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− | + | <br><br> | |
− | < | + | <center><img src="https://static.igem.org/mediawiki/2017/0/08/Igem_protocol.png" width="25%"></center> |
− | + | ||
+ | <h2>protocols</h2> | ||
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− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="transformationWithDigestMaterials"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#transformationWithDigestMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> |
<div> | <div> | ||
− | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | + | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><!--div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div--> |
</div> | </div> | ||
</a> | </a> | ||
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<div class="panel-body"> | <div class="panel-body"> | ||
<br> | <br> | ||
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<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="CloneVerificationMethods"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#CloneVerificationMethods-collapse" aria-expanded="false" aria-controls="CloneVerificationMethods-collapse"> |
<div> | <div> | ||
<div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
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<div class="panel-body"> | <div class="panel-body"> | ||
− | <center>< | + | <center><h6 style="text-align:left"> |
<ol type="a"> | <ol type="a"> | ||
<li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | <li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
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</div></div> | </div></div> | ||
− | <h3> | + | <h3>Digest Ligations</h3> |
<div class="text-center"> | <div class="text-center"> | ||
<div class="container"> | <div class="container"> | ||
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="DigestLigationsMaterials"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#DigestLigationsMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> |
<div> | <div> | ||
<div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
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</div> | </div> | ||
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<div class="panel-body"> | <div class="panel-body"> | ||
<br> | <br> | ||
− | < | + | <h6 style="text-align:left"> |
<ul> | <ul> | ||
− | <li>DNA | + | <li>T4 DNA Ligation Buffer. Invitrogen, Cat No. 46-0114</li> |
− | + | <li>T4 DNA Ligase, Thermo Scientific, Cat No. K1231</li> | |
− | <li> | + | <li>Reagent grade water, NERL, Cat No. 98555</li> |
− | <li> | + | |
<li>1.5 mL tube</li> | <li>1.5 mL tube</li> | ||
<li>Vortex</li> | <li>Vortex</li> | ||
<li>Pipet and tips</li> | <li>Pipet and tips</li> | ||
<li>Ice bucket and ice</li> | <li>Ice bucket and ice</li> | ||
− | |||
− | |||
</ul> | </ul> | ||
</h6> | </h6> | ||
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<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="DigestLigationsMethods"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#DigestLigationsMethods-collapse" aria-expanded="false" aria-controls="DigestLigationsMethods-collapse"> |
<div> | <div> | ||
<div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
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</div> | </div> | ||
− | <div id=" | + | <div id="DigestLigationsMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="DigestLigationsMethods"> |
<div class="panel-body"> | <div class="panel-body"> | ||
− | <center>< | + | <center><h6 style="text-align:left"> |
<ol type="a"> | <ol type="a"> | ||
− | <li> | + | <li>Set ligation as shown below in 1.5 mL tube.</li> |
− | <li> | + | <li>The tubes were incubated at room temperature for 1 hour.</li> |
<li>Turn on incubator-shaker at 37°C.</li> | <li>Turn on incubator-shaker at 37°C.</li> | ||
− | <li> | + | <li>The tubes were then transferred to ice.</li> |
− | <li> | + | <li>Ligation Condition (with insert) |
− | + | <table> | |
− | + | <tr><th>Component</th><th>Volume (μl)</th></tr> | |
− | + | <tr><td>Reagent grade water</td><td>4.5 μl</td></tr> | |
− | + | <tr><td>10X T4 ligation buffer</td><td>1.0 μl</td></tr> | |
− | + | <tr><td>EcoR1-Pst1<br>Linearized - pSB1C3</td><td>0.5 μl</td></tr> | |
− | + | <tr><td>EcoR1/SpeI - Linearized –promoter<br>(For us: <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_J23101">BBa_J23100, BBa_J23101, BBa_J23110</a>)</td><td>2.0 μl</td></tr> | |
− | + | <tr><td>XbaI/Pst1 - Linearized -Insert (Catabolic pathway)<br>(For us: Fluorene 1, or Fluorene 2, or Phenanthrene 1, or Phenanthrene 2) | |
− | + | </td><td>1.5 μl</td></tr> | |
− | + | <tr><td>T4 DNA Ligase (5Weiss/μl)</td><td>1.5 μl</td></tr> | |
− | <li> | + | |
− | + | </table> | |
− | + | </li> | |
+ | <li>Control Ligation Condition (no insert) | ||
+ | <table> | ||
+ | <tr><th>Component</th><th>Volume (μl)</th></tr> | ||
+ | <tr><td>Reagent grade water </td><td>8.0 μl</td></tr> | ||
+ | <tr><td>10X T4 ligation buffer</td><td>1.0 μl</td></tr> | ||
+ | <tr><td>EcoR1-Pst1 pSB1C3</td><td>0.5 μl</td></tr> | ||
+ | <tr><td>T4 DNA Ligase (5Weiss/μl)</td><td>0.5 μl</td></tr> | ||
+ | </table> | ||
+ | </li> | ||
+ | </ol></h6></center> | ||
+ | <br> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div></div> | ||
+ | <h3>OD Calibration</h3> | ||
+ | <div class="text-center"> | ||
+ | <div class="container"> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="ODCalibrationMaterials"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#ODCalibrationMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="ODCalibrationMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="ODCalibrationMaterials"> | ||
+ | <div class="panel-body"> | ||
+ | <br> | ||
+ | <h6 style="text-align:left"> | ||
+ | <ul> | ||
+ | <li>1 mL LUDOX (provided in kit)</li> | ||
+ | <li>Water</li> | ||
+ | <li>96 well plate with flat with transparent bottom</li> | ||
+ | </ul> | ||
+ | </h6> | ||
+ | <br> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="some-padding"></div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="ODCalibrationMethods"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#ODCalibrationMethods-collapse" aria-expanded="false" aria-controls="ODCalibrationMethods-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="ODCalibrationMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="ODCalibrationMethods"> | ||
+ | <div class="panel-body"> | ||
+ | <center><h6 style="text-align:left"> | ||
+ | <ol type="a"> | ||
+ | <li>Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)</li> | ||
+ | <li>Add 100 μl of H 2 O into wells A2, B2, C2, D2 (or 1 mL H 2 O into cuvette)</li> | ||
+ | <li>Measure absorbance 600 nm of all samples in all standard measurement modes in instrument</li> | ||
