Difference between revisions of "Team:NKU China/Notebook"
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| − | {{NKU_China/ | + | {{NKU_China/menubar}} |
<html> | <html> | ||
<head> | <head> | ||
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| − | <meta name="viewport" content="width=device-width, initial-scale=1"> | + | <meta charset="utf-8"> |
| + | <meta name="viewport" content="width=device-width, initial-scale=1"> | ||
<link rel="stylesheet" href="https://2016.igem.org/Template:NKU_China/css/jquery-ui?action=raw&ctype=text/css" /> | <link rel="stylesheet" href="https://2016.igem.org/Template:NKU_China/css/jquery-ui?action=raw&ctype=text/css" /> | ||
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<script src="https://2016.igem.org/Template:NKU_China/js/jquery-ui?action=raw&ctype=text/javascript"></script> | <script src="https://2016.igem.org/Template:NKU_China/js/jquery-ui?action=raw&ctype=text/javascript"></script> | ||
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<link rel="stylesheet" href="share.css"> | <link rel="stylesheet" href="share.css"> | ||
| − | <style> | + | <style> |
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| − | + | @media all and (max-width: 480px) { | |
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</style> | </style> | ||
| − | <style> | + | <style class="table-theme.css"> |
| − | /**/ | + | /*PCR system*/ |
| − | + | .table-theme-1 { | |
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| − | + | .table-theme-1 table tr:last-child td { | |
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| − | + | .table-theme-1 tr:nth-child(odd) { | |
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| − | . | + | .table-theme-1 tr:nth-child(even) { |
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| − | . | + | /*PCR reaction condition*/ |
| − | + | .table-theme-2 { | |
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| − | + | border-top-color: rgb(155, 187, 89); | |
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| − | + | .table-theme-2 table tr:last-child td { | |
| − | + | border-bottom-color: rgb(75,172,198); | |
| − | + | } | |
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| − | + | .table-theme-2 tr:nth-child(odd) { | |
| − | + | background-color: rgb(230,238,213); | |
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| − | + | .table-theme-2 tr:nth-child(even) { | |
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} | } | ||
| − | + | /*ligation system*/ | |
| − | + | .table-theme-3 { | |
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| − | + | .table-theme-3 caption { | |
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} | } | ||
| − | + | .table-theme-3 table tr:last-child td { | |
| − | + | border-bottom-color: rgb(128, 100, 162); | |
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| − | . | + | .table-theme-3 tr:nth-child(odd) { |
| − | + | background-color: rgb(223, 215, 232); | |
} | } | ||
| − | + | .table-theme-3 tr:nth-child(even) { | |
| − | . | + | |
| − | + | ||
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} | } | ||
| − | . | + | /*digestion system*/ |
| − | + | .table-theme-4 { | |
| + | color: rgb(148, 54, 52); | ||
} | } | ||
| − | . | + | .table-theme-4 caption { |
| − | + | border-top-color: rgb(192, 80, 77); | |
| − | + | border-bottom-color: rgb(192, 80, 77); | |
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| − | . | + | .table-theme-4 table tr:last-child td { |
| − | + | border-bottom-color: rgb(192, 80, 77); | |
| − | + | } | |
| − | + | ||
| − | } | + | |
| − | + | .table-theme-4 tr:nth-child(odd) { | |
| − | + | background-color: rgb(239, 211, 210); | |
| − | + | } | |
| − | + | ||
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| − | + | .table-theme-4 tr:nth-child(even) { | |
| − | + | } | |
| − | + | ||
| − | + | ||
| − | + | /*methylation system*/ | |
| − | + | .table-theme-5 { | |
| − | + | color: rgb(227, 108, 10); | |
| − | + | } | |
| − | + | ||
| − | + | .table-theme-5 caption { | |
| − | + | border-top-color: rgb(247, 150, 70); | |
| − | + | border-bottom-color: rgb(247, 150, 70); | |
| − | + | } | |
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| − | + | .table-theme-5 table tr:last-child td { | |
| − | + | border-bottom-color: rgb(247, 150, 70); | |
| − | + | } | |
| − | + | .table-theme-5 tr:nth-child(odd) { | |
| − | + | background-color: rgb(253, 228, 208); | |
| − | + | } | |
| − | } | + | |
| + | .table-theme-5 tr:nth-child(even) { | ||
| + | } | ||
| − | </style> | + | /*Groups divided*/ |
| + | .table-theme-6 { | ||
| + | color: rgb(0, 0, 0); | ||
| + | } | ||
| + | |||
| + | .table-theme-6 caption { | ||
| + | border-top-color: rgb(0, 0, 0); | ||
| + | border-bottom-color: rgb(0, 0, 0); | ||
| + | } | ||
| + | |||
| + | .table-theme-6 table tr:last-child td { | ||
| + | border-bottom-color: rgb(0, 0, 0); | ||
| + | } | ||
| + | |||
| + | .table-theme-6 tr:nth-child(odd) { | ||
| + | background-color: rgb(192, 192, 192); | ||
| + | } | ||
| + | |||
| + | .table-theme-6 tr:nth-child(even) { | ||
| + | } | ||
| + | </style> | ||
<script id="ready-js"> | <script id="ready-js"> | ||
$(function () { | $(function () { | ||
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heightStyle: 'content', | heightStyle: 'content', | ||
collapsible: true, | collapsible: true, | ||
| − | active: | + | active: 0, |
icons: false, | icons: false, | ||
}); | }); | ||
| − | + | ||
$('.tabs').each( | $('.tabs').each( | ||
function (index, value) { | function (index, value) { | ||
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event: "mouseover", | event: "mouseover", | ||
heightStyle: 'content', | heightStyle: 'content', | ||
| − | active: | + | active:0, |
}); | }); | ||
} | } | ||
| − | ) | + | ) |
| − | }) | + | }); |
| − | </script> | + | </script> |
</head> | </head> | ||
| − | <body> | + | <body id="dongzhuoer"> |
| − | + | <div style="display:none"> | |
| + | <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> | ||
| + | <table class="table-theme-2" id="table5-1-2"> | ||
| + | <caption>PCR reaction condition</caption> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>58℃</td> | ||
| + | <td>15 sec</td> | ||
| + | <td>30 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>16℃</td> | ||
| + | <td>∞</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <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℃ overnight</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <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℃ for 40 min</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <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℃ for 1 hour</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <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> | ||
| + | <figure> | ||
| + | <img src="image/notebook/notebook-6-13-1.png" width="347" height="284"> | ||
| + | <figcaption> | ||
| + | Selecting positive clones by colony PCR<br> | ||
| + | (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> | ||
| − | + | <main id="notebook"> | |
| − | + | <div class="h1">Laboratory Notes</div> | |
| − | < | + | <div id="accordion"> |
