|
|
Line 56: |
Line 56: |
| </div> | | </div> |
| <section class="popout protocols"> | | <section class="popout protocols"> |
− | <div class="collapsible" id="Agarplates"> Basic experiments</div> | + | <div class="collapsible" id="Agarplates"> Modification of chassis </div> |
| <div class="collapsibleContent"> | | <div class="collapsibleContent"> |
| <p class="MsoNormal"><span style="font-size:14.0pt; ">(1)Lysogeny Broth (LB) </span></p> | | <p class="MsoNormal"><span style="font-size:14.0pt; ">(1)Lysogeny Broth (LB) </span></p> |
Line 116: |
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| | | |
| <section class="popout protocols"> | | <section class="popout protocols"> |
− | <div class="collapsible" id="Competence">Plasmid Extraction</div> | + | <div class="collapsible" id="Competence">Verification of acid tolerant factors</div> |
| <div class="collapsibleContent"> | | <div class="collapsibleContent"> |
| <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Add ethanol (96-100%) to Buffer PW before use, check bottle tag for the adding volume. </span></p> | | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Add ethanol (96-100%) to Buffer PW before use, check bottle tag for the adding volume. </span></p> |
Line 137: |
Line 137: |
| | | |
| <section class="popout protocols"> | | <section class="popout protocols"> |
− | <div class="collapsible"> Bacterial Genome Extraction</div> | + | <div class="collapsible"> Verification of Toehold Switches</div> |
| <div class="collapsibleContent"> | | <div class="collapsibleContent"> |
| <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Ensure that Buffer GD and Buffer PW have been prepared with appropriate volume of ethanol (96-100%) as indicated on the bottle and shake thoroughly. </span></p> | | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Ensure that Buffer GD and Buffer PW have been prepared with appropriate volume of ethanol (96-100%) as indicated on the bottle and shake thoroughly. </span></p> |
Line 158: |
Line 158: |
| | | |
| <section class="popout protocols"> | | <section class="popout protocols"> |
− | <div class="collapsible" id="GenDNA">Primer Design</div> | + | <div class="collapsible" id="GenDNA">Improvement of T7 promoters</div> |
| <div class="collapsibleContent"> | | <div class="collapsibleContent"> |
| <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">All Primers used in experiments meet basic requirements below:</span></p> | | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">All Primers used in experiments meet basic requirements below:</span></p> |
Line 187: |
Line 187: |
| | | |
| <section class="popout protocols"> | | <section class="popout protocols"> |
− | <div class="collapsible" id="plasmidDNA">Polymerase Chain Reaction (PCR)</div> | + | <div class="collapsible" id="plasmidDNA">Construction of VerProS pool</div> |
| <div class="collapsibleContent"> | | <div class="collapsibleContent"> |
| <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Primers preparation: Carefully hold the lyophilized primer powder and centrifuge at 12000rmp for 2min. Dilute to 20 </span><span style="font-family:'微软雅黑',sans-serif; ">μM with ddH<sub>2</sub>O.</span></p> | | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Primers preparation: Carefully hold the lyophilized primer powder and centrifuge at 12000rmp for 2min. Dilute to 20 </span><span style="font-family:'微软雅黑',sans-serif; ">μM with ddH<sub>2</sub>O.</span></p> |
Line 502: |
Line 502: |
| | | |
| <section class="popout protocols"> | | <section class="popout protocols"> |
− | <div class="collapsible">DNA Agarose Gel Electrophoresis </div> | + | <div class="collapsible">Construction of working part </div> |
| <div class="collapsibleContent"> | | <div class="collapsibleContent"> |
| <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(1) Preparation of 1% gel</span></p> | | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(1) Preparation of 1% gel</span></p> |
Line 543: |
Line 543: |
| | | |
| <section class="popout protocols"> | | <section class="popout protocols"> |
− | <div class="collapsible"> DNA Fragments Recovery</div> | + | <div class="collapsible"> Test of VerProS system</div> |
| <div class="collapsibleContent"> | | <div class="collapsibleContent"> |
| <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(1) Gel Extraction</span></strong></p> | | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(1) Gel Extraction</span></strong></p> |
Line 577: |
Line 577: |
| </section> | | </section> |
| | | |
− | <section class="popout protocols">
| + | </div> |
− | <div class="collapsible">Gibson Assembly</div>
| + | |
− | <div class="collapsibleContent">
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(1) Prepare the assay according to the chart.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(2) Run at 50</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 1h.</span><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; "> </span></strong></p>
| + | |
− | </div>
| + | |
− | </section>
| + | |
− | | + | |
− | <section class="popout protocols">
| + | |
− | <div class="collapsible">Restriction Enzyme Digest</div>
| + | |
− | <div class="collapsibleContent">
| + | |
− | <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="568" style="border-collapse:collapse;border:none;">
| + | |
− | <tr>
| + | |
− | <td width="307" colspan="2" valign="top" style="width:230.55pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10</span><span style="font-family:'微软雅黑',sans-serif; ">μl assay for single digestion</span></p></td>
| + | |
− | <td width="261" colspan="2" valign="top" style="width:195.4pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">50</span><span style="font-family:'微软雅黑',sans-serif; ">μl assay for double digestion</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="176" valign="top" style="width:132.2pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">NEB CutSmart<sup>®</sup> Buffer</span></p></td>
| + | |
− | <td width="131" valign="top" style="width:98.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
| + | |
− | <td width="147" valign="top" style="width:110.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">NEB CutSmart<sup>®</sup> Buffer</span></p></td>
| + | |
