracted from DH5α. To prevent the plasmid from DAM&DCM methylation, we transformed it into E.coli JM110.
The plasmid pWH-C2-luxS was extracted from JM110,and then it was treated with BamH Ⅰ methylase.
10× BamH Ⅰ methyltransferase Buffer | 10μL |
BamH Ⅰ methyltransferase | 1μL |
S-adenosylmethionine | 0.5μL |
pWH-C2-luxS | 80μL |
ddH2O | 8.5μL |
Total | 100μL |
Reaction condition: 37oC for 1 hour |
The plasmid was transformed into GR286 by electroporation.[Failed]
☞☟ Week7 (Jun 27–Jul 03)
This week, we tried to use different voltages to transform the plasmid. Sadly, all of these attempts rendered negative results.
We considered whether the luxS gene is toxic for GR286, and the bacteria tends to reject the gene when a strong promoter is inserted upstream of it. So, we planned to use inducible promoter to reconstruct our expression vector.
The plasmid pWH1520 contains a strong xylA promoter originating from Bacillus megaterium, and transcription initiated by this promoter is xylose-inducible. Also, the gene of interest carries its own ribosome binding sequence (RBS) and translation initiation codon. Based on these points, we redesigned primers.
☞☟ Week8 (Jul 04–Jul 10)
luxS gene was cloned from GR286 using our new primers.
2× Taq Master Mix | 25μL |
YD-luxS-F | 2μL |
YD-luxS-R | 2μL |
Bacterium solution | 2μL |
ddH2O | 19μL |
Total | 50μL |
}
94oC | 10 min | |
94oC | 30 sec | |
58oC | 30 sec | 30 cycles |
72oC | 40 sec | |
72oC | 10 min | |
16oC | ∞ |
The luxS fragment was purified by gel extraction, and ligated into linearized pWH1520. Then the vector was transformed into DH5α.
10× FastDigest Buffer | 4μL |
BamH Ⅰ | 2μL |
pWH1520 | 25μL |
ddH2O | 9μL |
Total | 40μL |
Reaction condition: 37oC for 40 min |
10× DNA Ligase Buffer | 2μL |
T4 DNA Ligase | 1μL |
linearized pWH1520 | 1μL |
luxS | 3μL |
ddH2O | 13μL |
Total | 20μL |
Reaction condition: 16oC overnight |
Positive clones were selected by colony PCR.
2× Taq Master Mix | 10μL |
pWH-F | 1μL |
pWH-R | 1μL |
Bacterium solution | 1μL |
ddH2O | 7μL |
Total | 20μL |
}
94oC | 10 min | |
94oC | 30 sec | |
58oC | 30 sec | 30 cycles |
72oC | 40 sec | |
72oC | 10 min | |
16oC | ∞ |
4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.
10× FastDigest Buffer | 2μL |
BamH Ⅰ | 1μL |
pWH-luxs | 10μL |
ddH2O | 7μL |
Total | 20μL |
Reaction condition: 37oC for 40 min |
☞☟ Week9 (Jul 11–Jul 17)
The sequencing result showed there's three positive strains. So one positive strain was chosen to be used for the following experiments. The pWH-luxS plasmid was extracted from the chosen strain. To prevent the plasmid from DAM&DCM methylation, we transformed it into E.coli JM110.
the plasmid pWH-luxS was extracted from JM110,and it was treated withBamH Ⅰ methylase.
10× BamH Ⅰ methyltransferase Buffer | 10μL |
BamH Ⅰ methyltransferase | 1μL |
S-adenosylmethionine | 0.5μL |
pWH-C2-luxS | 80μL |
ddH2O | 8.5μL |
Total | 100μL |
Reaction condition: 37oC for 1 hour |
The plasmid was transformed into GR286 by electroporation, and positive clones were selected.
The construction of supplier was accomplished!
☞☟ Week10 (Jul 18–Jul 24)
We have received the product of synthetic lsrACDB gene. We first used restriction-ligation method to ligate lsrACDB to plasmid pWH1520, but we failed to select positive after several tries.