+ | <li>Record the data in the table below or in your notebook</li> | ||
+ | <li>Import data into Excel ( OD600 reference point tab )</li> | ||
+ | |||
</ol></h6></center> | </ol></h6></center> | ||
<br> | <br> | ||
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− | </div></div><h3> | + | </div></div> |
+ | |||
+ | |||
+ | <h3>Biotransformation Measurement</h3> | ||
<div class="text-center"> | <div class="text-center"> | ||
<div class="container"> | <div class="container"> | ||
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="BiotransformationMeasurementMaterials"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#BiotransformationMeasurementMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> |
<div> | <div> | ||
<div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
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</div> | </div> | ||
− | <div id=" | + | <div id="BiotransformationMeasurementMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="BiotransformationMeasurementMaterials"> |
<div class="panel-body"> | <div class="panel-body"> | ||
<br> | <br> | ||
− | < | + | <h6 style="text-align:left"> |
<ul> | <ul> | ||
− | <li> | + | <li>Cultures of clones in transformed in Escherichia coli strain BL21(DE3)</li> |
− | <li>LB | + | <li>LB (Luria Bertani) media</li> |
− | <li> | + | <li>Minimal medium</li> |
− | <li> | + | <li>Testing substance (Fluorene and Phenanthrene)</li> |
− | <li> | + | <li>Appropriate Antibiotics (Chloramphenicol, Tetracycline and Ampicillin)</li> |
− | <li> | + | <li>IPTG</li> |
− | <li> | + | <li>Baffled flasks</li> |
− | + | <li>50 mL Falcon tube with vented cap</li> | |
− | + | <li>Incubator at 37℃</li> | |
− | + | <li>1.5mL Eppendorf tubes for sample storage</li> | |
+ | <li>Ice bucket with ice</li> | ||
+ | <li>Pipettes</li> | ||
+ | <li>96 well plate with flat transparent bottom devices</li> | ||
+ | <li>Shaker</li> | ||
</ul> | </ul> | ||
</h6> | </h6> | ||
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<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="BiotransformationMeasurementMethods"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#BiotransformationMeasurementMethods-collapse" aria-expanded="false" aria-controls="BiotransformationMeasurementMethods-collapse"> |
<div> | <div> | ||
− | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | + | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Instrumental Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> |
</div> | </div> | ||
</a> | </a> | ||
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</div> | </div> | ||
− | <div id=" | + | <div id="BiotransformationMeasurementMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="BiotransformationMeasurementMethods"> |
<div class="panel-body"> | <div class="panel-body"> | ||
− | <center>< | + | <center><h6 style="text-align:left"> |
<ol type="a"> | <ol type="a"> | ||
− | <li> | + | <li>Set instrument to read OD600 (as OD calibration setting)</li> |
− | <li> | + | <li>Measure OD600 of the overnight cultures</li> |
− | <li> | + | <li>Record data in notebook</li> |
− | <li> | + | <li>Import data into Excel <b>Dilution Calculation</b></li> |
− | + | <li>Dilute the cultures to a target OD 600 of 0.02 (see the volume of preloading culture and media in Excel) (<b>Dilution Calculation</b> Sheet_1) in 4 mL LB medium + correct antibiotic combinations (for us Chloramphenicol or tetracycline or both) in 14 mL falcon tube</li> | |
− | <li> | + | <li>Incubate the cultures at 37℃ and 220 rpm</li> |
− | + | <li>Take 450 μL samples of the cultures at various time points. At each time point, take a sample from each clone, two colonies per clone</li> | |
− | <li> | + | <li>Place samples on ice.</li> |
− | <li> | + | <li>At the end of sampling point, measure samples</li> |
− | + | <li>Use the same instrument setting for all time point</li> | |
− | + | <li>Pipette 100 μL of each sample into each well.</li> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</ol></h6></center> | </ol></h6></center> | ||
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</div></div> | </div></div> | ||
− | + | <h3>DNA Preparations</h3> | |
− | + | ||
− | <h3> | + | |
<div class="text-center"> | <div class="text-center"> | ||
<div class="container"> | <div class="container"> | ||
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="DNAPreparationsMaterials"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#DNAPreparationsMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> |
<div> | <div> | ||
<div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
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</div> | </div> | ||
− | <div id=" | + | <div id="DNAPreparationsMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="DNAPreparationsMaterials"> |
<div class="panel-body"> | <div class="panel-body"> | ||
<br> | <br> | ||
− | < | + | <h6 style="text-align:left"> |
<ul> | <ul> | ||
− | <li> | + | <li><u>Promoters (100, 101, 110):</u> |
− | <li> | + | <li>Reagent grade water</li> |
+ | <li>The promoter areas were designed by CCA-IGEM-Team 2017.</li> | ||
+ | <li>The promoter areas were synthesized by IDT.</li> | ||
+ | <li>The synthetic promoter areas were delivered to us lyophilized.</li> | ||
+ | </li> | ||
+ | <li><u>Polycistronic codons (F1, F2, P1, P2):</u> | ||
+ | <ul><li>Reagent grade water</li> | ||
+ | <li>The mini-genes were designed by CCA-IGEM-Team 2017.</li> | ||
+ | <li>The mini-genes were synthesized by Genscript.</li> | ||
+ | <li>The genes were delivered to us lyophilized.</li></ul> | ||
+ | </li> | ||
<li>DH5a competent cells, Invitrogen, Cat 18265-017</li> | <li>DH5a competent cells, Invitrogen, Cat 18265-017</li> | ||
<li>SOC (Recovery Medium), Lucigen, Cat No. F98226</li> | <li>SOC (Recovery Medium), Lucigen, Cat No. F98226</li> | ||
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<div class="some-padding"></div> | <div class="some-padding"></div> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="DNAPreparationsMethods"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#DNAPreparationsMethods-collapse" aria-expanded="false" aria-controls="DNAPreparationsMethods-collapse"> |
<div> | <div> | ||
<div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
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</div> | </div> | ||
− | <div id=" | + | <div id="DNAPreparationsMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="DNAPreparationsMethods"> |
<div class="panel-body"> | <div class="panel-body"> | ||
− | <center>< | + | <center><h6 style="text-align:left"> |
<ol type="a"> | <ol type="a"> | ||
<li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | <li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