− | <td width="114" valign="top" style="width:85.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">5</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr style="height:7.55pt;">
| + | |
− | <td width="176" rowspan="2" valign="top" style="width:132.2pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;height:7.55pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Restriction Enzyme</span></p></td>
| + | |
− | <td width="131" rowspan="2" valign="top" style="width:98.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:7.55pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">0.5</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
| + | |
− | <td width="147" valign="top" style="width:110.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:7.55pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Restriction Enzyme 1</span></p></td>
| + | |
− | <td width="114" valign="top" style="width:85.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:7.55pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr style="height:7.55pt;">
| + | |
− | <td width="147" valign="top" style="width:110.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:7.55pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Restriction Enzyme 2</span></p></td>
| + | |
− | <td width="114" valign="top" style="width:85.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:7.55pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="176" valign="top" style="width:132.2pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Template</span></p></td>
| + | |
− | <td width="131" valign="top" style="width:98.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Depends on original concentration</span></p></td>
| + | |
− | <td width="147" valign="top" style="width:110.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Template</span></p></td>
| + | |
− | <td width="114" valign="top" style="width:85.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal" style="margin-left:10.5pt;text-indent:-10.5pt;"><span style="font-family:'微软雅黑',sans-serif; ">Depends on original concentration</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="176" valign="top" style="width:132.2pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span></p></td>
| + | |
− | <td width="131" valign="top" style="width:98.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | <td width="147" valign="top" style="width:110.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span></p></td>
| + | |
− | <td width="114" valign="top" style="width:85.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="176" valign="top" style="width:132.2pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Final concentration of template</span></p></td>
| + | |
− | <td width="131" valign="top" style="width:98.3pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">500ng/</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
| + | |
− | <td width="147" valign="top" style="width:110.05pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Final concentration of template</span></p></td>
| + | |
− | <td width="114" valign="top" style="width:85.35pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">3000ng/</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="568" colspan="4" valign="top" style="width:425.9pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Incubate at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for several hours.</span></p></td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | </div>
| + | |
− | </section>
| + | |
− | | + | |
− | <section class="popout protocols">
| + | |
− | <div class="collapsible">Annealing stitching</div>
| + | |
− | <div class="collapsibleContent">
| + | |
− | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(1) Annealing</span></strong></p>
| + | |
− | <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="342" style="border-collapse:collapse;border:none;">
| + | |
− | <tr>
| + | |
− | <td width="158" colspan="2" valign="top" style="width:118.4pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">20</span><span style="font-family:'微软雅黑',sans-serif; ">μl assay</span></p></td>
| + | |
− | <td width="184" colspan="3" valign="top" style="width:138.25pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Procedure</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="110" valign="top" style="width:82.75pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10</span><span style="font-family:'微软雅黑',sans-serif; ">×PNK buffer</span></p></td>
| + | |
− | <td width="48" valign="top" style="width:35.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
| + | |
− | <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Temperature</span></p></td>
| + | |
− | <td width="46" valign="top" style="width:34.7pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Time</span></p></td>
| + | |
− | <td width="46" valign="top" style="width:34.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Cycle</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="110" valign="top" style="width:82.75pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Forward primer</span></p></td>
| + | |
− | <td width="48" valign="top" style="width:35.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
| + | |
− | <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">95</span><span style="font-family:'微软雅黑',sans-serif; ">℃ </span></p></td>
| + | |
− | <td width="46" valign="top" style="width:34.7pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">3min</span></p></td>
| + | |
− | <td width="46" valign="top" style="width:34.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="110" valign="top" style="width:82.75pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Reverse primer</span></p></td>
| + | |
− | <td width="48" valign="top" style="width:35.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
| + | |
− | <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">94</span><span style="font-family:'微软雅黑',sans-serif; ">℃ </span></p></td>
| + | |
− | <td width="46" valign="top" style="width:34.7pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1min</span></p></td>
| + | |
− | <td width="46" rowspan="2" valign="top" style="width:34.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">57</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="110" valign="top" style="width:82.75pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span></p></td>