10× DNA Ligase Buffer | 2μL |
T4 DNA Ligase | 1μL |
linearized pWH1520 | 1μL |
lsrACDB | 3μL |
ddH2O | 13μL |
Total | 20μL |
Reaction condition: 16oC overnight |
Considering that the lsrACDB gene is a large fragment (4500bp), we used ClonExpress technique to clone the gene again to improve the efficiency of ligation. The lsrACDB sequence was divided into two parts and they were cloned separately. Then the two segments were ligated to the plasmid pWH1520 and the recombinant vector was transformed into DH5α. After that, verification PCR was used to select the positive clones. However, we didn't get a good result.
☞☟ Week11 (Jul 25–Jul 31)
We learnt a new method called circular polymerase extension cloning (CPEC) for high-throughput cloning of complex and combinatorial DNA libraries, and we decided to use this method to try to ligate our lsrACDB gene. It's encouraging that we succeeded to ligate the lsrACDB gene to the plasmid pHT-01.
☞☟ Week12 (Aug 1–Aug 7)
Since we have already successfully constructed "supplier" and part of "consumer", we decided to measure the growth curve to explore the function of our "controller".
GR286 | wild strain as control group |
GR286ΔluxS | GR286 without luxS gene |
pWH-luxS | luxS overexpression plasmid in GR286; without induced by xylose |
pWH-luxS + xyl | luxS overexpression plasmid in GR286; induced by xylose |
pWH1520 | empty plasmid in GR286 as control group |
pHT-lsrACDB | lsrACDB overexpression plasmid in GR286ΔluxS |
pHT-01 | empty plasmid in GR286ΔluxS as control group |
Cultured media of our supplier was tested for the presence of AI-2 by inducing luminescence of Vibrio harveyi reporter strain BB170.
☞☟ Week13 (Aug 8–Aug 14)
For our consumer, we should also overexpress the lsrK and lsrFG gene for phosphorylating and degrading phosphorylated AI-2. We used ClonExpress technique to clone the two genes and ligate them to plasmid pHT-01 successfully.
We co-cultured the supplier with BB170 and tested the fluorescence intensity to explore the function of supplier. (negative result)
☞☟ Week14 (Aug 15–Aug 21)
Since the controller we constructed with GR286 did not demonstrate positive results, we decided to substitute GR286 with Escherichia coli whose AI-2 metabolic pathway had been elucidated as the chassis of our controller.
-
The strains to be constructed are shown in the tables below.
AI-2 supplier LuxS Mtn pTrcHisB Native Native pLuxS Induced Native pLuxS-Mtn Induced Induced AI-2 consumer LsrACDB LsrK LsrFG pTrcHisB Native Native Native pLsrACDB Induced Native Native pLsrFG Native Native Induced pLsrK Native Induced Native pLsrACDBFG Induced Native Induced pLsrACDBK Induced Constitutive Native plsrACDBFGK Induced Constitutive Induced -
The sequences of lsrACDB, lsrFG, lsrK, luxS, mtn gene in E.coli K12 were searched in NCBI, and primers were designed according to the gene sequences. The primers used in the construction of AI-2 controller are shown in the table below.