| − | <div class=" | + | <div class="accordion-fragment" id="week1"> |
| + | <div class="accordion-header"> | ||
| + | <span class="default">☞</span><span class="active">☟</span> Week1 (May 16–May 22) | ||
| + | </div> | ||
| + | <div class="accordion-content"> | ||
<div class="p"> | <div class="p"> | ||
| − | + | In order to make sure our "consumer" efficient, 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 class=" | + | <div class="tabs" id="tabs1"> |
| − | + | <ul> | |
| − | + | <li><a href="#fragment1-1">➀</a></li> | |
| − | + | <li><a href="#fragment1-2">➁</a></li> | |
| − | + | <li><a href="#fragment1-3">➂</a></li> | |
| − | + | </ul> | |
| − | + | <div id="fragment1-1"> | |
| − | + | <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="fragment1-2"> | |
| − | + | <div class="p"> | |
| − | < | + | Transformed pKSU-Δ<i>luxS</i> into GR286, and selected out positive clones. |
| − | + | ||
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| − | + | ||
| − | + | ||
| − | + | ||
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| − | + | ||
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| − | + | ||
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| − | + | ||
</div> | </div> | ||
| − | <div | + | <div class="flex-container"> |
| − | < | + | <div class="table-wrapper"> |
| − | < | + | <table class="table-theme-1" id="table1-2-1"> |
| − | < | + | <caption>20μL PCR system</caption> |
| − | + | <tr> | |
| − | <div | + | <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> | ||
| + | </div> | ||
| + | <div class="table-wrapper"> | ||
| + | <table class="table-theme-2" id="table1-2-2"> | ||
| + | <caption>PCR reaction condition</caption> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>58℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td>30 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>1 min 30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>16℃</td> | ||
| + | <td>∞</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <figure style="max-width:15rem"> | ||
| + | <a href="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"></a> | ||
| + | <figcaption>Selecting positive clones by PCR</figcaption> | ||
| + | </figure> | ||
</div> | </div> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | |||
| − | + | </div> | |
| − | + | <div id="fragment1-3"> | |
| − | + | <div class="p"> | |
| + | The transformants were cultured at 42℃ with chloramphenicol to select single-crossover clones. | ||
</div> | </div> | ||
| + | <div class="flex-container"> | ||
| + | <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> | ||
| + | </div> | ||
| + | <div class="table-wrapper"> | ||
| + | <table class="table-theme-2" id="table1-3-2"> | ||
| + | <caption>PCR reaction condition</caption> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>56℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td>30 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>2 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>16℃</td> | ||
| + | <td>∞</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <figure style="max-width:20rem"> | ||
| + | <a href="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"></a> | ||
| + | <figcaption> | ||
| + | Selecting single-crossover clones using PCR<br> | ||
| + | (No.1-4 are single-crossover strains, No.5 is positive control.) | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="accordion-fragment" id="week2"> | |
| − | + | <div class="accordion-header"> | |
| + | <span class="default">☞</span><span class="active">☟</span> Week2 (May 23–May 29) | ||
| + | </div> | ||
| + | <div class="accordion-content"> | ||
| + | <div class="tabs" id="tabs2"> | ||
| + | <ul> | ||
| + | <li><a href="#fragment2-1">➀</a></li> | ||
| + | <li><a href="#fragment2-2">➁</a></li> | ||
| + | </ul> | ||
| + | <div id="fragment2-1"> | ||
| + | <div class="p"> | ||
| + | The single-crossover strains were then cultured at LB medium and passaged every 12 hours for 4 generation. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div id="fragment2-2"> | ||
| + | <div class="p"> | ||
| + | Cultured the last generation at medium with 5-fluorouracil to select double-crossover clones. Regretfully, we didn't get the double-crossover clones. | ||
| + | </div> | ||
| + | <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"> | ||
| + | <table class="table-theme-2" id="table2-2-2"> | ||
| + | <caption>PCR reaction condition</caption> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>56℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td>30 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>2 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>16℃</td> | ||
| + | <td>∞</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <figure style="max-width:20rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/3/3d/T--NKU_China--notebook-1-5-3.png"><img src="https://static.igem.org/mediawiki/2016/3/3d/T--NKU_China--notebook-1-5-3.png"></a> | ||
| + | <figcaption> | ||
| + | Selecting double-crossover clones using PCR<br> | ||
| + | (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 class="accordion-content"> | + | </div> |
| − | + | </div> | |
| − | + | <div class="accordion-fragment" id="week3"> | |
| − | + | <div class="accordion-header"> | |
| − | + | <span class="default">☞</span><span class="active">☟</span> Week3 (May 30–Jun 05) | |
| − | + | </div> | |
| − | + | <div class="accordion-content"> | |
| − | + | <div class="tabs" id="tabs3"> | |
| − | + | <ul> | |
| − | + | <li><a href="#fragment3-1">➀</a></li> | |
| − | + | <li><a href="#fragment3-2">➁</a></li> | |
| − | + | <li><a href="#fragment3-3">➂</a></li> | |
| − | + | </ul> | |
| − | + | <div id="fragment3-1"> | |
| − | + | <div class="p"> | |
| − | + | We cultured transformants at 42℃ with chloramphenicol again and selected the single-crossover clones successfully. | |
| − | + | ||
</div> | </div> | ||
| − | <div id=" | + | <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"> | ||
| + | <table class="table-theme-2" id="table3-1-2"> | ||
| + | <caption>PCR reaction condition</caption> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>56℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td>30 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>2 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>16℃</td> | ||
| + | <td>∞</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <figure style="max-width:20rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/9/93/T--NKU_China--notebook-1-5-4.png"><img src="https://static.igem.org/mediawiki/2016/9/93/T--NKU_China--notebook-1-5-4.png"></a> | ||
| + | <figcaption> | ||
| + | Selecting single-crossover clones using PCR<br> | ||
| + | (No.1&2&4 are single-crossover strains,No.5 is positive control. ) | ||
| + | </figcaption> | ||
| + | </figure> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div id="fragment3-2"> | |
| − | + | <div class="p"> | |
| − | + | The single-crossover strains were then cultured at LB medium and passaged every 12 hours for 4 generations. | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</div> | </div> | ||
| + | </div> | ||
| + | <div id="fragment3-3"> | ||