| + | |
− | <td width="48" valign="top" style="width:35.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">16</span><span style="font-family:'微软雅黑',sans-serif; ">μl</span></p></td>
| + | |
− | <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">-1</span><span style="font-family:'微软雅黑',sans-serif; ">℃/Cycle</span></p></td>
| + | |
− | <td width="46" valign="top" style="width:34.7pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="110" valign="top" style="width:82.75pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | <td width="48" valign="top" style="width:35.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">12</span><span style="font-family:'微软雅黑',sans-serif; ">℃ </span></p></td>
| + | |
− | <td width="46" valign="top" style="width:34.7pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">∞ </span></p></td>
| + | |
− | <td width="46" valign="top" style="width:34.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">× </span></p></td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(2) Phosphorylation</span></strong></p>
| + | |
− | <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="179" style="border-collapse:collapse;border:none;">
| + | |
− | <tr>
| + | |
− | <td width="179" colspan="2" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">20</span><span style="font-family:'微软雅黑',sans-serif; ">μl assay</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="132" valign="top" style="width:99.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing product</span></p></td>
| + | |
− | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="132" valign="top" style="width:99.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10</span><span style="font-family:'微软雅黑',sans-serif; ">×PNK buffer</span></p></td>
| + | |
− | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="132" valign="top" style="width:99.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10mM ATP</span></p></td>
| + | |
− | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="132" valign="top" style="width:99.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">T4 PNK enzyme</span></p></td>
| + | |
− | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="132" valign="top" style="width:99.0pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span></p></td>
| + | |
− | <td width="47" valign="top" style="width:35.45pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">14</span></p></td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Incubate at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 45min.</span></p>
| + | |
− | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(3) T4 Ligation</span></strong></p>
| + | |
− | <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="387" style="border-collapse:collapse;border:none;">
| + | |
− | <tr>
| + | |
− | <td width="387" colspan="2" valign="top" style="width:290.35pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10</span><span style="font-family:'微软雅黑',sans-serif; ">μl assay</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="208" valign="top" style="width:155.7pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Vector plasmid</span></p></td>
| + | |
− | <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">depends on concentration</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="208" valign="top" style="width:155.7pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing product of promotor</span></p></td>
| + | |
− | <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">μL</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="208" valign="top" style="width:155.7pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing product of trigger</span></p></td>
| + | |
− | <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">μL</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="208" valign="top" style="width:155.7pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Annealing product of terminator</span></p></td>
| + | |
− | <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">μL</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="208" valign="top" style="width:155.7pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">T4 ligase</span></p></td>
| + | |
− | <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">μL</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="208" valign="top" style="width:155.7pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10</span><span style="font-family:'微软雅黑',sans-serif; ">×T4 PNK buffer</span></p></td>
| + | |
− | <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">μL</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="208" valign="top" style="width:155.7pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH<sub>2</sub>O</span></p></td>
| + | |
− | <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">*The final concentration of vector in the assay is 100ng/</span><span style="font-family:'微软雅黑',sans-serif; ">μL.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Incubate at 16</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 12h.</span></p>
| + | |
− | </div>
| + | |
− | </section>
| + | |
− | | + | |
− | <section class="popout protocols">
| + | |
− | <div class="collapsible">Golden Gate </div>
| + | |
− | <div class="collapsibleContent">
| + | |
− | <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="549" style="border-collapse:collapse;border:none;">
| + | |
− | <tr>
| + | |
− | <td width="359" colspan="2" valign="top" style="width:269.1pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">20</span><span style="font-family:'微软雅黑',sans-serif; ">μl assay</span></p></td>
| + | |
− | <td width="190" colspan="3" valign="top" style="width:142.4pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Procedure</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Plasmid ccdB</span></p></td>
| + | |
− | <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">depends on concentration</span></p></td>
| + | |