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<li>Turn on incubator for plates at 37°C.</li> | <li>Turn on incubator for plates at 37°C.</li> | ||
<li>Set up water bath at 42°C.</li> | <li>Set up water bath at 42°C.</li> | ||
− | + | ||
− | <li> | + | <li>The vials containing the lyophilized DNA(~4 µg) were spun down before opening the vials for the first time</li> |
− | + | <li>16 µL of 0.2µm filtered water was added to the lyophilized powder using a P20 pipet.</li> | |
− | + | <li>After closing the tubes, they were vortexed for 2-3 minutes and were allowed to sit at 60-65°C for 15 minutes to resuspend the DNA. The tubes were then spun.</li> | |
− | + | <li>Aliquots were taken to start cloning.</li> | |
− | + | <li>All stocks are stored at -20°C.</li> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
+ | |||
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− | </div></div> | + | </div></div><h3>Gel</h3> |
− | <h3> | + | |
<div class="text-center"> | <div class="text-center"> | ||
<div class="container"> | <div class="container"> | ||
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="GelMaterials"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#GelMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> |
<div> | <div> | ||
<div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
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</div> | </div> | ||
− | <div id=" | + | <div id="GelMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="GelMaterials"> |
<div class="panel-body"> | <div class="panel-body"> | ||
<br> | <br> | ||
− | < | + | <h6 style="text-align:left"> |
<ul> | <ul> | ||
− | <li> | + | <li>Agarose</li> |
− | <li> | + | <li>1xTAE</li> |
− | + | <li>Microwave</li> | |
− | + | <li>EtBr</li> | |
− | + | <li>Tray and comb</li> | |
− | + | </ul> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | </ul> | + | |
</h6> | </h6> | ||
<br> | <br> | ||
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<div class="some-padding"></div> | <div class="some-padding"></div> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="GelMethods"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#GelMethods-collapse" aria-expanded="false" aria-controls="GelMethods-collapse"> |
<div> | <div> | ||
<div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
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</div> | </div> | ||
− | <div id=" | + | <div id="GelMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="GelMethods"> |
<div class="panel-body"> | <div class="panel-body"> | ||
− | <center>< | + | <center><h6 style="text-align:left"> |
<ol type="a"> | <ol type="a"> | ||
− | <li> | + | <li>Measure 1 g of agarose</li> |
− | + | <li>Mix agarose powder with 100 mL 1xTAE in a microwavable flask.</li> | |
− | + | <li>Microwave for 1-3 min until the agarose is completely dissolved </li> | |
− | + | <li>Let agarose solution cool down to about 50°C (about when you can comfortably keep your hand on the flask), about 5 mins.</li> | |
− | + | <li>Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL</li> | |
− | + | <li>Pour the agarose into a gel tray with the well comb in place.</li> | |
− | + | <li>Place newly poured gel at 4°C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely solidified.</li> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
Line 441: | Line 493: | ||
− | </div></div><h3> | + | </div></div><h3>Gel Purification</h3> |
<div class="text-center"> | <div class="text-center"> | ||
<div class="container"> | <div class="container"> | ||
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="GelPurificationMaterials"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#GelPurificationMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> |
<div> | <div> | ||
<div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
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</div> | </div> | ||
− | <div id=" | + | <div id="GelPurificationMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="GelPurificationMaterials"> |
<div class="panel-body"> | <div class="panel-body"> | ||
<br> | <br> | ||
− | < | + | <h6 style="text-align:left"> |
<ul> | <ul> | ||
− | <li>DNA | + | <li>Finished Gel</li> |
− | <li>LB agar plates, Cat No. Teknova, | + | <li>Microtube</li> |
− | + | <li>DNA Purification Kit</li> | |
− | + | </ul> | |
− | + | </h6> | |
− | + | <br> | |
− | + | </div> | |
− | + | ||
− | + | </div> | |
− | + | </div> | |
+ | |||
+ | <div class="some-padding"></div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="GelPurificationMethods"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#GelPurificationMethods-collapse" aria-expanded="false" aria-controls="GelPurificationMethods-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="GelPurificationMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="GelPurificationMethods"> | ||
+ | <div class="panel-body"> | ||
+ | <center><h6 style="text-align:left"> | ||
+ | <ol type="a"> | ||
+ | <li>Once you have run your gel, move it to an open UV box.</li> | ||
+ | <li>Remove it from any gel tray as plastic will block much of the UV and with a clean, sterile razor blade, slice the desired DNA fragment from the gel.</li> | ||
+ | <li>Place the gel in a labeled microfuge tube.</li> | ||
+ | <li>Using a scale, weigh the tube with the gel fragment. The weight of the gel is directly proportional to its liquid volume and this is used to determine how much of each buffer to add during the DNA isolation step.</li> | ||
+ | <li>Isolate the DNA from the gel using any gel purification kit. Follow kit instructions.</li> | ||
+ | |||
+ | </ol></h6></center> | ||
+ | <br> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | </div></div><h3>Creating Constructs</h3> | ||
+ | <div class="text-center"> | ||
+ | <div class="container"> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="CreatingConstructsMaterials"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#CreatingConstructsMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="CreatingConstructsMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="CreatingConstructsMaterials"> | ||
+ | <div class="panel-body"> | ||
+ | <br> | ||
+ | <h6 style="text-align:left"> | ||
+ | <ul> | ||
+ | <li>DNA samples</li> | ||
+ | <li>Transformation efficiency DNA, specific plasmid (for us: pUC 19, 10 pg/ µL), Lucigen, Cat No. F92078-1</li> | ||
+ | <li>LB Carbenicillin 50 agar plates, Cat No. Teknova, L1801</li> | ||
+ | <li>E coli BL21 DE3, Life Technology, Cat No. 60106-1</li> | ||
+ | <li>SOC (Recovery Medium), Lucigen, Cat No. F98226</li> | ||
+ | <li>15 mL culture tube</li> | ||
+ | <li>Vortex</li> | ||
+ | <li>Tooth pick</li> | ||
+ | <li>Incubator shaker</li> | ||