| + | <div class="p"> | ||
| + | Cultured the last generation at medium with 5-fluorouracil to select double-crossover clones. We finally got 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"> | ||
| + | <table class="table-theme-2" id="table3-3-2"> | ||
| + | <caption>PCR reaction condition</caption> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>56℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td>30 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>2 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>16℃</td> | ||
| + | <td>∞</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <figure style="max-width:23rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/a/aa/T--NKU_China--notebook-1-5-5.png"><img src="https://static.igem.org/mediawiki/2016/a/aa/T--NKU_China--notebook-1-5-5.png"></a> | ||
| + | <figcaption> | ||
| + | Selecting the strain lacking of <i>luxS</i> gene using PCR<br> | ||
| + | (The No.4 is the aimed strain<U+00A1><U+00AA>GR286Δ<i>luxS</i>) | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="accordion-fragment" id="week4"> | |
| − | + | <div class="accordion-header"> | |
| + | <span class="default">☞</span><span class="active">☟</span> Week4 (Jun 06–Jun 12) | ||
| + | </div> | ||
| + | <div class="accordion-content"> | ||
| + | <div class="tabs" id="tabs4"> | ||
| + | <ul> | ||
| + | <li><a href="#fragment4-1">➀</a></li> | ||
| + | <li><a href="#fragment4-2">➁</a></li> | ||
| + | <li><a href="#fragment4-3">➂</a></li> | ||
| + | <li><a href="#fragment4-4">➃</a></li> | ||
| + | <li><a href="#fragment4-5">➄</a></li> | ||
| + | <li><a href="#fragment4-6">➅</a></li> | ||
| + | </ul> | ||
| + | <div id="fragment4-1"> | ||
| + | <div class="p"> | ||
| + | Cultured the GR286Δ<i>luxS</i> strain and made it competence for future use. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div id="fragment4-2"> | ||
| + | <div class="p"> | ||
| + | Cloned the <i>lsrACDB</i> gene from <i>Bacillus thuringiensis</i> and ligated it 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"> | ||
| + | <table class="table-theme-2" id="table4-2-2"> | ||
| + | <caption>PCR reaction condition</caption> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>57℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td>30 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>4 min 30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>16℃</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℃ overnight</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div id="fragment4-3"> | ||
| + | <div class="p"> | ||
| + | Transformed the T-<i>lsrACDB</i> into DH5α and coated plate, and then selected positive clones by colony PCR. | ||
| + | </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"> | ||
| + | <table class="table-theme-2" id="table4-3-2"> | ||
| + | <caption>PCR reaction condition</caption> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>59℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td>30 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>4 min 30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>16℃</td> | ||
| + | <td>∞</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <figure style="max-width:15rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/7/72/T--NKU_China--notebook-1-5-6.png"><img src="https://static.igem.org/mediawiki/2016/7/72/T--NKU_China--notebook-1-5-6.png"></a> | ||
| + | <figcaption> | ||
| + | Selecting positive clones by PCR<br> | ||
| + | (No. 3&4 are positive results) | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div id="fragment4-4"> | ||
| + | <div class="p"> | ||
| + | After restriction enzyme digestion verification, we sent them to sequencing. Unfortunately, the sequencing result showed some mutations in cloning 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℃ for 40 min</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <figure style="max-width:19rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/9/91/T--NKU_China--notebook-1-5-7.png"><img src="https://static.igem.org/mediawiki/2016/9/91/T--NKU_China--notebook-1-5-7.png"></a> | ||
| + | <figcaption> | ||
| + | Restriction enzyme digestion verification<br> | ||
| + | (No.1 are <i>lsrACDB</i> fragement, No.2 are linearized T-vector.) | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div id="fragment4-5"> | ||
| + | <div class="p"> | ||
| + | We repeated the process of gene cloning but there were still some mutations. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div id="fragment4-6"> | ||
| + | <div class="p"> | ||
| + | We finally decided to request the gene company to synthesize the lsrACDB gene. | ||
| + | </div> | ||
| + | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | </div> | |
| − | + | <div class="accordion-fragment" id="week5"> | |
| − | + | <div class="accordion-header"> | |
| − | + | <span class="default">☞</span><span class="active">☟</span> Week5 (Jun 13–Jun 19) | |
| − | + | </div> | |
| − | + | <div class="accordion-content"> | |
| − | + | <div class="p"> | |
| − | + | This week, we started to construct another controller―supplier. | |
| − | + | </div> | |
| − | + | <div class="tabs" id="tabs5"> | |
| − | + | <ul> | |
| − | + | <li><a href="#fragment5-1">➀</a></li> | |
| − | < | + | <li><a href="#fragment5-2">➁</a></li> |
| − | + | <li><a href="#fragment5-3">➂</a></li> | |
| − | + | <li><a href="#fragment5-4">➃</a></li> | |
| − | + | </ul> | |
| − | + | <div id="fragment5-1"> | |
| − | + | <div class="p"> | |
| − | + | We cloned a strong promoter <i>C2</i> from former kit and cloned <i>luxS</i> gene from GR286. | |
| − | + | ||
</div> | </div> | ||
| − | <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> | |
| − | <div class=" | + | <td>2× Taq Master Mix</td> |
| − | + | <td>25μL</td> | |
| − | </div> | + | </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"> | ||
| + | <table class="table-theme-2" id="table5-1-2"> | ||
| + | <caption>PCR reaction condition</caption> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>58℃</td> | ||
| + | <td>15 sec</td> | ||
| + | <td>30 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>16℃</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"> | ||
| + | <table class="table-theme-2" id="table5-1-4"> | ||
| + | <caption>PCR reaction condition</caption> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>59℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td>30 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>16℃</td> | ||
| + | <td>∞</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <figure style="max-width:19rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/d/dc/T--NKU_China--notebook-1-5-8.png"><img src="https://static.igem.org/mediawiki/2016/d/dc/T--NKU_China--notebook-1-5-8.png"></a> | ||
| + | <figcaption> | ||
| + | Cloning promoter <i>C2</i> and <i>luxS</i> using PCR<br> | ||
| + | (NO.1&2 are <i>C2</i>, No.3&4 are <i>luxS</i>) | ||
| + | </figcaption> | ||
| + | </figure> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div id="fragment5-2"> | |
| − | + | <div class="p"> | |