− | <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Temperature</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | <td width="52" valign="top" style="width:38.85pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Time</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | <td width="46" valign="top" style="width:34.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Cycle</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Plasmid with Trigger A mix</span></p></td>
| + | |
− | <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(As above)</span></p></td>
| + | |
− | <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">37</span><span style="font-family:'微软雅黑',sans-serif; ">℃</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | <td width="52" valign="top" style="width:38.85pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10min</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | <td width="46" rowspan="2" valign="top" style="width:34.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">50</span><span style="font-family:'微软雅黑',sans-serif; ">×</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Plasmid with Trigger B mix</span></p></td>
| + | |
− | <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(As above)</span></p></td>
| + | |
− | <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">16</span><span style="font-family:'微软雅黑',sans-serif; ">℃</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | <td width="52" valign="top" style="width:38.85pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">7min</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Plasmid with Trigger C mix</span></p></td>
| + | |
− | <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(As above)</span></p></td>
| + | |
− | <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">50</span><span style="font-family:'微软雅黑',sans-serif; ">℃</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | <td width="52" valign="top" style="width:38.85pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">10min</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | <td width="46" valign="top" style="width:34.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">×</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Plasmid with Trigger D mix</span></p></td>
| + | |
− | <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(As above)</span></p></td>
| + | |
− | <td width="92" valign="top" style="width:68.8pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">12</span><span style="font-family:'微软雅黑',sans-serif; ">℃</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | <td width="52" valign="top" style="width:38.85pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">∞</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | <td width="46" valign="top" style="width:34.75pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span><span style="font-family:'微软雅黑',sans-serif; ">×</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Bsal enzyme</span></p></td>
| + | |
− | <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span></p></td>
| + | |
− | <td style="border:none;padding:0cm 0cm 0cm 0cm;" width="190" colspan="3"><p class="MsoNormal"> </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">T4 DNA ligase </span></p></td>
| + | |
− | <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">1</span></p></td>
| + | |
− | <td style="border:none;padding:0cm 0cm 0cm 0cm;" width="190" colspan="3"><p class="MsoNormal"> </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">T4 DNA ligase buffer</span></p></td>
| + | |
− | <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">2</span></p></td>
| + | |
− | <td style="border:none;padding:0cm 0cm 0cm 0cm;" width="190" colspan="3"><p class="MsoNormal"> </td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="179" valign="top" style="width:134.45pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">ddH2O</span></p></td>
| + | |
− | <td width="180" valign="top" style="width:134.65pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; "> </span></strong></p></td>
| + | |
− | <td style="border:none;padding:0cm 0cm 0cm 0cm;" width="190" colspan="3"><p class="MsoNormal"> </td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">*The final concentration of each plasmid in the assay is 100ng/</span><span style="font-family:'微软雅黑',sans-serif; ">μL.</span></p>
| + | |
− | </div>
| + | |
− | </section>
| + | |
− | | + | |
− | <section class="popout protocols">
| + | |
− | <div class="collapsible" id="GelElectro">Preparation of Competent Cells</div>
| + | |
− | <div class="collapsibleContent">
| + | |
− | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(1) For Chemical Transformation</span></strong><span style="font-family:'微软雅黑',sans-serif; "> </span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><1> Inoculate the bacteria by streaking and culture for 12h.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><2> Pick a single colony and dissolve it in in 2mL LB medium. Culture overnight at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220rpm. </span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><3> Precool the centrifuge to 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃. Transfer appropriate amounts of bacteria solution to fresh medium in the proportion of 1:100. Culture at 220rpm for some time until OD<sub>600</sub> is between 0.4 and 0.6.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><4> Transfer the bacterial cells to cold centrifuge tubes and ice them for 10 minutes to reach 0</span><span style="font-family:'微软雅黑',sans-serif; ">℃. Rotate the tubes sometimes to ensure complete cooling. </span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><5> Centrifuge at 4000 rpm and 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 15min. Discard the supernatant and centrifuge again at 4000rpm and 4℃ for 2 minutes to thoroughly remove the medium.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><6> Resuspend cells with 30mL cold and sterilized MgCl<sub>2</sub>-CaCl<sub>2</sub> solution mildly.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><7> Centrifuge at 4000rpm and 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃</span><span style="font-family:'微软雅黑',sans-serif; "> for 15min. Discard the supernatant and centrifuge again at 4000rpm and 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 2 minutes.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><8> Resuspend cells with 800</span><span style="font-family:'微软雅黑',sans-serif; ">μL 0.1M cold CaCl<sub>2</sub> mildly. Add 200μL 0.2M 60% glycerol and mix them thoroughly. </span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><9> Distribute the bacteria solution to tubes every 100</span><span style="font-family:'微软雅黑',sans-serif; ">μL.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><10> Froze in liquid nitrogen for 1min.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><11> Store at -80</span><span style="font-family:'微软雅黑',sans-serif; ">℃.</span></p>