+ | <li>42°C Water bath</li> | ||
+ | <li>Ice and ice bucket</li> | ||
+ | <li>Pipet</li> | ||
</ul> | </ul> | ||
</h6> | </h6> | ||
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<div class="some-padding"></div> | <div class="some-padding"></div> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="CreatingConstructsMethods"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#CreatingConstructsMethods-collapse" aria-expanded="false" aria-controls="CreatingConstructsMethods-collapse"> |
<div> | <div> | ||
<div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
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</div> | </div> | ||
− | <div id=" | + | <div id="CreatingConstructsMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="CreatingConstructsMethods"> |
<div class="panel-body"> | <div class="panel-body"> | ||
− | <center>< | + | <center><h6 style="text-align:left"> |
<ol type="a"> | <ol type="a"> | ||
<li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | <li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
− | + | <li>Transform 1 µL of DNA of plasmid (for us: pUC19) as transformation efficiency control</li> | |
− | + | <li>Turn on incubator-shaker at 37°C.</li> | |
− | + | <li>Turn on incubator for plates at 37°C.</li> | |
− | + | <li>Set up water bathat 42°C.</li> | |
− | + | <li>Bring to room temperature S.O.C medium.</li> | |
− | + | <li>Bring LB plates supplemented with appropriate antibiotic at room temperature.</li> | |
− | + | <li>Thaw competent cells on ice.</li> | |
− | + | <li>Aliquots competent cells in as many tubes as needed.</li> | |
− | + | <li>Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix</li> | |
− | + | <li>Incubate on ice for 20 minutes</li> | |
− | + | <li>Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.</li> | |
− | + | <li>Add 200 µl of 18-25°C SOCmedium and transfer mixture to 15mL Falcon tube</li> | |
− | + | <li>Incubate in shaker at 37°C, 225 rpm for 30 min</li> | |
− | + | <li>Plate 2 volumes (50 µL and ~100µL) of the mixture onto 2 different plates of LB agar plates supplemented with Carbenicillin 100 µg/mL</li> | |
− | + | <li>Incubate plates at 37°C overnight</li> | |
− | + | <li>Count colonies and estimate transformation efficiency</li> | |
+ | <li>Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion</li> | ||
+ | |||
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− | </div></div><h3> | + | </div></div><h3>Cloning with pUC Plasmid of Lac Operation (CCA_San_Diego Specific)</h3> |
<div class="text-center"> | <div class="text-center"> | ||
<div class="container"> | <div class="container"> | ||
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="CloningWPucMaterials"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#CloningWPucMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> |
<div> | <div> | ||
<div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
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</div> | </div> | ||
− | <div id=" | + | <div id="CloningWPucMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="CloningWPucMaterials"> |
<div class="panel-body"> | <div class="panel-body"> | ||
<br> | <br> | ||
− | < | + | <h6 style="text-align:left"> |
<ul> | <ul> | ||
− | <li>DNA | + | <li>Synthetic DNA<ul> |
− | <li> | + | <li>Synthetic flnB, dbfA1, dbfA2: 3219 bp</li> |
− | + | <li>Synthetic flnE, flnD1, ORF16, flnC: 3545 bp</li> | |
− | + | <li>Synthetic phnF, phnE, phnC, phnD: 4227 bp</li> | |
− | + | <li>Synthetic phnAc, phnAd, phnB: 3174 bp</li></ul> | |
− | + | </li> | |
− | + | ||
− | + | <li>HindIII restriction enzyme, Thermo Fisher, Cat No. ER0501</li> | |
− | + | <li>Restriction digest Buffer R, Thermo Fisher, Cat No. 00267554</li> | |
− | + | <li>Antarctic Phosphatase, New England Biolabs Cat No. M0289G</li> | |
+ | <li>Agarose, 100 g, Fisher, Cat No. BP-164-100</li> | ||
+ | <li>50XTAE Electrophoresis Buffer, 1L, (1X: 40 mMTris, 20mM Acetic Acid, 1 mM EDTA), Thermofisher, Cat No. B49</li> | ||
+ | <li>Sybr Safe DNA Gel Stain, Invitrogen, Cat No. S33102</li> | ||
+ | <li>100 bp DNA marker, Invitrogen, Cat No. 15628-019</li> | ||
+ | <li>10X Blue Juice DNA loading buffer, Invitrogen, 10816-015</li> | ||
+ | <li>Reagent grade water</li> | ||
+ | <li>Gel DNA Recovery Kit, Zymo Research, Cat No. D4007</li> | ||
+ | <li>1.5 mL tubes</li> | ||
+ | <li>Vortex</li> | ||
+ | <li>Ice bucket and ice</li> | ||
</ul> | </ul> | ||
</h6> | </h6> | ||
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<div class="some-padding"></div> | <div class="some-padding"></div> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="CloningWPucMethods"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#CloningWPucMethods-collapse" aria-expanded="false" aria-controls="CloneVerificationMethods-collapse"> |
<div> | <div> | ||
<div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
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</div> | </div> | ||
− | <div id=" | + | <div id="CloningWPucMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="CloningWPucMethods"> |
<div class="panel-body"> | <div class="panel-body"> | ||
− | <center>< | + | <center><h6 style="text-align:left"> |
<ol type="a"> | <ol type="a"> | ||
− | < | + | <h3><i>Preparation of Recipient Vector (Insert 1)</i></h3> |
− | + | <li>This insert contains the vector (origin of replication of pUC and the first part of the catabolic pathway under the control of the T7 promoter + inducible lacI +RBS)</li> | |
− | + | <li>Digest clone containing synthetic gene containing insert 1 with the restriction enzyme HindIII that cuts only at one site.</li> | |
− | + | <li>Restriction Digest Set-up (20µl reaction) in a 1.5 mL tube</li> | |
− | + | <li>Add the reagents as described below.</li> | |
− | + | <li>Incubate at 37°C for 1 hours</li> | |
− | <li> | + | <li>Add 2 µl of phosphatase buffer</li> |
− | + | <li>Add 1 µl of phosphatase</li> | |
− | + | <li>Incubate at 37°C for 30 minutes</li> | |
− | + | <li>Add 2.3 µl of loading buffer at the end of the reaction</li> | |
− | + | <li>Load the reaction on a 1% Agarose gel</li> | |
− | + | <li>After running the electrophoresis, cut the linearized band.</li> | |
− | + | <li>Purify the DNA from the gel using the Zymoclean Gel DNA Recovery Kit</li> | |
− | + | <li>Elute the DNA from the Zymoclean column with 12 µl of sterile filtered H2O</li> | |
− | + | <table> | |
− | + | <tr><th>Component</th><th>Volume (μl)</th><th>Final condition</th></tr> | |
− | + | <tr><td>Reagent grade water</td><td>8.0 μl</td></tr> | |
+ | <tr><td>10X Buffer R</td><td>2.0 μl</td><td>1x</td></tr> | ||
+ | <tr><td>Vector-Insert 1</td><td>1.0 μl</td><td>0.5 µg</td></tr> | ||
+ | <tr><td>HindIII (10U/µl)</td><td>1.0 μl</td><td>10U</td></tr> | ||