| − | + | Fuse the two segments together by fusion PCR, and ligated it into T-vector. Then, transformed the vector into DH5α. | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</div> | </div> | ||
| − | <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"> | ||
| + | <table class="table-theme-2" id="table5-2-2"> | ||
| + | <caption>PCR reaction condition</caption> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>59℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td>30 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>40 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>16℃</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℃ overnight</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div id="fragment5-3"> | |
| − | + | <div class="p"> | |
| − | + | Selected the positive clones by colony PCR. | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</div> | </div> | ||
| − | <div | + | <div class="flex-container"> |
| − | < | + | <div class="table-wrapper"> |
| − | < | + | <table class="table-theme-1" id="table5-3-1"> |
| − | < | + | <caption>20μL PCR system</caption> |
| − | + | <tr> | |
| − | <div | + | <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"> | ||
| + | <table class="table-theme-2" id="table5-3-2"> | ||
| + | <caption>PCR reaction condition</caption> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>59℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td>30 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>40 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>16℃</td> | ||
| + | <td>∞</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <figure style="max-width:18rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/c/c9/T--NKU_China--notebook-1-5-9.png"><img src="https://static.igem.org/mediawiki/2016/c/c9/T--NKU_China--notebook-1-5-9.png"></a> | ||
| + | <figcaption> | ||
| + | Selecting positive clones by colony PCR | ||
| + | </figcaption> | ||
| + | </figure> | ||
</div> | </div> | ||
| − | <div id=" | + | </div> |
| − | < | + | <div id="fragment5-4"> |
| − | + | <div class="p"> | |
| − | < | + | We chose 4 positive strains to culture overnight and extracted the plasmid. After restriction enzyme digestion verification, we sent them to sequencing. |
| − | + | </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℃ for 40 min</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <figure style="max-width:19rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/f/fb/T--NKU_China--notebook-1-5-10.png"><img src="https://static.igem.org/mediawiki/2016/f/fb/T--NKU_China--notebook-1-5-10.png"></a> | ||
| + | <figcaption> | ||
| + | Restriction enzyme digestion verification<br> | ||
| + | (No.1 are linearized T-vector, No.2 are <i>C2-luxS</i> fragment.) | ||
| + | </figcaption> | ||
| + | </figure> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="accordion-fragment" id="week6"> | |
| − | + | <div class="accordion-header"> | |
| + | <span class="default">☞</span><span class="active">☟</span> Week6 (Jun 20–Jun 26) | ||
| + | </div> | ||
| + | <div class="accordion-content"> | ||
| + | <div class="tabs" id="tabs6"> | ||
| + | <ul> | ||
| + | <li><a href="#fragment6-1">➀</a></li> | ||
| + | <li><a href="#fragment6-2">➁</a></li> | ||
| + | <li><a href="#fragment6-3">➂</a></li> | ||
| + | <li><a href="#fragment6-4">➃</a></li> | ||
| + | <li><a href="#fragment6-5">➄</a></li> | ||
| + | </ul> | ||
| + | <div id="fragment6-1"> | ||
| + | <div class="p"> | ||
| + | The sequencing result showed there's a correct strain. So we can use the strain for the following experiment. We obtained the correct plasmid T-<i>C2-luxS</i> from DH5α. Then we got the fragment <i>C2-luxS</i> 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℃ for 40 min</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div id="fragment6-2"> | ||
| + | <div class="p"> | ||
| + | Ligated the <i>C2-luxS</i> to linearized plasmid pWH1520, and transformed it 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℃ overnight</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div id="fragment6-3"> | ||
| + | <div class="p"> | ||
| + | Extracted the plasmid pWH-<i>C2-luxS</i> from DH5α. To prevent the plasmid from DAM&DCM methylation, we transformed it into <i>E.coli</i> JM110. | ||
| + | </div> | ||
| + | </div> | ||
| + | <div id="fragment6-4"> | ||
| + | <div class="p"> | ||
| + | Extracted the plasmid pWH-<i>C2-luxS</i> from JM110,and dealt with it by 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℃ for 1 hour</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div id="fragment6-5"> | ||
| + | <div class="p"> | ||
| + | Transformed the plasmid into GR286 by electroporation.[Failed] | ||
| + | </div> | ||
| + | <div class="flex-container"> | ||
| + | <figure style="max-width:18rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/e/e0/T--NKU_China--notebook-6-13-1.png"><img src="https://static.igem.org/mediawiki/2016/e/e0/T--NKU_China--notebook-6-13-1.png"></a> | ||
| + | <figcaption> | ||
| + | Selecting positive clones by colony PCR<br> | ||
| + | (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> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | </div> | |
| − | + | <div class="accordion-fragment" id="week7"> | |
| − | + | <div class="accordion-header"> | |
| − | + | <span class="default">☞</span><span class="active">☟</span> Week7 (Jun 27–Jul 03) | |
| − | + | </div> | |
| − | + | <div class="accordion-content"> | |
| − | + | <div class="tabs" id="tabs7"> | |
| − | + | <ul> | |
| − | + | <li><a href="#fragment7-1">➀</a></li> | |
| − | + | <li><a href="#fragment7-2">➁</a></li> | |
| − | + | <li><a href="#fragment7-3">➂</a></li> | |
| − | + | </ul> | |
| − | + | <div id="fragment7-1"> | |
| − | + | <div class="p"> | |
| − | + | This week, we tried to use different voltages to transform the plasmid. Sadly, all of these tries got bad results. | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div id="fragment7-2"> | |
| − | + | <div class="p"> | |
| − | + | We considered whether the <i>luxS</i> gene is a little toxic for GR286, and the bacteria tends to refuse the gene when we added a strong promoter in front of it. So, we planned to use induced expression to reconstruction our expression vector. | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div id="fragment7-3"> | |
| − | + | <div class="p"> | |
| − | + | The plasmid pWH1520 contains the strong <i>xylA</i> promoter originating, and transcription from this promoter is xylose inducible. So, the gene of interest carries its own ribosome binding sequence (RBS) and translation initiation codon. Based on these points, we redesigned primers. | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</div> | </div> | ||
| − | <div | + | <div class="flex-container"> |
| − | < | + | <figure style="max-width:30rem"> |
| − | + | <a href="https://static.igem.org/mediawiki/2016/e/eb/T--NKU_China--notebook-6-13-2.png"><img src="https://static.igem.org/mediawiki/2016/e/eb/T--NKU_China--notebook-6-13-2.png"></a> | |
| − | + | <figcaption> | |
| − | + | </figcaption> | |
| − | < | + | </figure> |
| − | + | ||
| − | </ | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | </ | + | |
</div> | </div> | ||
| − | + | </div> | |
| − | + | </div> | |