| + | |
− | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(2) For Electrotransformation</span></strong></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><1> Inoculate the bacteria by streaking and culture for 12h.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><2> Pick a single colony and dissolve it in in 10mL LB medium. Culture for 8-12h at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220rpm. </span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><3> Precool the centrifuge to 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃. Transfer appropriate amounts of bacteria culture to fresh medium in the proportion of 1:100. Culture at 37℃ and 220rpm until OD<sub>600</sub> is between 0.4 and 0.6. </span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><4> Transfer the bacterial cells to cold centrifuge tubes and ice them for 20-30min. Rotate the tubes sometimes to ensure complete cooling. </span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><5> Centrifuge at 4000 rpm and 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 15min. Discard the supernatant and centrifuge again at 4000rpm and 4℃ for 2min to thoroughly remove the medium. </span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><6> Suspend and wash bacterial cells with 25mL cold ultrapure water every two tubes. Centrifuge at 4000rpm and 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 20min. Discard the supernatant. </span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><7> Resuspend cells with 25mL cold 10% glycerol. Centrifuge at 4000 and 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃for 20min. Discard the supernatant.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><8> Repeat Step <7>.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><9> Resuspend cells with 1mL cold 10% glycerol. Dilute the solution by 1:200. Measure the concentration and distribute to small tubes every 100</span><span style="font-family:'微软雅黑',sans-serif; ">μL when OD<sub>600</sub> is between 0.5 and 0.7.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><10> Froze in liquid nitrogen for 1min.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><11> Store at -80</span><span style="font-family:'微软雅黑',sans-serif; ">℃.</span></p>
| + | |
− | </div>
| + | |
− | </section>
| + | |
− | | + | |
− | <section class="popout protocols">
| + | |
− | <div class="collapsible" id="Transformation">Transformation</div>
| + | |
− | <div class="collapsibleContent">
| + | |
− | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(1) Chemical Method</span></strong></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><1> Take competent cells out of -80</span><span style="font-family:'微软雅黑',sans-serif; ">℃ refrigerator and set on ice for 5-10min to melt them.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><2> Add plasmids to competent cells solution and flick the tube slightly to mix them up. </span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><3> Set still on ice for 30min without any vibration.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><4> Heat shock at 42</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 90s.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><5> Set back on ice immediately and sit for 2-3min.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><6> Add 800</span><span style="font-family:'微软雅黑',sans-serif; ">μl SOC medium (preheated to 42℃).</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><7> Culture at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220rpm for 45-60min.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><8> Centrifuge at 7000rpm for 3min. Leave 100</span><span style="font-family:'微软雅黑',sans-serif; ">μl </span><span style="font-family:'微软雅黑',sans-serif; ">supernatant and resuspend the cells.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><9> Spread the bacteria solution on a plate with antibiotic.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><10> Culture at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 12h.</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p>
| + | |
− | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(2) Electroporation</span></strong></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><1> Take competent cells out of -80</span><span style="font-family:'微软雅黑',sans-serif; ">℃ refrigerator and unfreeze them for 5-10min on ice.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><2> Add 100-200 ng plasmids to 100</span><span style="font-family:'微软雅黑',sans-serif; ">μL competent cells solution and flick the tube slightly to mix them up. Set still on ice for 1 min. </span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><3> Suck out all solution and transfer to a prechilled cuvette. Set still on ice for 5 min.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><4> Set the cuvette size (2 mm) and voltage (2.5 kV) on the instrument. Insert the cuvette and deliver the pulse.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><5> Add 1 mL SOC medium to the cuvette and mix evenly. Transfer all solution to a centrifuge tube.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><6> Culture at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220rpm for 45-60min.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><7> Centrifuge at 7000rpm for 3min. Leave 100</span><span style="font-family:'微软雅黑',sans-serif; ">μl </span><span style="font-family:'微软雅黑',sans-serif; ">supernatant and resuspend the cells.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><8> Spread the bacteria solution on a plate with antibiotic.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><9> Culture at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 12h.</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p>