+ | |||
+ | </table> | ||
+ | </li> | ||
+ | </li> | ||
+ | |||
+ | <h3><i>Preparation of Insert (Insert 2)</i></h3><ol type="a"> | ||
+ | <li>This insert contains the second part of the catabolic pathway under the control of the T7 promoter + inducible lacI +RBS)</li> | ||
+ | <li>Digest plasmid containing synthetic gene designated as insert 2 with the restriction enzyme HindIII that cuts at 2 sites (HindIII sites are flanking the genes)</li> | ||
+ | <li>Set-up restriction digestion (20µl reaction) in a 1.5 mL tube</li> | ||
+ | <li>Turn on water bath at 37°C </li> | ||
+ | <li>Add the reagents in the order and with volume described in the table below.</li> | ||
+ | <li>Spin the tube briefly for 15 seconds at 10,000 rpm</li> | ||
+ | <li>Incubate the tube at 37°C for 1 hours</li> | ||
+ | <li>Add 2 µl of loading buffer in the tube at the end of the reaction</li> | ||
+ | <li>Load the digestion reaction on a 1% Agarose gel, TAE</li> | ||
+ | <li>After running the electrophoresis for 2 hours at 80V, cut out with a razor blade the linearized band of the desired size.</li> | ||
+ | <li>Purify the DNA from the gel using the Zymoclean Gel DNA Recovery Kit.</li> | ||
+ | <li>Elute the DNA from the Zymoclean column with 12 µl of sterile filtered H2O</li> | ||
+ | |||
+ | <table> | ||
+ | <tr><th>Component</th><th>Volume (μl)</th><th>Final condition</th></tr> | ||
+ | <tr><td>Reagent grade water</td><td>12.5 μl</td></tr> | ||
+ | <tr><td>10X Buffer R</td><td>1.5 μl</td><td>1x</td></tr> | ||
+ | <tr><td>Vector-Insert 2</td><td>1.0 μl</td><td> ~0.4 µg</td></tr> | ||
+ | <tr><td>HindIII (10U/µl)</td><td>1.0 μl</td><td>10U</td></tr> | ||
+ | |||
+ | </table> | ||
+ | </li> | ||
+ | </li> | ||
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− | </div></div><h3> | + | </div></div> |
+ | |||
+ | |||
+ | </div></div> | ||
+ | <h3>Cloning with Constructive Promoter of Different Strengths (CCA_San_Diego Specific)</h3> | ||
+ | <!--div class="text-center"> | ||
+ | <div class="container"> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="ClonePromoterMaterials"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#ClonePromoterMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="ClonePromoter-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="ClonePromoterMaterials"> | ||
+ | <div class="panel-body"> | ||
+ | <br> | ||
+ | <h6 style="text-align:left"> | ||
+ | <ul> | ||
+ | <li>Synthetic DNA [Fluorene F1, F2] and [Phenanthrene P1 and P2] | ||
+ | <ul><li>Synthetic F1 : flnB, dbfA1, dbfA2: 3219 bp</li> | ||
+ | <li>Synthetic F2: flnE, flnD1, ORF16, flnC: 3545 bp</li> | ||
+ | <li>Synthetic P1: phnF, phnE, phnC, phnD: 4227 bp</li> | ||
+ | <li>Synthetic P2: phnAc, phnAd, phnB: 3174 bp</li> | ||
+ | </li> | ||
+ | <li>Synthetic DNA [Promoter areas 100, 101, 110] | ||
+ | <li>Synthetic Promoter 100: 88 bp | ||
+ | Synthetic Promoter 101: 88 bp | ||
+ | Synthetic Promoter 110: 88 bp </li></li> | ||
+ | <li>Agarose, 100 g, Fisher, Cat No. BP-164-100</li> | ||
+ | <li>50XTAE Electrophoresis Buffer, 1L, (1X: 40 mMTris, 20mM Acetic Acid, 1 mM EDTA), Thermofisher, Cat No. B49</li> | ||
+ | <li>Sybr Safe DNA Gel Stain, Invitrogen, Cat No. S33102</li> | ||
+ | <li>1kb plus DNA ladder DNA marker, Thermo Scientific, Cat No. SM1334</li> | ||
+ | <li>10X Blue Juice DNA loading buffer, Invitrogen, 10816-015</li> | ||
+ | <li>Reagent grade water</li> | ||
+ | <li>Gel DNA Recovery Kit, Zymo Research, Cat No. D4007</li> | ||
+ | <li>1.5 mL tubes</li> | ||
+ | <li>Vortex</li> | ||
+ | <li>Ice bucket and ice</li> | ||
+ | <li>Cryo box for restriction enzyme</li> | ||
+ | <li>P1000 and P200 with corresponding tips</li> | ||
+ | |||
+ | </ul> | ||
+ | </h6> | ||
+ | <br> | ||
+ | </div> | ||
+ | |||
+ | </div--> | ||
+ | |||
+ | |||
+ | |||
+ | |||
<div class="text-center"> | <div class="text-center"> | ||
<div class="container"> | <div class="container"> | ||
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="ClonePromoter"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#ClonePromoter-collapse" aria-expanded="false" aria-controls="signup-collapse"> |
<div> | <div> | ||
<div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
Line 622: | Line 838: | ||
</div> | </div> | ||
− | <div id=" | + | <div id="ClonePromoter-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="ClonePromoter"> |
<div class="panel-body"> | <div class="panel-body"> | ||
<br> | <br> | ||
− | <h3 style="text-align:left"> | + | <h6 style="text-align:left"> |
+ | <ul> | ||
+ | <li>Synthetic DNA [Fluorene F1, F2] and [Phenanthrene P1 and P2] | ||
+ | <ul><li>Synthetic F1 : flnB, dbfA1, dbfA2: 3219 bp</li> | ||
+ | <li>Synthetic F2: flnE, flnD1, ORF16, flnC: 3545 bp</li> | ||
+ | <li>Synthetic P1: phnF, phnE, phnC, phnD: 4227 bp</li> | ||
+ | <li>Synthetic P2: phnAc, phnAd, phnB: 3174 bp</li></ul> | ||
+ | </li> | ||
+ | <li>Synthetic DNA [Promoter areas 100, 101, 110] | ||
+ | <ul><li>Synthetic Promoter 100: 88 bp </li> | ||
+ | <li>Synthetic Promoter 101: 88 bp</li> | ||
+ | <li>Synthetic Promoter 110: 88 bp </li></ul></li> | ||
+ | <li>Agarose, 100 g, Fisher, Cat No. BP-164-100</li> | ||
+ | <li>50XTAE Electrophoresis Buffer, 1L, (1X: 40 mMTris, 20mM Acetic Acid, 1 mM EDTA), Thermofisher, Cat No. B49</li> | ||
+ | <li>Sybr Safe DNA Gel Stain, Invitrogen, Cat No. S33102</li> | ||
+ | <li>1kb plus DNA ladder DNA marker, Thermo Scientific, Cat No. SM1334</li> | ||
+ | <li>10X Blue Juice DNA loading buffer, Invitrogen, 10816-015</li> | ||
+ | <li>Reagent grade water</li> | ||
+ | <li>Gel DNA Recovery Kit, Zymo Research, Cat No. D4007</li> | ||
+ | <li>1.5 mL tubes</li> | ||
+ | <li>Vortex</li> | ||
+ | <li>Ice bucket and ice</li> | ||
+ | <li>Cryo box for restriction enzyme</li> | ||
+ | <li>P1000 and P200 with corresponding tips</li> | ||
+ | |||
+ | </ul> | ||
+ | </h6><br> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <div class="some-padding"></div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="CloningWPromoterMethods"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#CloningWPromoterMethods-collapse" aria-expanded="false" aria-controls="CloningWPromoterMethods-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="CloningWPromoterMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="CloningWPromoterMethods"> | ||
+ | <div class="panel-body"> | ||
+ | <center><h6 style="text-align:left"> | ||
+ | <ol type="a"> | ||
+ | <h3 style="color:black"><i>Preparation of Promoter Areas with RBS</i></h3> | ||
+ | <li>The promoters/RBS regions were designed by the CCA_IGEM team and synthetized by IDT.</li> | ||
+ | <li>For the writing on plates, the designations for the promoters + RBS were as follows: | ||