Primers used in the construction of AI-2 controller Primer name Sequence lsrACDBFG1-F CGACGATGACGATAAGGATCCATGCAAACGAGTGATACCCGC lsrACDBFG1-R TGTTTGGCGTTTCCGGCAGCGGTGCGGAGAGC lsrACDBFG2-F TGTTTGGCGTTTCCGCGATTG lsrACDBFG2-R GCACTCTCACACCACGTTGCATGGCGCGTTTC lsrACDBFG3-F GTGGTGTGAGAGTGCTGAC lsrACDBFG3-R ACCAGCTGCAGATCTCGAGCTCGTCACGGCATCAACCCATTGAAC lsrACDB1-F CGACGATGACGATAAGGATCCATGCAAACGAGTGATACCCGC lsrACDB1-R ACCGGAACCGCCGAGCAAACTAATGCCGCCCAGCAC lsrACDB2-F CTCGGCGGTTCCGGTGCGAT lsrACDB2-R ACCAGCTGCAGATCTCGAGCTCGTCAGAAATCGTATTTGCCG luxS-F ATGACGATAAGGATCCGAGCTCGATGCCGTTGTTAGATAGCTTC luxS-R GGACTCCCCCGGGGGACTAAATGTGCAGTTCCTGCAACTTC mtn-F TCCCCCGGGGGAGTCCTCTCCCGCGTGAGAAATAC mtn-R TATGGTACCAGCTGCAGATCTCTTAGCCATGTGCAAGTTTCTG luxS-Fd GGAATTCCCTAAATGTGCAGTTCCTGCAACTTC luxS-Rv GGGGTACCCCATGCCGTTGTTAGATAGCTTCACAG lsrK-Fd GGGGTACCCCATGGCTCGACTCTTTACC lsrK-Rv GGAATTCCCTATAACCCAGGCGCTTTCC lsrFG-Fd CGGGATCCCGATGGCAGATTTAGACGATATTAAAGATGG lsrFG-Rv GAAGATCTTCTCACGGCATCAACCCATTGAAC lsrK-F AACTGCAGAACCAATGCATTGGTTTACGGCTAGCTCAGTCCTAGGTATAGTGCTAGCAAAGAGGAGAAAATGGCTCGACTCTTTACC lsrK-R TTCGAACTATAACCCAGGCGCTTTCC BK-JC-F TTTCAGTGTATGTCGCGGATGC GK-JC-F TTGCGCTTCGATGTCTTACAGG pTrc-JC-F TGGGCACTCGACCGGAATTATC pTrc-JC-R GCTACTGCCGCCAGGCAAATTC
- Construction of pLuxS-Mtn
luxS gene was cloned from E.coli K12.
PrimeSTAR Max Premix(2×) | 25μL |
luxS-F | 2μL |
luxS-R | 2μL |
E.coli K12 | 2μL |
ddH2O | 19μL |
Total | 50μL |
}
94oC | 10 min | |
94oC | 30 sec | |
63oC | 30 sec | 30 cycles |
72oC | 30 sec | |
72oC | 10 min | |
16oC | ∞ |
PrimeSTAR Max Premix(2×) | 25μL |
mtn-F | 2μL |
mtn-R | 2μL |
E.coli K12 | 2μL |
ddH2O | 19μL |
Total | 50μL |
}
94oC | 10 min | |
94oC | 30 sec | |
63oC | 30sec | 30 cycles |
72oC | 45 sec | |
72oC | 10 min | |
16oC | ∞ |
ClonExpress technique was used to ligate luxS and mtn genes to plasmid pTrcHisB. The recombinant vector pTrc-luxS-mtn was transformed into E.coli K12. After that, verification PCR was performed to select the positive clones.
PrimeSTAR Max Premix(2×) | 10μL |
pTrc-JC-F | 1μL |
pTrc-JC-R | 1μL |
Bacterium solution | 1μL |
ddH2O | 7μL |
Total | 20μL |
}
94oC | 10 min | |
94oC | 30 sec | |
58oC | 30sec | 30 cycles |
72oC | 1 min 30 sec | |
72oC | 10 min | |
16oC | ∞ |
3 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.
10× FastDigest Buffer | 2μL |
Sac Ⅰ | 1μL |
Bgl Ⅱ | 1μL |
pTrc-luxS-mtn | 6μL |
ddH2O | 10μL |
Total | 20μL |
Reaction condition: 37oC for 40 min |
☞☟ Week15 (Aug 22–Aug 28)
- Construction of pLuxS
- Construction of pLsrACDB
- Construction of pLsrACDBFG
luxS gene was cloned from E.coli K12.
PrimeSTAR Max Premix(2×) | 25μL |
luxS-Fd | 2μL |
luxS-Rv | 2μL |
E.coli K12 | 2μL |
ddH2O | 19μL |
Total | 50μL |
}
94oC | 10 min | |
94oC | 30 sec | |
60oC | 30 sec | 30 cycles |
72oC | 30 sec | |
72oC | 10 min | |
16oC | ∞ |
The luxS gene was purified by gel extraction. Then, both the luxS segment and pTrcHisB vector were treated with restriction enzyme. After that, luxS gene was ligated to linearized pTrcHisB, and the ligation product was transformed into E.coli K12.