| − | + | </div> | |
| − | + | </div> | |
| − | + | <div class="accordion-fragment" id="week8"> | |
| − | + | <div class="accordion-header"> | |
| − | + | <span class="default">☞</span><span class="active">☟</span> Week8 (Jul 04–Jul 10) | |
| − | + | </div> | |
| − | + | <div class="accordion-content"> | |
| − | </ | + | <div class="tabs" id="tabs8"> |
| + | <ul> | ||
| + | <li><a href="#fragment8-1">➀</a></li> | ||
| + | <li><a href="#fragment8-2">➁</a></li> | ||
| + | <li><a href="#fragment8-3">➂</a></li> | ||
| + | <li><a href="#fragment8-4">➃</a></li> | ||
| + | </ul> | ||
| + | <div id="fragment8-1"> | ||
| + | <div class="p"> | ||
| + | We cloned <i>luxS</i> gene from GR286 using our new primers. | ||
</div> | </div> | ||
| − | <div id=" | + | <div class="flex-container"> |
| − | <img src="https://static.igem.org/mediawiki/2016/ | + | <div class="table-wrapper"> |
| − | < | + | <table class="table-theme-1" id="table8-1-1"> |
| − | <div class=" | + | <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"> | ||
| + | <table class="table-theme-2" id="table8-1-2"> | ||
| + | <caption>PCR reaction condition</caption> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>58℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td>30 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>40 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>16℃</td> | ||
| + | <td>∞</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <figure style="max-width:12rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/3/3a/T--NKU_China--notebook-6-13-3.png"><img src="https://static.igem.org/mediawiki/2016/3/3a/T--NKU_China--notebook-6-13-3.png"></a> | ||
| + | <figcaption> | ||
| + | Cloning <i>luxS</i> gene by PCR | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div id="fragment8-2"> | ||
| + | <div class="p"> | ||
| + | Purified the <i>luxS</i> fragment by gel extraction, and ligated it into linearized pWH1520. Then, transformed the vector 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℃ 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℃ overnight</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div id="fragment8-3"> | ||
| + | <div class="p"> | ||
| + | Selected the positive clones 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"> | ||
| + | <table class="table-theme-2" id="table8-3-2"> | ||
| + | <caption>PCR reaction condition</caption> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>94℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>58℃</td> | ||
| + | <td>30 sec</td> | ||
| + | <td>30 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>40 sec</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72℃</td> | ||
| + | <td>10 min</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>16℃</td> | ||
| + | <td>∞</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <figure style="max-width:17rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/4/49/T--NKU_China--notebook-6-13-4.png"><img src="https://static.igem.org/mediawiki/2016/4/49/T--NKU_China--notebook-6-13-4.png"></a> | ||
| + | <figcaption> | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div id="fragment8-4"> | ||
| + | <div class="p"> | ||
| + | We chose 4 positive strains to culture overnight and extracted the plasmid. After restriction enzyme digestion verification, we sent them to sequencing. | ||
| + | </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℃ for 40 min</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | <figure style="max-width:19rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/3/35/T--NKU_China--notebook-6-13-5.png"><img src="https://static.igem.org/mediawiki/2016/3/35/T--NKU_China--notebook-6-13-5.png"></a> | ||
| + | <figcaption> | ||
| + | Restriction enzyme digestion verification<br> | ||
| + | (No.1 are linearized pWH1520, No.2 are <i>luxS</i> fragments.) | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </div> | ||
| + | </div> | ||
| − | + | </div> | |
| − | + | </div> | |
| − | + | </div> | |
| − | + | <div class="accordion-fragment" id="week9"> | |
| − | </ | + | <div class="accordion-header"> |
| + | <span class="default">☞</span><span class="active">☟</span> Week9 (Jul 11–Jul 17) | ||
| + | </div> | ||
| + | <div class="accordion-content"> | ||
| + | <div class="tabs" id="tabs9"> | ||
| + | <ul> | ||
| + | <li><a href="#fragment9-1">➀</a></li> | ||
| + | <li><a href="#fragment9-2">➁</a></li> | ||
| + | <li><a href="#fragment9-3">➂</a></li> | ||
| + | </ul> | ||
| + | <div id="fragment9-1"> | ||
| + | <div class="p"> | ||
| + | The sequencing result showed there's three correct strains. So we can choose a correct strain for the following experiment. We extracted the correct plasmid pWH-<i>luxS</i> from DH5α. To prevent the plasmid from DAM&DCM methylation, we transformed it into E.coli JM110. | ||
</div> | </div> | ||
| − | |||
| − | |||
| − | |||
| − | |||
| − | + | </div> | |
| − | + | <div id="fragment9-2"> | |
| − | + | <div class="p"> | |
| − | + | Extracted the plasmid pWH -<i>luxS</i> from JM110,and dealt with it by 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℃ for 1 hour</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| + | <div id="fragment9-3"> | ||
| + | <div class="p"> | ||
| + | Transformed the plasmid into GR286 by electroporation, and selected positive clones. | ||
| + | </div> | ||
| + | <div class="flex-container"> | ||
| + | <figure style="max-width:18rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/a/aa/T--NKU_China--notebook-6-13-6.png"><img src="https://static.igem.org/mediawiki/2016/a/aa/T--NKU_China--notebook-6-13-6.png"></a> | ||
| + | <figcaption> | ||
| + | Selecting positive clones by colony PCR | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="p"> | ||
| + | The construction of supplier was complished! | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="accordion-fragment" id="week10"> | |
| − | + | <div class="accordion-header"> | |
| + | <span class="default">☞</span><span class="active">☟</span> Week10 (Jul 18–Jul 24) | ||
| + | </div> | ||
| + | <div class="accordion-content"> | ||
| + | <div class="tabs" id="tabs10"> | ||
| + | <ul> | ||
| + | <li><a href="#fragment10-1">➀</a></li> | ||
| + | <li><a href="#fragment10-2">➁</a></li> | ||
| + | </ul> | ||
| + | <div id="fragment10-1"> | ||
| + | <div class="p"> | ||
| + | Our synthetic <i>lsrACDB</i> gene came back. 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℃ overnight</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div id="fragment10-2"> | ||
| + | <div class="p"> | ||
| + | Considering the <i>lsrACDB</i> gene is a large fragment (4500bp), we used ClonExpress technique cloning the gene again to improve the efficiency of ligation. We divided the <i>lsrACDB</i> sequence into two parts and cloned them separately. Then we ligated the two segments to the plasmid pWH1520 and transformed it into DH5α. After that, we used PCR to select the positive clones. However, we didn't get a good result. | ||
| + | </div> | ||
| + | <div class="flex-container"> | ||
| + | <figure style="max-width:18rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/1/17/T--NKU_China--notebook-6-13-7.png"><img src="https://static.igem.org/mediawiki/2016/1/17/T--NKU_China--notebook-6-13-7.png"></a> | ||