+ | <ol><li>BBa_J23100 + RBS =100</li> | ||
+ | <li>BBa_J23101 + RBS =101</li> | ||
+ | <li>BBa_J23110 + RBS =110</li></ol></li> | ||
+ | <li>Double Digestion of promoter + RBS region with restriction enzymes EcoRI and SpeI<li> | ||
+ | <li>Restriction Digest Set-up (20µl reaction) in a 1.5 mL tube</li> | ||
+ | <li>Add the reagents as described below.</li> | ||
+ | <li>Incubate the tube at 37°C for 15 min</li> | ||
+ | <li>Note: there is no need to add a loading buffer because the digestion buffer already includes it.</li></li> | ||
+ | <li>Load the reaction on a 1.5 % Agarose gel, TAE</li> | ||
+ | <li>After running the electrophoresis for 1 hour at 80V, cut the linearized band.</li> | ||
+ | <li>Purify the DNA from the gel using the Zymoclean Gel DNA Recovery Kit</li> | ||
+ | <li>Elute the DNA from the Zymoclean column with 12 µl of sterile filtered H2O</li> | ||
+ | <table> | ||
+ | <tr><th>Component</th><th>Volume (μl)</th><th>Final condition</th></tr> | ||
+ | <tr><td>Reagent grade water</td><td>12.0 μl</td></tr> | ||
+ | <tr><td>10X Buffer R</td><td>2.0 μl</td><td>1x</td></tr> | ||
+ | <tr><td>Promoter+RBS Region</td><td>4.0 μl</td><td> ~1 µg</td></tr> | ||
+ | <tr><td>EcoRI (10U/µl)</td><td>1.0 μl</td><td></td></tr> | ||
+ | <tr><td>SpeI (10U/µl)</td><td>1.0 μl</td><td></td></tr> | ||
+ | </table> | ||
+ | </li> | ||
+ | </ol> | ||
+ | <h3 style="color:black">Preparation of insert F1, F2, P1, and P2</h3> | ||
+ | <ol> | ||
+ | <li>The following polycistronic sequences are cloned behind the 3 different promoter regions: | ||
+ | <ol> | ||
+ | <li>For fluorene catabolic pathway | ||
+ | <ol><li>FLUO-Insert 1 or F1 [Synthetic flnB, dbfA1, dbfA2]</li> | ||
+ | <li>FLUO-Insert 2 or F2 [Synthetic flnE, flnD1, ORF16, flnC]</li> | ||
+ | </ol> | ||
+ | </li> | ||
+ | |||
+ | <li>For phenanthrene catabolic pathway | ||
+ | <ol> | ||
+ | <li>PHE-Insert 1 or P1 [Synthetic phnF, phnE, phnC, phnD]</li> | ||
+ | <li>PHE-Insert 2 or P2 [Synthetic phnAc, phnAd, phnB]</li> | ||
+ | </ol> | ||
+ | </li> | ||
+ | <li>The fragments are digested with 2 enzymes: XbaI and Pst I</li> | ||
+ | <li>Digest plasmid containing synthetic gene designated F1, F2, P1, and P2 with the restriction enzyme XbaI and PstI</li> | ||
+ | <li>Set-up restriction digestion (15 µl reaction) in a 1.5 mL tube</li> | ||
+ | <li>Turn on water bath at 37°C </li> | ||
+ | <li>Add the reagents in the order and with volume described in the table below.</li> | ||
+ | <li>Spin the tube briefly for 15 seconds at 10,000 rpm</li> | ||
+ | <li>Incubate the tube at 37°C for 15 min</li> | ||
+ | <li>Note: there is no need to add a loading buffer because the digestion buffer already has it.</li> | ||
+ | <li>Load the digestion reaction on a 1% Agarose gel, TAE</li> | ||
+ | <li>After running the electrophoresis for 2 hours at 80V, cut out with a razor blade the linearized band of the desired size.</li> | ||
+ | <li>Purify the DNA from the gel using the Zymoclean Gel DNA Recovery Kit.</li> | ||
+ | <li>Elute the DNA from the Zymoclean column with 12 µl of sterile filtered H2O | ||
+ | |||
+ | <table> | ||
+ | <tr><th>Component</th><th>Volume (μl)</th><th>Final condition</th></tr> | ||
+ | <tr><td>Reagent grade water</td><td>10.0 μl</td><td></td></tr> | ||
+ | <tr><td>10X Buffer</td><td>1.5 μl</td><td>1X</td></tr> | ||
+ | <tr><td>Preparation of Insert<br>F1, F2, P1 and P2 | ||
+ | </td><td>1.5 μl</td><td>~0.4 µg</td></tr> | ||
+ | <tr><td>Xbal</td><td>1.0 μl</td><td></td></tr> | ||
+ | <tr><td>Pstl</td><td>1.0 μl</td><td></td></tr> | ||
+ | </table> | ||
+ | |||
+ | </li> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </ol> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </li> | ||
+ | <li>For fluorene catabolic pathway | ||
+ | FLUO-Insert 1 or F1 [Synthetic flnB, dbfA1, dbfA2] | ||
+ | FLUO-Insert 2 or F2 [Synthetic flnE, flnD1, ORF16, flnC] | ||
+ | For phenanthrene catabolic pathway | ||
+ | PHE-Insert 1 or P1 [Synthetic phnF, phnE, phnC, phnD] | ||
+ | PHE-Insert 2 or P2 [Synthetic phnAc, phnAd, phnB] | ||
+ | The fragments are digested with 2 enzymes: XbaI and Pst I | ||
+ | Digest plasmid containing synthetic gene designated F1, F2, P1, and P2 with the restriction enzyme XbaI and PstI | ||
+ | Set-up restriction digestion (15 µl reaction) in a 1.5 mL tube | ||
+ | Turn on water bath at 37°C | ||
+ | Add the reagents in the order and with volume described in the table below. | ||
+ | Spin the tube briefly for 15 seconds at 10,000 rpm | ||
+ | Incubate the tube at 37°C for 15 min | ||
+ | Note: there is no need to add a loading buffer because the digestion buffer already has it. | ||
+ | Load the digestion reaction on a 1% Agarose gel, TAE | ||
+ | After running the electrophoresis for 2 hours at 80V, cut out with a razor blade the linearized band of the desired size. | ||
+ | Purify the DNA from the gel using the Zymoclean Gel DNA Recovery Kit. | ||
+ | Elute the DNA from the Zymoclean column with 12 µl of sterile filtered H2O | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | </ol></h6></center> | ||
+ | <br> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | </div></div> | ||
+ | |||
+ | |||
+ | </div></div><h3>Cloning for depository (CCA_San_Diego Specific)</h3> | ||
+ | <div class="text-center"> | ||
+ | <div class="container"> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="Cloning4DepositoryMaterials"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#Cloning4DepositoryMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="Cloning4DepositoryMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="Cloning4DepositoryMaterials"> | ||
+ | <div class="panel-body"> | ||
+ | <br> | ||
+ | <h6 style="text-align:left"> | ||
<ul> | <ul> | ||
<li>DNA miniprrep</li> | <li>DNA miniprrep</li> | ||
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<div class="some-padding"></div> | <div class="some-padding"></div> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="Cloning4DepositoryMethods"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#Cloning4DepositoryMethods-collapse" aria-expanded="false" aria-controls="Cloning4DepositoryMethods-collapse"> |
<div> | <div> | ||
<div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
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</div> | </div> | ||
− | <div id=" | + | <div id="Cloning4DepositoryMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="Cloning4DepositoryMethods"> |
<div class="panel-body"> | <div class="panel-body"> | ||
− | <center>< | + | <center><h6 style="text-align:left"> |
<ol type="a"> | <ol type="a"> | ||
<li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | <li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