10× FastDigest Buffer | 4μL |
Kpn Ⅰ | 2μL |
EcoR Ⅰ | 2μL |
luxS | 25μL |
ddH2O | 7μL |
Total | 40μL |
Reaction condition: 37oC for 40 min |
10× FastDigest Buffer | 4μL |
Kpn Ⅰ | 2μL |
EcoR Ⅰ | 2μL |
pTrcHisB | 20μL |
ddH2O | 12μL |
Total | 40μL |
Reaction condition: 37oC for 40 min |
10× DNA Ligase Buffer | 2μL |
T4 DNA Ligase | 1μL |
linearized pTrcHisB | 1μL |
luxS | 3μL |
ddH2O | 13μL |
Total | 20μL |
Reaction condition: 16oC overnight |
Verification PCR was performed to select the positive clones.
2× Taq Master Mix | 10μL |
pTrc-JC-F | 1μL |
pTrc-JC-R | 1μL |
Bacterium solution | 1μL |
ddH2O | 7μL |
Total | 20μL |
}
94oC | 10 min | |
94oC | 30 sec | |
58oC | 30sec | 30 cycles |
72oC | 50 sec | |
72oC | 10 min | |
16oC | ∞ |
One positive strain was chosen to be cultured overnight and plasmid was extracted. After restriction enzyme digestion verification, the positive clone was sequenced.
10× FastDigest Buffer | 2μL |
Kpn Ⅰ | 1μL |
EcoR Ⅰ | 1μL |
pTrc-luxS | 6μL |
ddH2O | 10μL |
Total | 20μL |
Reaction condition: 37oC for 40 min |
The lsrACDBFG sequence was divided into three parts and they were cloned separately. Then the three segments were ligated to the linearized pTrcHisB by ClonExpress technique, and the recombinant vector was transformed into DH5α.
PrimeSTAR Max Premix(2×) | 25μL |
lsrACDB1-F | 2μL |
lsrACDB1-R | 2μL |
E.coli K12 | 2μL |
ddH2O | 19μL |
Total | 50μL |
PrimeSTAR Max Premix(2×) | 25μL |
lsrACDB2-F | 2μL |
lsrACDB2-R | 2μL |
E.coli K12 | 2μL |
ddH2O | 19μL |
Total | 50μL |
}
94oC | 10 min | |
94oC | 30 sec | |
60oC | 30 sec | 30 cycles |
72oC | 2 min 20 sec | |
72oC | 10 min | |
16oC | ∞ |
Verification PCR was performed to select the positive clones.
4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.
10× FastDigest Buffer | 2μL |
BamH Ⅰ | 1μL |
Sac Ⅰ | 1μL |
pTrc-lsrACDB | 6μL |
ddH2O | 10μL |
Total | 20μL |
Reaction condition: 37oC for 40 min |
The lsrACDBFG sequence was divided into three parts and they were cloned separately. Then the three segments were ligated to the linearized pTrcHisB by ClonExpress technique, and the recombinant vector was transformed into DH5α.
PrimeSTAR Max Premix(2×) | 25μL |
lsrACDBFG1-F | 2μL |
lsrACDBFG1-R | 2μL |
E.coli K12 | 2μL |
ddH2O | 19μL |
Total | 50μL |
PrimeSTAR Max Premix(2×) | 25μL |
lsrACDBFG2-F | 2μL |
lsrACDBFG2-R | 2μL |
E.coli K12 | 2μL |
ddH2O | 19μL |
Total | 50μL |
PrimeSTAR Max Premix(2×) | 25μL |
lsrACDBFG3-F | 2μL |
lsrACDBFG3-R | 2μL |
E.coli K12 | 2μL |
ddH2O | 19μL |
Total | 50μL |
}
94oC | 10 min | |
94oC | 30 sec | |
58oC | 30 sec | 30 cycles |
72oC | 2 min | |
72oC | 10 min | |
16oC | ∞ |
Verification PCR was performed to select the positive clones.