| + | <figcaption> | ||
| + | Selecting positive clones by colony PCR<br> | ||
| + | (No.6 is positive control, No.1-5 are experimental groups) | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </div> | ||
| + | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | </div> | |
| − | + | <div class="accordion-fragment" id="week11"> | |
| − | + | <div class="accordion-header"> | |
| − | + | <span class="default">☞</span><span class="active">☟</span> Week11 (Jul 25–Jul 31) | |
| − | + | </div> | |
| − | + | <div class="accordion-content"> | |
| − | + | <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 ligating 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 style="max-width:18rem"> | |
| − | < | + | <a href="https://static.igem.org/mediawiki/2016/c/c2/T--NKU_China--notebook-6-13-8.png"><img src="https://static.igem.org/mediawiki/2016/c/c2/T--NKU_China--notebook-6-13-8.png"></a> |
| − | + | <figcaption> | |
| − | + | Selecting positive clones by colony PCR<br> | |
| − | + | (No.3-6 are positive clones) | |
| − | + | </figcaption> | |
| − | + | </figure> | |
| − | + | <figure style="max-width:19rem"> | |
| − | + | <a href="https://static.igem.org/mediawiki/2016/7/7a/T--NKU_China--notebook-6-13-9.png"><img src="https://static.igem.org/mediawiki/2016/7/7a/T--NKU_China--notebook-6-13-9.png"></a> | |
| + | <figcaption> | ||
| + | Restriction enzyme digestion verification<br> | ||
| + | (No.2&3 are positive results.) | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="accordion-fragment" id="week12"> | ||
| + | <div class="accordion-header"> | ||
| + | <span class="default">☞</span><span class="active">☟</span> Week12 (Aug 1–Aug 7) | ||
| + | </div> | ||
| + | <div class="accordion-content"> | ||
| + | <div class="tabs" id="tabs12"> | ||
| + | <ul> | ||
| + | <li><a href="#fragment12-1">➀</a></li> | ||
| + | <li><a href="#fragment12-2">➁</a></li> | ||
| + | </ul> | ||
| + | <div id="fragment12-1"> | ||
| + | <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". | ||
</div> | </div> | ||
| − | <div id=" | + | <div class="flex-container"> |
| − | <img src="https://static.igem.org/mediawiki/2016/ | + | <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 style="max-width:40rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/a/ad/T--NKU_China--notebook-6-13-10.png"><img src="https://static.igem.org/mediawiki/2016/a/ad/T--NKU_China--notebook-6-13-10.png"></a> | ||
| + | <figcaption> | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | <figure style="max-width:25rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/0/00/T--NKU_China--notebook-6-13-11.png"><img src="https://static.igem.org/mediawiki/2016/0/00/T--NKU_China--notebook-6-13-11.png"></a> | ||
| + | <figcaption> | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | <figure style="max-width:25rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/a/a9/T--NKU_China--notebook-6-13-12.png"><img src="https://static.igem.org/mediawiki/2016/a/a9/T--NKU_China--notebook-6-13-12.png"></a> | ||
| + | <figcaption> | ||
| + | </figcaption> | ||
| + | </figure> | ||
| + | <figure style="max-width:25rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/6/6b/T--NKU_China--notebook-6-13-13.png"><img src="https://static.igem.org/mediawiki/2016/6/6b/T--NKU_China--notebook-6-13-13.png"></a> | ||
| + | <figcaption> | ||
| + | </figcaption> | ||
| + | </figure> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div id="fragment12-2"> | |
| − | + | <div class="p"> | |
| − | + | Cultured media of our supplier was tested for the presence of AI-2 by inducing luminescence in <i>Vibrio harveyi</i> reporter strain BB170. | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</div> | </div> | ||
| − | <div | + | <div class="flex-container"> |
| − | < | + | <figure style="max-width:30rem"> |
| − | + | <a href="https://static.igem.org/mediawiki/2016/a/a4/T--NKU_China--notebook-6-13-14.png"><img src="https://static.igem.org/mediawiki/2016/a/a4/T--NKU_China--notebook-6-13-14.png"></a> | |
| − | < | + | <figcaption> |
| − | + | </figcaption> | |
| − | + | </figure> | |
| − | + | <figure style="max-width:40rem"> | |
| − | < | + | <a href="https://static.igem.org/mediawiki/2016/c/c5/T--NKU_China--notebook-6-13-15.png"><img src="https://static.igem.org/mediawiki/2016/c/c5/T--NKU_China--notebook-6-13-15.png"></a> |
| − | + | <figcaption> | |
| − | </ | + | </figcaption> |
| + | </figure> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | </div> | |
| − | + | </div> | |
| − | + | </div> | |
| − | + | <div class="accordion-fragment" id="week13"> | |
| − | + | <div class="accordion-header"> | |
| − | + | <span class="default">☞</span><span class="active">☟</span> Week13 (Aug 8–Aug 14) | |
| − | + | </div> | |
| − | + | <div class="accordion-content"> | |
| − | </ | + | <div class="tabs" id="tabs12"> |
| + | <ul> | ||
| + | <li><a href="#fragment12-1">➀</a></li> | ||
| + | <li><a href="#fragment12-2">➁</a></li> | ||
| + | </ul> | ||
| + | <div id="fragment12-1"> | ||
| + | <div class="p"> | ||
| + | For our consumer, we should also clone the <i>lsrK</i> and <i>lsrFG</i> gene for phosphorylating and degrading AI-2. We used ClonExpress technique cloning the two genes and ligated them to plasmid pHT-01 successfully. | ||
</div> | </div> | ||
| − | <div | + | <div class="flex-container"> |
| − | <img src="https://static.igem.org/mediawiki/2016/ | + | <figure style="max-width:19rem"> |
| − | < | + | <a href="https://static.igem.org/mediawiki/2016/c/c5/T--NKU_China--notebook-6-13-16.png"><img src="https://static.igem.org/mediawiki/2016/c/c5/T--NKU_China--notebook-6-13-16.png"></a> |
| − | + | <figcaption> | |
| − | + | Restriction enzyme digestion verification<br> | |
| − | + | (No.1&3&4 are positive results. ) | |
| − | + | </figcaption> | |
| − | </ | + | </figure> |
| − | + | </div> | |
| − | </ | + | </div> |
| + | <div id="fragment12-2"> | ||
| + | <div class="p"> | ||
| + | We cocultured the supplier with BB170 and tested the fluoresent intensity to explore the function of supplier. (negative result) | ||
| + | </div> | ||
| + | <div class="flex-container"> | ||
| + | <figure style="max-width:25rem"> | ||
| + | <a href="https://static.igem.org/mediawiki/2016/f/f1/T--NKU_China--notebook-6-13-17.png"><img src="https://static.igem.org/mediawiki/2016/f/f1/T--NKU_China--notebook-6-13-17.png"></a> | ||
| + | <figcaption> | ||
| + | </figcaption> | ||
| + | </figure> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | </div> | + | </div> |
| − | </main> | + | <div class="accordion-fragment" id="week14"> |
| + | <div class="accordion-header"> | ||
| + | <span class="default">☞</span><span class="active">☟</span> Week14 (Aug 15–Aug 21) | ||
| + | </div> | ||
| + | <div class="accordion-content"> | ||
| + | |||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </main> | ||
</body> | </body> | ||
</html> | </html> | ||
Revision as of 05:35, 26 August 2016
Laboratory Notes
☞☟ Week1 (May 16–May 22)
In order to make sure our "consumer" efficient, 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.