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<li>Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion</li> | <li>Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion</li> | ||
− | + | ||
− | + | ||
</ol></h6></center> | </ol></h6></center> | ||
<br> | <br> | ||
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− | </div></div><h3> | + | </div></div> |
+ | |||
+ | |||
+ | </div></div><h3>Clone verification</h3> | ||
<div class="text-center"> | <div class="text-center"> | ||
<div class="container"> | <div class="container"> | ||
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="CloneVerificationMaterials"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#CloneVerificationMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> |
<div> | <div> | ||
<div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
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</div> | </div> | ||
− | <div id=" | + | <div id="CloneVerificationMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="CloneVerificationMaterials"> |
<div class="panel-body"> | <div class="panel-body"> | ||
<br> | <br> | ||
− | < | + | <h6 style="text-align:left"> |
<ul> | <ul> | ||
<li>DNA miniprrep</li> | <li>DNA miniprrep</li> | ||
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<div class="some-padding"></div> | <div class="some-padding"></div> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="CloneVerificationMethods"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#CloneVerificationMethods-collapse" aria-expanded="false" aria-controls="CloneVerificationMethods-collapse"> |
<div> | <div> | ||
<div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
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</div> | </div> | ||
− | <div id=" | + | <div id="CloneVerificationMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="CloneVerificationMethods"> |
<div class="panel-body"> | <div class="panel-body"> | ||
− | <center>< | + | <center><h6 style="text-align:left"> |
<ol type="a"> | <ol type="a"> | ||
<li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | <li>Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells</li> | ||
Line 763: | Line 1,162: | ||
<li>Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion</li> | <li>Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion</li> | ||
+ | |||
+ | </ol></h6></center> | ||
+ | <br> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | </div></div> | ||
+ | |||
+ | |||
+ | </div></div><h3>Glycerol Stock</h3> | ||
+ | <div class="text-center"> | ||
+ | <div class="container"> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="CloneVerificationMaterials"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#GlycerolStockMaterials-collapse" aria-expanded="false" aria-controls="signup-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center; font-size: 30px;color:#56cfff;"><center>Materials</center></h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="GlycerolStockMaterials-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="GlycerolStockMaterials"> | ||
+ | <div class="panel-body"> | ||
+ | <br> | ||
+ | <h6 style="text-align:left"> | ||
+ | <ul> | ||
+ | <li>Glycerol</li> | ||
+ | <li>LB liquid medium</li> | ||
+ | <li>Antibiotic stock solution</li> | ||
+ | <li>15 mL culture tube</li> | ||
+ | <li>Incubator-Shaker</li> | ||
+ | <li>10 mL pipette</li> | ||
+ | <li>Pipet aid</li> | ||
+ | <li>Vortex</li> | ||
+ | <li>Rack</li> | ||
+ | <li>Toothpick</li> | ||
+ | </ul> | ||
+ | </h6> | ||
+ | <br> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="some-padding"></div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="GlycerolStockMethods"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#GlycerolStockMethods-collapse" aria-expanded="false" aria-controls="GlycerolStockMethods-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11"><h3 style="text-align:center;position:1000px; font-size: 30px;color:#56cfff;">Methods</h3></div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="GlycerolStockMethods-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="GlycerolStockMethods"> | ||
+ | <div class="panel-body"> | ||
+ | <h3 style="color:black"><i>Set-up Culture and Prepare Glycerol Stocks</i></h3> | ||
+ | <center><h6 style="text-align:left"> | ||
+ | <ol type="a"> | ||
+ | <li>Grow selected clones that have been checked (so they are correct) in 3 mL LB medium supplemented with appropriate antibiotics overnight at 37°C in the incubator/shaker at 220 rpm.</li> | ||
+ | <li>Prepare LB medium with 40% glycerol and add 0.5 mL to a cryogenic vial</li> | ||
+ | <li>Add 0.5 mL of culture sample to be stored</li> | ||
+ | <li>Gently vortex the cryogenic vial to ensure the culture and glycerol is well mixed</li> | ||
+ | <li>Label tube with date and identifier</li> | ||
+ | <li>Organize in a freezer box and label box</li> | ||
+ | <li>Prepare excel spreadsheet with all information</li> | ||
+ | <li>Store freezer box at -80°C</li> | ||
+ | |||
+ | |||
</ol></h6></center> | </ol></h6></center> | ||
<br> | <br> | ||
Line 774: | Line 1,249: | ||
</div></div> | </div></div> | ||
+ | |||
Latest revision as of 03:09, 2 November 2017
protocols
Transformation with Digest
- DNA miniprrep
- LB agar plates, Cat No. Teknova, appropriate antibiotic
- DH5a competent cells, Invitrogen, Cat 18265-017
- SOC (Recovery Medium), Lucigen, Cat No. F98226
- 1.5 mL tube
- Vortex
- Pipet and tips
- Ice bucket and ice
- Water bath (42°C)
- Incubator (37°C) and shaker
- Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells
- For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells
- Turn on incubator-shaker at 37°C.
- Turn on incubator for plates at 37°C.
- Set up water bath at 42°C.
- Bring to room temperature S.O.C medium.
- Bring LB plates supplemented with appropriate antibiotic at room temperature.
- Thaw competent cells on ice.
- Aliquots competent cells in as many tubes as needed.
- Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix
- Incubate on ice for 20 minutes
- Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.
- Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube
- Incubate in shaker at 37°C, 225 rpm for 30 min
- Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)
- Incubate plates at 37°C overnight
- Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion
Digest Ligations
- T4 DNA Ligation Buffer. Invitrogen, Cat No. 46-0114
- T4 DNA Ligase, Thermo Scientific, Cat No. K1231
- Reagent grade water, NERL, Cat No. 98555
- 1.5 mL tube
- Vortex
- Pipet and tips
- Ice bucket and ice
- Set ligation as shown below in 1.5 mL tube.
- The tubes were incubated at room temperature for 1 hour.
- Turn on incubator-shaker at 37°C.
- The tubes were then transferred to ice.