4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.
10× FastDigest Buffer | 2μL |
BamH Ⅰ | 1μL |
Sac Ⅰ | 1μL |
pTrc-lsrACDB | 6μL |
ddH2O | 10μL |
Total | 20μL |
Reaction condition: 37oC for 40 min |
☞☟ Week16 (Aug 29–Sep 04)
- Construction of pLsrFG
- Construction of pLsrACDBK
- Construction of pLsrACDBFGK
lsrFG gene was cloned from E.coli K12.
PrimeSTAR Max Premix(2×) | 25μL |
lsrFG-Fd | 2μL |
lsrFG-Rv | 2μL |
E.coli K12 | 2μL |
ddH2O | 19μL |
Total | 50μL |
}
94oC | 10 min | |
94oC | 30 sec | |
58oC | 30 sec | 30 cycles |
72oC | 1 min 15 sec | |
72oC | 10 min | |
16oC | ∞ |
The lsrFG gene was purified by gel extraction. Then, both the lsrFG segment and pTrcHisB vector were treated with restriction enzyme. After that, lsrFG gene was ligated to linearized pTrcHisB, and the ligation product was transformed into E.coli K12.
10× FastDigest Buffer | 4μL |
BamH Ⅰ | 2μL |
Bgl Ⅱ | 2μL |
lsrFG | 25μL |
ddH2O | 7μL |
Total | 40μL |
Reaction condition: 37oC for 40 min |
10× FastDigest Buffer | 4μL |
BamH Ⅰ | 2μL |
Bgl Ⅱ | 2μL |
pTrcHisB | 20μL |
ddH2O | 12μL |
Total | 40μL |
Reaction condition: 37oC for 40 min |
10× DNA Ligase Buffer | 2μL |
T4 DNA Ligase | 1μL |
pMD19 T-Simple Vector | 1μL |
luxSFG | 3μL |
ddH2O | 13μL |
Total | 20μL |
Reaction condition: 16oC overnight |
Verification PCR was performed to select the positive clones.
2× Taq Master Mix | 10μL |
pTrc-JC-F | 1μL |
pTrc-JC-R | 1μL |
Bacterium solution | 1μL |
ddH2O | 7μL |
Total | 20μL |
}
94oC | 10 min | |
94oC | 30 sec | |
58oC | 30 sec | 30 cycles |
72oC | 1 min 30 sec | |
72oC | 10 min | |
16oC | ∞ |
Two positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.
10× FastDigest Buffer | 2μL |
BamH Ⅰ | 1μL |
Bgl Ⅱ | 1μL |
pTrc-lsrFG | 6μL |
ddH2O | 10μL |
Total | 20μL |
Reaction condition: 37oC for 40 min |
lsrK gene was cloned from E.coli K12.
PrimeSTAR Max Premix(2×) | 25μL |
lsrK-F | 2μL |
lsrK-R | 2μL |
E.coli K12 | 2μL |
ddH2O | 19μL |
Total | 50μL |
}
94oC | 10 min | |
94oC | 30 sec | |
55oC | 30 sec | 30 cycles |
72oC | 1 min 40 sec | |
72oC | 10 min | |
16oC | ∞ |
The lsrK gene was purified by gel extraction. Then, both the lsrK segment and pTrc-lsrACDB vector were treated with restriction enzyme. After that, lsrK gene was ligated to linearized pTrc-lsrACDB, and the ligation product was transformed into E.coli K12.
10× FastDigest Buffer | 4μL |
Pst Ⅰ | 2μL |
BstB Ⅰ | 2μL |
lsrK | 25μL |
ddH2O | 7μL |
Total | 40μL |
Reaction condition: 37oC for 40 min |
10× FastDigest Buffer | 4μL |
Pst Ⅰ | 2μL |
BstB Ⅰ | 2μL |
pTrc-lsrACDB | 25μL |
ddH2O | 7μL |
Total | 40μL |
Reaction condition: 37oC for 40 min |
10× DNA Ligase Buffer | 2μL |
T4 DNA Ligase | 1μL |
linearized pTrc-lsrACDB | 1μL |
lsrK | 3μL |
ddH2O | 13μL |
Total | 20μL |
Reaction condition: 16oC overnight |
Verification PCR was performed to select the positive clones.