Transformed pKSU-ΔluxS into GR286, and selected out positive clones.
| 2× Taq Master Mix | 10μL |
| pKSU-F | 1μL |
| pKSU-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| 94℃ | 10 min | |
| 94℃ | 30 sec | |
| 58℃ | 30 sec | 30 cycles |
| 72℃ | 1 min 30 sec | |
| 72℃ | 10 min | |
| 16℃ | ∞ |
The transformants were cultured at 42℃ 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 |
| 94℃ | 10 min | |
| 94℃ | 30 sec | |
| 56℃ | 30 sec | 30 cycles |
| 72℃ | 2 min | |
| 72℃ | 10 min | |
| 16℃ | ∞ |
(No.1-4 are single-crossover strains, No.5 is positive control.)
☞☟ Week2 (May 23–May 29)
The single-crossover strains were then cultured at LB medium and passaged every 12 hours for 4 generation.
Cultured the last generation at 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 |
| 94℃ | 10 min | |
| 94℃ | 30 sec | |
| 56℃ | 30 sec | 30 cycles |
| 72℃ | 2 min | |
| 72℃ | 10 min | |
| 16℃ | ∞ |
(No.1-5 are experimental groups, No.6 is wild GR286. The result showed that we failed to get the double-crossover clones.)
☞☟ Week3 (May 30–Jun 05)
We cultured transformants at 42℃ with chloramphenicol again and selected the single-crossover clones successfully.
| 2× Taq Master Mix | 10μL |
| luxS-up-F | 1μL |
| luxS-dn-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| 94℃ | 10 min | |
| 94℃ | 30 sec | |
| 56℃ | 30 sec | 30 cycles |
| 72℃ | 2 min | |
| 72℃ | 10 min | |
| 16℃ | ∞ |
(No.1&2&4 are single-crossover strains,No.5 is positive control. )
The single-crossover strains were then cultured at LB medium and passaged every 12 hours for 4 generations.
Cultured the last generation at medium with 5-fluorouracil to select double-crossover clones. We finally got our aimed strain—GR286ΔluxS.
| 2× Taq Master Mix | 10μL |
| luxS-up-F | 1μL |
| luxS-dn-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| 94℃ | 10 min | |
| 94℃ | 30 sec | |
| 56℃ | 30 sec | 30 cycles |
| 72℃ | 2 min | |
| 72℃ | 10 min | |
| 16℃ | ∞ |
(The No.4 is the aimed strain
☞☟ Week4 (Jun 06–Jun 12)
Cultured the GR286ΔluxS strain and made it competence for future use.
Cloned the lsrACDB gene from Bacillus thuringiensis and ligated it to T-vector.
| 2× Taq Master Mix | 25μL |
| lsrACDB-F | 2μL |
| lsrACDB-R | 2μL |
| Bacterium solution | 2μL |
| ddH2O | 19μL |
| Total | 50μL |
| 94℃ | 10 min | |
| 94℃ | 30 sec | |
| 57℃ | 30 sec | 30 cycles |
| 72℃ | 4 min 30 sec | |
| 72℃ | 10 min | |
| 16℃ | ∞ |
| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| pMD19 T-Simple Vector | 1μL |
| lsrACDB | 3μL |
| ddH2O | 13μL |
| Total | 20μL |
| Reaction condition: 16℃ overnight | |
Transformed the T-lsrACDB into DH5α and coated plate, and then selected positive clones by colony PCR.
| 2× Taq Master Mix | 10μL |
| M13F | 1μL |
| M13R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| 94℃ | 10 min | |
| 94℃ | 30 sec | |
| 59℃ | 30 sec | 30 cycles |
| 72℃ | 4 min 30 sec | |
| 72℃ | 10 min | |
| 16℃ | ∞ |
(No. 3&4 are positive results)
After restriction enzyme digestion verification, we sent them to sequencing. Unfortunately, the sequencing result showed some mutations in cloning gene.
| 10× FastDigest Buffer | 2μL |
| BamH Ⅰ | 1μL |
| T-lsrACDB | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| Reaction condition: 37℃ for 40 min | |
(No.1 are lsrACDB fragement, No.2 are linearized T-vector.)
We repeated the process of gene cloning 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.
We cloned a strong promoter C2 from former kit and cloned luxS gene from GR286.
| 2× Taq Master Mix | 25μL |
| C2-F | 2μL |
| C2-R | 2μL |
| p-C2 | 2μL |
| ddH2O | 19μL |
| Total | 50μL |
| 94℃ | 10 min | |
| 94℃ | 30 sec | |
| 58℃ | 15 sec | 30 cycles |
| 72℃ | 30 sec | |
| 72℃ | 10 min | |
| 16℃ | ∞ |
| 2× Taq Master Mix | 25μL |
| luxS-F | 2μL |
| luxS-R | 2μL |
| Bacterium solution | 1μL |
| GR286 | 2μL |
| ddH2O | 19μL |
| Total | 50μL |
| 94℃ | 10 min | |
| 94℃ | 30 sec | |
| 59℃ | 30 sec | 30 cycles |
| 72℃ | 30 sec | |
| 72℃ | 10 min | |
| 16℃ | ∞ |
(NO.1&2 are C2, No.3&4 are luxS)
Fuse the two segments together by fusion PCR, and ligated it into T-vector. Then, transformed the vector into DH5α.