| + | |
− | </div></section>
| + | |
− | | + | |
− | <section class="popout protocols">
| + | |
− | <div class="collapsible"> Plasmids Discard</div>
| + | |
− | <div class="collapsibleContent">
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(1) Pick single colonies from the plate and inoculate in LB liquid medium without antibiotics. Culture at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220rpm for 12h.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(2) Dilute the bacteria solution by 10<sup>4</sup>, 10<sup>5 </sup>and 10<sup>6</sup> times successively. For each gradient, spread the solution on two plates with and without antibiotic respectively.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(3) </span><span style="font-family:'微软雅黑',sans-serif; ">Culture at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ for 12h.</span><span style="font-family:'微软雅黑',sans-serif; "> </span></p>
| + | |
− | </div>
| + | |
− | </section>
| + | |
− | | + | |
− | <section class="popout protocols">
| + | |
− | <div class="collapsible"> Red Fluorescence Detection</div>
| + | |
− | <div class="collapsibleContent">
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">TECAN<sup>®</sup> infinite M200 PRO microplate spectrophotometer is used to measure red fluorescence intensity. The stimulated UV wavelength is 580nm.<strong></strong></span></p>
| + | |
− | </div>
| + | |
− | </section>
| + | |
− | | + | |
− | <section class="popout protocols">
| + | |
− | <div class="collapsible">Optical Density (OD) Measurement</div>
| + | |
− | <div class="collapsibleContent">
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">YOKE<sup>®</sup> UV755B ultraviolet-visible pectrophotometer is used to measure OD under 600nm tungsten light.</span></p>
| + | |
− | </div>
| + | |
− | </section>
| + | |
− | | + | |
− | <section class="popout protocols">
| + | |
− | <div class="collapsible"> Growth Curve Measurement <i>Streptomyces</i></div>
| + | |
− | </section>
| + | |
− | | + | |
− | <section class="popout protocols">
| + | |
− | <div class="collapsible">Screening of the Most Acid-Resistant Strain</div>
| + | |
− | <div class="collapsibleContent">
| + | |
− | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(1) Test Under Acid Pressure</span></strong></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><1> Pick monoclones from LB plate and inoculate in 2 mL LBG liquid medium (pH 7.0) containing antibiotic. Cultivate overnight at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220 rpm.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><2> After growth entering stationary phase, measure OD<sub>600</sub>. Dilute bacteria solution with fresh LBG liquid medium (pH 4.5) until OD<sub>600</sub> value is 1. Transfer 15</span><span style="font-family:'微软雅黑',sans-serif; ">μL diluted liquid to 285 μL LBG liquid medium (pH 4.5) containing antibiotic on 100-well Honeycomb 2 culture plate.</span><span style="font-family:'微软雅黑',sans-serif; "> Culture in the automatic growth curve analyzer Bioscreen C at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and a medium oscillation frequency and amplitude for 24h. Measure real-time OD<sub>600</sub> every 30min. </span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><3> Dilute bacteria solution with certain amounts of LBG fresh liquid medium (pH4.5) according to the formula: 100 </span><span style="font-family:'微软雅黑',sans-serif; ">× OD<sub>600</sub> value = (100+V) × 1.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><4> Conduct 3-5 parallel experiments.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Procedure:</span></p>
| + | |
− | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(2) Test Under Acid Shock</span></strong></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><1> Pick monoclones from LB plate and inoculate in 2 mL LBG liquid medium (pH 7.0) containing antibiotic. Cultivate overnight at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220 rpm.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><2> Transfer the solution to 10 mL LBG liquid medium (pH 4.5) containing antibiotic. Incubate at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220rpm until OD<sub>600 </sub>value is between 0.5 and 0.6.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><3> Place the bacteria suspension on ice to stop the growth. Use fresh LBG liquid medium (pH 4.5) to dilute it until OD<sub>600</sub> value is 0.5. Transfer 20</span><span style="font-family:'微软雅黑',sans-serif; ">μL diluted liquid to 180 μL LBG liquid medium (pH 2.5) containing antibiotic on 96-well plate. Culture at 37℃ and 700rpm in microplate thermostatic oscillator or at 37℃ and 250rpm for 1 h in shaker , and do the control experiment at the same time. </span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><4> Dilute the bacteria solution with fresh LBG liquid medium (pH 4.5) by 10, 10<sup>2</sup>, 10<sup>3</sup> and 10<sup>4</sup> times.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><5> Spread 4 </span><span style="font-family:'微软雅黑',sans-serif; ">μL diluted and undiluted solution on plates respectively. Culture at room temperature overnight.</span></p>
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− | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(3) Glutamate Decarboxylase (GAD) Activity Test</span></strong></p>
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− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><1> Pick monoclones from LB plate and inoculate in 2 mL LB-2% (w/v) glucose liquid medium containing antibiotic. Cultivate overnight at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220 rpm. </span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><2> Transfer the solution to 10 mL LBG liquid medium (pH 4.5) containing antibiotic. Incubate at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220rpm until OD<sub>600 </sub>value is between 0.5 and 0.6 or incubate for about 16h directly.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><3> Place the bacteria suspension on ice for 10min to stop the growth. Centrifuge at 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 3500rpm for 10mim. Discard the supernatant. Centrifuge again for 2min and remove the medium completely with a pipette.</span></p>