2× Taq Master Mix | 10μL |
BK-JC-F | 1μL |
pTrc-JC-R | 1μL |
Bacterium solution | 1μL |
ddH2O | 7μL |
Total | 20μL |
}
94oC | 10 min | |
94oC | 30 sec | |
58oC | 30 sec | 30 cycles |
72oC | 2 min | |
72oC | 10 min | |
16oC | ∞ |
Two positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.
10× FastDigest Buffer | 2μL |
Pst Ⅰ | 1μL |
BstB Ⅰ | 1μL |
pTrc-lsrACDBK | 6μL |
ddH2O | 10μL |
Total | 20μL |
Reaction condition: 37oC for 40 min |
lsrK gene was cloned from E.coli K12.
PrimeSTAR Max Premix(2×) | 25μL |
lsrK-F | 2μL |
lsrK-R | 2μL |
E.coli K12 | 2μL |
ddH2O | 19μL |
Total | 50μL |
}
94oC | 10 min | |
94oC | 30 sec | |
55oC | 30 sec | 30 cycles |
72oC | 1 min 40 sec | |
72oC | 10 min | |
16oC | ∞ |
The lsrK gene was purified by gel extraction. Then, both the lsrK segment and pTrc-lsrACDBFG vector were treated with restriction enzyme. After that, lsrK gene was ligated to linearized pTrc-lsrACDBFG, and the ligation product was transformed into E.coli K12.
10× FastDigest Buffer | 4μL |
Pst Ⅰ | 2μL |
BstB Ⅰ | 2μL |
lsrK | 25μL |
ddH2O | 7μL |
Total | 40μL |
Reaction condition: 37oC for 40 min |
10× FastDigest Buffer | 4μL |
Pst Ⅰ | 2μL |
BstB Ⅰ | 2μL |
pTrc-lsrACDBFG | 20μL |
ddH2O | 12μL |
Total | 40μL |
Reaction condition: 37oC for 40 min |
10× DNA Ligase Buffer | 2μL |
T4 DNA Ligase | 1μL |
linearized pTrc-lsrACDFGB | 1μL |
lsrK | 3μL |
ddH2O | 13μL |
Total | 20μL |
Reaction condition: 16oC overnight |
Verification PCR was performed to select the positive clones.
2× Taq Master Mix | 10μL |
GK-JC-F | 1μL |
pTrc-JC-R | 1μL |
Bacterium solution | 1μL |
ddH2O | 7μL |
Total | 20μL |
}
94oC | 10 min | |
94oC | 30 sec | |
58oC | 30 sec | 30 cycles |
72oC | 2 min | |
72oC | 10 min | |
16oC | ∞ |
Two positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.
10× FastDigest Buffer | 2μL |
Pst Ⅰ | 1μL |
BstB Ⅰ | 1μL |
pTrc-lsrACDBKFGK | 6μL |
ddH2O | 10μL |
Total | 20μL |
Reaction condition: 37oC for 40 min |
☞☟ Week17 (Sep 05–Sep 11)
- Construction of pLsrK
lsrK gene was cloned from E.coli K12.
PrimeSTAR Max Premix(2×) | 25μL |
lsrK-Fd | 2μL |
lsrK-Rv | 2μL |
E.coli K12 | 2μL |
ddH2O | 19μL |
Total | 50μL |
}
94oC | 10 min | |
94oC | 30 sec | |
55oC | 30 sec | 30 cycles |
72oC | 1 min 40 sec | |
72oC | 10 min | |
16oC | ∞ |
The lsrK gene was purified by gel extraction. Then, both the lsrK segment and pTrc-lsrACDBFG vector were treated with restriction enzyme. After that, lsrK gene was ligated to linearized pTrc-lsrACDBFG, and the ligation product was transformed into E.coli K12.