| 2× Taq Master Mix | 25μL |
| C2-F | 2μL |
| luxS-R | 2μL |
| C2 | 2μL |
| luxS | 2μL |
| ddH2O | 17μL |
| Total | 50μL |
| 94℃ | 10 min | |
| 94℃ | 30 sec | |
| 59℃ | 30 sec | 30 cycles |
| 72℃ | 40 sec | |
| 72℃ | 10 min | |
| 16℃ | ∞ |
| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| pMD19 T-Simple Vector | 1μL |
| C2-luxS | 4μL |
| ddH2O | 12μL |
| Total | 20μL |
| Reaction condition: 16℃ overnight | |
Selected the positive clones by colony PCR.
| 2× Taq Master Mix | 10μL |
| M13-F | 1μL |
| M13-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| 94℃ | 10 min | |
| 94℃ | 30 sec | |
| 59℃ | 30 sec | 30 cycles |
| 72℃ | 40 sec | |
| 72℃ | 10 min | |
| 16℃ | ∞ |
We chose 4 positive strains to culture overnight and extracted the plasmid. After restriction enzyme digestion verification, we sent them to sequencing.
| 10× FastDigest Buffer | 2μL |
| BamH Ⅰ | 1μL |
| T-lsrACDB | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| Reaction condition: 37℃ for 40 min | |
(No.1 are linearized T-vector, No.2 are C2-luxS fragment.)
☞☟ Week6 (Jun 20–Jun 26)
The sequencing result showed there's a correct strain. So we can use the strain for the following experiment. We obtained the correct plasmid T-C2-luxS from DH5α. Then we got the fragment C2-luxS by digestion and gel extraction.
| 10× FastDigest Buffer | 4μL |
| BamH Ⅰ | 2μL |
| T-C2-luxS | 25μL |
| ddH2O | 9μL |
| Total | 20μL |
| Reaction condition: 37℃ for 40 min | |
Ligated the C2-luxS to linearized plasmid pWH1520, and transformed it 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: 16℃ overnight | |
Extracted the plasmid pWH-C2-luxS from DH5α. To prevent the plasmid from DAM&DCM methylation, we transformed it into E.coli JM110.
Extracted the plasmid pWH-C2-luxS from JM110,and dealt with it by 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: 37℃ for 1 hour | |
Transformed the plasmid into GR286 by electroporation.[Failed]
(No.1 is positive control, No.2-6 are experimental groups. The result showed that we failed to transformed the plasmid pWH-C2-luxS into GR286)
☞☟ Week7 (Jun 27–Jul 03)
This week, we tried to use different voltages to transform the plasmid. Sadly, all of these tries got bad results.
We considered whether the luxS gene is a little toxic for GR286, and the bacteria tends to refuse the gene when we added a strong promoter in front of it. So, we planned to use induced expression to reconstruction our expression vector.
The plasmid pWH1520 contains the strong xylA promoter originating, and transcription from this promoter is xylose inducible. So, 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)
We cloned luxS gene 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 |
| 94℃ | 10 min | |
| 94℃ | 30 sec | |
| 58℃ | 30 sec | 30 cycles |
| 72℃ | 40 sec | |
| 72℃ | 10 min | |
| 16℃ | ∞ |
Purified the luxS fragment by gel extraction, and ligated it into linearized pWH1520. Then, transformed the vector into DH5α.
| 10× FastDigest Buffer | 4μL |
| BamH Ⅰ | 2μL |
| pWH1520 | 25μL |
| ddH2O | 9μL |
| Total | 40μL |
| Reaction condition: 37℃ for 40 min | |
| 10× DNA Ligase Buffer | 2μL |
| T4 DNA Ligase | 1μL |
| linearized pWH1520 | 1μL |
| luxS | 3μL |
| ddH2O | 13μL |
| Total | 20μL |
| Reaction condition: 16℃ overnight | |
Selected the positive clones by colony PCR.
| 2× Taq Master Mix | 10μL |
| pWH-F | 1μL |
| pWH-R | 1μL |
| Bacterium solution | 1μL |
| ddH2O | 7μL |
| Total | 20μL |
| 94℃ | 10 min | |
| 94℃ | 30 sec | |
| 58℃ | 30 sec | 30 cycles |
| 72℃ | 40 sec | |
| 72℃ | 10 min | |
| 16℃ | ∞ |
We chose 4 positive strains to culture overnight and extracted the plasmid. After restriction enzyme digestion verification, we sent them to sequencing.
| 10× FastDigest Buffer | 2μL |
| BamH Ⅰ | 1μL |
| pWH-luxs | 10μL |
| ddH2O | 7μL |
| Total | 20μL |
| Reaction condition: 37℃ for 40 min | |
(No.1 are linearized pWH1520, No.2 are luxS fragments.)
☞☟ Week9 (Jul 11–Jul 17)
The sequencing result showed there's three correct strains. So we can choose a correct strain for the following experiment. We extracted the correct plasmid pWH-luxS from DH5α. To prevent the plasmid from DAM&DCM methylation, we transformed it into E.coli JM110.
Extracted the plasmid pWH -luxS from JM110,and dealt with it by 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: 37℃ for 1 hour | |
Transformed the plasmid into GR286 by electroporation, and selected positive clones.
The construction of supplier was complished!
☞☟ Week10 (Jul 18–Jul 24)
Our synthetic lsrACDB gene came back. 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: 16℃ overnight | |
Considering the lsrACDB gene is a large fragment (4500bp), we used ClonExpress technique cloning the gene again to improve the efficiency of ligation. We divided the lsrACDB sequence into two parts and cloned them separately. Then we ligated the two segments to the plasmid pWH1520 and transformed it into DH5α. After that, we used PCR to select the positive clones. However, we didn't get a good result.
(No.6 is positive control, No.1-5 are experimental groups)
☞☟ 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 ligating our lsrACDB gene. It's encouraging that we succeeded to ligate the lsrACDB gene to the plasmid pHT-01.
(No.3-6 are positive clones)
(No.2&3 are positive results.)
☞☟ Week12 (Aug 1–Aug 7)
Since we have 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 in Vibrio harveyi reporter strain BB170.
☞☟ Week13 (Aug 8–Aug 14)
For our consumer, we should also clone the lsrK and lsrFG gene for phosphorylating and degrading AI-2. We used ClonExpress technique cloning the two genes and ligated them to plasmid pHT-01 successfully.
(No.1&3&4 are positive results. )
We cocultured the supplier with BB170 and tested the fluoresent intensity to explore the function of supplier. (negative result)
☞☟ Week14 (Aug 15–Aug 21)