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− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><4> Suspend cells with 1mL cold 20 mM K<sub>2</sub>HPO<sub>4</sub>-KH<sub>2</sub>PO<sub>4</sub> (pH7.4) buffer. Centrifuge at 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 12000rpm for 2min. Suck out supernatant with a pipette. Centrifuge again, and remove the supernatant completely with a pipette.</span></p>
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− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><5> Suspend the cells with 1mL cold 20 mM K<sub>2</sub>HPO<sub>4</sub>-KH<sub>2</sub>PO<sub>4</sub> (pH7.4) buffer. </span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><6> Break up the cells with a ultrasonic cell grinder. Centrifuge at 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 12000rpm for 2 min. Take the supernatant crude enzyme solution for enzyme activity measurement.</span></p>
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− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><7> Measure the standard growth curves: Mix 10 </span><span style="font-family:'微软雅黑',sans-serif; ">μL NaOH standard solution preheated to 37℃ and 90 μL of L GAD reaction solution (or blank solution) fully in 96-well plate. Measure the absorbance with a microplate reader at 37℃. The indicator and corresponding wavelengths are: </span></p>
| + | |
− | <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="192" style="border-collapse:collapse;border:none;">
| + | |
− | <tr>
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− | <td width="129" valign="top" style="width:96.85pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Bromocresol green</span></p></td>
| + | |
− | <td width="63" valign="top" style="width:47.5pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">620 nm</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="129" valign="top" style="width:96.85pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Bromcresol purple</span></p></td>
| + | |
− | <td width="63" valign="top" style="width:47.5pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">590 nm</span></p></td>
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− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="129" valign="top" style="width:96.85pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Bromothymol blue </span></p></td>
| + | |
− | <td width="63" valign="top" style="width:47.5pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">615 nm</span></p></td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><8> Measurement of enzyme activity: Mix 20 </span><span style="font-family:'微软雅黑',sans-serif; ">μL crude enzyme solution preheated to 37℃ and 180 μL of GAD reaction solution (or blank solution) fully in 96-well plate. Measure the abosorbance with microplate reader at 37℃ in dynamic detection mode every 1 min for 2 h in the linear range. The wavelengths are the same as above. Calculate enzyme activity with the formula below: </span></p>
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− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">Enzyme activity (nmol/min/OD<sub>600</sub>) =<span style="position:relative; top:14.0pt; "><img src="notebook_clip_image002.gif" alt="" width="164" height="46"></span></span></p>
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− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">When:</span></p>
| + | |
− | <table class="MsoTableGrid" border="1" cellspacing="0" cellpadding="0" width="568" style="border-collapse:collapse;border:none;">
| + | |
− | <tr>
| + | |
− | <td width="57" valign="top" style="width:42.85pt;border:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">OD<sub>600</sub></span></p></td>
| + | |
− | <td width="511" valign="top" style="width:383.25pt;border:solid windowtext 1.0pt;border-left:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">The OD<sub>600 </sub>of the bacteria solution before breaking down.</span></p></td>
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− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="57" valign="top" style="width:42.85pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">v<sub>reaction</sub></span></p></td>
| + | |
− | <td width="511" valign="top" style="width:383.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">The change of light absorption value of crude enzyme solution at 620/590/615 nm) after reacting with different experimental glutamic acid decarboxylase solution for 1min</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="57" valign="top" style="width:42.85pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">v<sub>blank</sub></span></p></td>
| + | |
− | <td width="511" valign="top" style="width:383.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">The change of light absorption value of crude enzyme solution at 620/590/615 nm) after reacting with different blank glutamic acid decarboxylase solution for 1min</span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="57" valign="top" style="width:42.85pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">k<sub>reaction</sub></span></p></td>
| + | |
− | <td width="511" valign="top" style="width:383.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(In the measurement of standard curves of different experimental glutamic acid decarboxylase reaction solution) The change of light absorption value at 620/590/615 nm when NaOH standard solution consumed 1 nmol H<sup>+</sup></span></p></td>
| + | |
− | </tr>
| + | |
− | <tr>
| + | |
− | <td width="57" valign="top" style="width:42.85pt;border:solid windowtext 1.0pt;border-top:none;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">k<sub>blank</sub></span></p></td>
| + | |
− | <td width="511" valign="top" style="width:383.25pt;border-top:none;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;"><p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">(In the measurement of standard curves of different blank glutamic acid decarboxylase reaction solution) The change of light absorption value at 620/590/615 nm when NaOH standard solution consumed 1 nmol H<sup>+</sup></span></p></td>
| + | |
− | </tr>
| + | |
− | </table>
| + | |
− | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(4) </span></strong><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">γ-aminobutyrate (GABA) Release Test </span></strong></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><1> Pick monoclones from LB plate and inoculate in 2 mL LBG liquid medium (pH 7.0) containing antibiotic. Cultivate overnight at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220 rpm.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><2> Transfer the solution to fresh LBG liquid medium (pH4.5) by 1:100. Culture the cells until OD<sub>600</sub> is between 0.5 and 0.6 (to logarithmic phase) or culture for about 16h directly to stationary phase and measure the accurate OD<sub>600 </sub>(h</span><span style="font-family:'微软雅黑',sans-serif; ">ereinafter </span><span style="font-family:'微软雅黑',sans-serif; ">denoted as OD).</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><3> For cells in logarithmic phase, transfer 4OD bacterial solution to a 15 mL BD tube and centrifuge at 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 3500rpm for 10 min. For cells in stationary phase, transfer 4OD bacterial solution to a 1.5 mL EP tube and centrifuge at 4℃ and 8000rpm for 2 min. Discard the supernatant. Suspend the cells with 1mL cool 10mM sodium glutamate citric acid buffer. Centrifuge at 4℃ and 12000rpm for 2 min. Discard the supernatant with a pipette. Centrifuge again for 30s and remove the supernatant completely with a pipette. Resuspend the cells to 4 OD/mL with cool citric acid buffer containing 10mM monosodium glutamate. Perform the whole cell reaction at 37℃ for 1 h in order that the bacteria release sufficient GABA.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><4> Centrifuge at 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 8000rpm for 2 min. Take 10μL supernatant and add to 90μL GABase reaction buffer. React at 37℃. Measure the final OD<sub>600</sub>.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><5> Draw the standard growth curves: prepare standard solutions of 0 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, 0.8 mM, 1 mM, 2 mM and 4 mM. Measure the light absorption at 340 nm wave after reacting for 1 h. Do three series of parallel experiments.</span></p>
| + | |
− | <p class="MsoNormal"><strong><span style="font-family:'微软雅黑',sans-serif; font-size:14.0pt; ">(5) NH<sub>4</sub><sup>+</sup> Release Test</span></strong></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><1> For stationary phase: pick monoclones from LB plate and inoculate in 2 mL LB-2% (w/v) glucose liquid medium containing antibiotic. Cultivate overnight at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220 rpm. </span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">For logarithmic phase: pick monoclones from LB plate and inoculate in 5 mL LB-2% (w/v) glucose liquid medium containing antibiotic. Cultivate overnight at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220 rpm.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><2> For stationary phase: Prepare pH standard solution (pH 4.01, 7.00, 9.21) and balance them at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃. Balance LBG liquid medium (pH 4.5) at room temperature. Measure OD<sub>600</sub>s of overnight-cultured bacteria solution and the bacteria solution diluted 10 times. Calculate the OD<sub>600</sub> value of 10 times diluted solution with the formula in test (1). Add corresponding amounts of bacteria solution to 20 mL LBG liquid medium (pH 4.5) to make the initial OD<sub>600</sub> value 0.05. </span><span style="font-family:'微软雅黑',sans-serif; ">Transfer the solution to 10 mL LBG liquid medium (pH 4.5) containing antibiotic. Incubate at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 220rpm until OD<sub>600 </sub>value is between 0.5 and 0.6. </span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">For logarithmic phase: Prepare pH standard solution (pH 4.01, 7.00, 9.21) and balance them at 37</span><span style="font-family:'微软雅黑',sans-serif; ">℃. Balance LBG liquid medium (pH 4.5) at room temperature. Measure OD<sub>600</sub> of overnight-cultured bacteria solution. Add 50mL LBG liqiud medium (pH 4.5) to the 1000mL empty conical flask and antibiotic subsequently. Calculate the OD<sub>600</sub> value of 10 times diluted solution with the formula in test (1). Add corresponding amounts of bacteria solution to 20 mL LBG liquid medium (pH 4.5) to make the initial OD<sub>600</sub> value 0.05. Culture at 37℃ and 220rpm for 2-3 hours. Stop the growth with ice bath when OD<sub>600</sub> value is between 0.5 and 0.6.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><3> Place the cells on ice for at least 10 min to cool down. Transfer 20OD<sub>600</sub> bacterium solution to a 50 mL centrifuge tube. Place back on ice to inhibit growth. Centrifuge at 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 3500rpm for 10 min. Discard the supernatant. Centrifuge again for 2 min and remove the supernatant completely with a pipette. Suspend the cells with 10 mL cool sodium citrate solution. Centrifuge at 4℃ and 3500rpm for 10 min. Place the cells on ice.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><4> Culture the cells in the LBG liquid medium (pH 4.5) to logarithmic phase. Centrifuge at 4</span><span style="font-family:'微软雅黑',sans-serif; ">℃ and 3500rpm for 10 min. Resuspend the cells with 20 mL 25 mM sodium citrate solution containing 10 mM glutamine (pH 4.5) to a concentration of 10OD<sub>600</sub>/mL.</span></p>
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; "><5> Use a DX218-NH<sub>4</sub><sup>+ </sup>ion selective electrode to continuously measure changes of NH<sub>4</sub><sup>+</sup> concentration in the buffer for 30min with a time interval of 1 min.</span></p>
| + | |
− | </div></section>
| + | |
− | | + | |
− | <section class="popout protocols">
| + | |
− | <div class="collapsible"> Sequencing Results Analysis</div>
| + | |
− | <div class="collapsibleContent">
| + | |
− | <p class="MsoNormal"><span style="font-family:'微软雅黑',sans-serif; ">The software MEGA 7 is used to align sequences.</span></p>
| + | |
− | </div>
| + | |
− | </section>
| + | |
− | </div> | + | |
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