10× FastDigest Buffer | 4μL |
Kpn Ⅰ | 2μL |
EcoR Ⅰ | 2μL |
lsrK | 25μL |
ddH2O | 7μL |
Total | 40μL |
Reaction condition: 37oC for 40 min |
10× FastDigest Buffer | 4μL |
Pst Ⅰ | 2μL |
BstB Ⅰ | 2μL |
pTrc-HisB | 20μL |
ddH2O | 12μL |
Total | 40μL |
Reaction condition: 37oC for 40 min |
10× DNA Ligase Buffer | 2μL |
T4 DNA Ligase | 1μL |
linearized pTrcHisB | 1μL |
lsrK | 3μL |
ddH2O | 13μL |
Total | 20μL |
Reaction condition: 16oC overnight |
Verification PCR was performed to select the positive clones.
2× Taq Master Mix | 10μL |
pTrc-JC-F | 1μL |
pTrc-JC-R | 1μL |
Bacterium solution | 1μL |
ddH2O | 7μL |
Total | 20μL |
}
94oC | 10 min | |
94oC | 30 sec | |
58oC | 30 sec | 30 cycles |
72oC | 2 min | |
72oC | 10 min | |
16oC | ∞ |
4 positive strains were chosen to be cultured overnight and plasmids were extracted. After restriction enzyme digestion verification, the positive clones were sequenced.
10× FastDigest Buffer | 2μL |
Kpn Ⅰ | 1μL |
EcoR Ⅰ | 1μL |
pTrc-lsrK | 6μL |
ddH2O | 10μL |
Total | 20μL |
Reaction condition: 37oC for 40 min |
☞☟ Week18 (Seq 12–Seq 18)
☞☟ Week19 (Seq 19–Seq 25)
☞☟ Week20 (Seq 26–Oct 02)
Biofilm studies and evaluation:
pTrcHisB, pLuxS, pLuxSMtn, pLsrACDB, pLsrACDBFG, pLsrACDBK, pLsrACDBFGK were diluted to OD~0.05 and reinoculated at a total volume of 200 μL at a 1:1 (v/v) ratio. IPTG (1 mM) was added at OD ~ 0.4, and biofilms were cultured for ~24 h (+/−30 min) at 30 oC in static conditions. After incubation, optical density was read on a plate reader at 600 nm. The supernatant was gently decanted, and each well was washed 3 times with 300 μL of sterile PBS to detach loosely adhered cells. The plate was then incubated at 60 oC with the lid off for 60 min, and afterwards, 250 μL of 0.1% crystal violet was added to each well and incubated for 15 min at room temperature. Crystal violet stain was aspirated with a pipette and excess stain was washed off by gently submerging and mixing in a tray filled with distilled water until washings were free of the stain. After the microplate was airdried, the dye was resolubilized by adding 250 μL of 95% ethanol, and incubated at room temperature with shaking for 30 min. The optical density of each well stained with crystal violet was measured at 540 nm.
pTrcHisB, pLuxS, pLuxSMtn, pLsrACDB, pLsrACDBFG, pLsrACDBK, pLsrACDBFGK were diluted to OD~0.05 and reinoculated at a total volume of 200 μL at a 1:1 (v/v) ratio. IPTG (1 mM) was added at OD ~ 0.4, and biofilms were cultured for ~24 h (+/−30 min) at 30 oC in static conditions. After incubation, optical density was read on a plate reader at 600 nm. The supernatant was gently decanted, and each well was washed 3 times with 300 μL of sterile PBS to detach loosely adhered cells. The plate was then incubated at 60 oC with the lid off for 60 min, and afterwards, 250 μL of 0.1% crystal violet was added to each well and incubated for 15 min at room temperature. Crystal violet stain was aspirated with a pipette and excess stain was washed off by gently submerging and mixing in a tray filled with distilled water until washings were free of the stain. After the microplate was airdried, the dye was resolubilized by adding 250 μL of 95% ethanol, and incubated at room temperature with shaking for 30 min. The optical density of each well stained with crystal violet was measured at 540 nm.
Cultured media of controllers were tested for the presence of AI-2 by HPLC. AI-2 output and uptake profiles of controllers were drawn according to the HPLC results.
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