- Ligation Condition (with insert)
Component Volume (μl)
Reagent grade water 4.5 μl
10X T4 ligation buffer 1.0 μl
EcoR1-Pst1
Linearized - pSB1C3 0.5 μl
EcoR1/SpeI - Linearized –promoter
(For us: BBa_J23100, BBa_J23101, BBa_J23110) 2.0 μl
XbaI/Pst1 - Linearized -Insert (Catabolic pathway)
(For us: Fluorene 1, or Fluorene 2, or Phenanthrene 1, or Phenanthrene 2)
1.5 μl
T4 DNA Ligase (5Weiss/μl) 1.5 μl
- Control Ligation Condition (no insert)
Component Volume (μl)
Reagent grade water 8.0 μl
10X T4 ligation buffer 1.0 μl
EcoR1-Pst1 pSB1C3 0.5 μl
T4 DNA Ligase (5Weiss/μl) 0.5 μl
Component | Volume (μl) |
---|---|
Reagent grade water | 4.5 μl |
10X T4 ligation buffer | 1.0 μl |
EcoR1-Pst1 Linearized - pSB1C3 | 0.5 μl |
EcoR1/SpeI - Linearized –promoter (For us: BBa_J23100, BBa_J23101, BBa_J23110) | 2.0 μl |
XbaI/Pst1 - Linearized -Insert (Catabolic pathway) (For us: Fluorene 1, or Fluorene 2, or Phenanthrene 1, or Phenanthrene 2) | 1.5 μl |
T4 DNA Ligase (5Weiss/μl) | 1.5 μl |
Component | Volume (μl) |
---|---|
Reagent grade water | 8.0 μl |
10X T4 ligation buffer | 1.0 μl |
EcoR1-Pst1 pSB1C3 | 0.5 μl |
T4 DNA Ligase (5Weiss/μl) | 0.5 μl |
OD Calibration
- 1 mL LUDOX (provided in kit)
- Water
- 96 well plate with flat with transparent bottom
- Add 100 μl LUDOX into wells A1, B1, C1, D1 (or 1 mL LUDOX into cuvette)
- Add 100 μl of H 2 O into wells A2, B2, C2, D2 (or 1 mL H 2 O into cuvette)
- Measure absorbance 600 nm of all samples in all standard measurement modes in instrument
- Record the data in the table below or in your notebook
- Import data into Excel ( OD600 reference point tab )
Biotransformation Measurement
- Cultures of clones in transformed in Escherichia coli strain BL21(DE3)
- LB (Luria Bertani) media
- Minimal medium
- Testing substance (Fluorene and Phenanthrene)
- Appropriate Antibiotics (Chloramphenicol, Tetracycline and Ampicillin)
- IPTG
- Baffled flasks
- 50 mL Falcon tube with vented cap
- Incubator at 37℃
- 1.5mL Eppendorf tubes for sample storage
- Ice bucket with ice
- Pipettes
- 96 well plate with flat transparent bottom devices
- Shaker
- Set instrument to read OD600 (as OD calibration setting)
- Measure OD600 of the overnight cultures
- Record data in notebook
- Import data into Excel Dilution Calculation
- Dilute the cultures to a target OD 600 of 0.02 (see the volume of preloading culture and media in Excel) (Dilution Calculation Sheet_1) in 4 mL LB medium + correct antibiotic combinations (for us Chloramphenicol or tetracycline or both) in 14 mL falcon tube
- Incubate the cultures at 37℃ and 220 rpm
- Take 450 μL samples of the cultures at various time points. At each time point, take a sample from each clone, two colonies per clone
- Place samples on ice.
- At the end of sampling point, measure samples
- Use the same instrument setting for all time point
- Pipette 100 μL of each sample into each well.
DNA Preparations
- Promoters (100, 101, 110):
- Reagent grade water
- The promoter areas were designed by CCA-IGEM-Team 2017.
- The promoter areas were synthesized by IDT.
- The synthetic promoter areas were delivered to us lyophilized.
- Polycistronic codons (F1, F2, P1, P2):
- Reagent grade water
- The mini-genes were designed by CCA-IGEM-Team 2017.
- The mini-genes were synthesized by Genscript.
- The genes were delivered to us lyophilized.
- DH5a competent cells, Invitrogen, Cat 18265-017
- SOC (Recovery Medium), Lucigen, Cat No. F98226
- 1.5 mL tube
- Vortex
- Pipet and tips
- Ice bucket and ice
- Water bath (42°C)
- Incubator (37°C) and shaker
- Reagent grade water
- The mini-genes were designed by CCA-IGEM-Team 2017.
- The mini-genes were synthesized by Genscript.
- The genes were delivered to us lyophilized.
- Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells
- For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells
- Turn on incubator-shaker at 37°C.
- Turn on incubator for plates at 37°C.
- Set up water bath at 42°C.
- The vials containing the lyophilized DNA(~4 µg) were spun down before opening the vials for the first time
- 16 µL of 0.2µm filtered water was added to the lyophilized powder using a P20 pipet.
- After closing the tubes, they were vortexed for 2-3 minutes and were allowed to sit at 60-65°C for 15 minutes to resuspend the DNA. The tubes were then spun.
- Aliquots were taken to start cloning.
- All stocks are stored at -20°C.
Gel
- Agarose
- 1xTAE
- Microwave
- EtBr
- Tray and comb
- Measure 1 g of agarose
- Mix agarose powder with 100 mL 1xTAE in a microwavable flask.
- Microwave for 1-3 min until the agarose is completely dissolved
- Let agarose solution cool down to about 50°C (about when you can comfortably keep your hand on the flask), about 5 mins.
- Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL
- Pour the agarose into a gel tray with the well comb in place.
- Place newly poured gel at 4°C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely solidified.
Gel Purification
- Finished Gel
- Microtube
- DNA Purification Kit
- Once you have run your gel, move it to an open UV box.
- Remove it from any gel tray as plastic will block much of the UV and with a clean, sterile razor blade, slice the desired DNA fragment from the gel.
- Place the gel in a labeled microfuge tube.
- Using a scale, weigh the tube with the gel fragment. The weight of the gel is directly proportional to its liquid volume and this is used to determine how much of each buffer to add during the DNA isolation step.
- Isolate the DNA from the gel using any gel purification kit. Follow kit instructions.
Creating Constructs
- DNA samples
- Transformation efficiency DNA, specific plasmid (for us: pUC 19, 10 pg/ µL), Lucigen, Cat No. F92078-1
- LB Carbenicillin 50 agar plates, Cat No. Teknova, L1801
- E coli BL21 DE3, Life Technology, Cat No. 60106-1
- SOC (Recovery Medium), Lucigen, Cat No. F98226
- 15 mL culture tube
- Vortex
- Tooth pick
- Incubator shaker
- 42°C Water bath
- Ice and ice bucket
- Pipet
- Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells
- Transform 1 µL of DNA of plasmid (for us: pUC19) as transformation efficiency control
- Turn on incubator-shaker at 37°C.
- Turn on incubator for plates at 37°C.
- Set up water bathat 42°C.
- Bring to room temperature S.O.C medium.
- Bring LB plates supplemented with appropriate antibiotic at room temperature.
- Thaw competent cells on ice.
- Aliquots competent cells in as many tubes as needed.
- Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix
- Incubate on ice for 20 minutes
- Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.
- Add 200 µl of 18-25°C SOCmedium and transfer mixture to 15mL Falcon tube
- Incubate in shaker at 37°C, 225 rpm for 30 min
- Plate 2 volumes (50 µL and ~100µL) of the mixture onto 2 different plates of LB agar plates supplemented with Carbenicillin 100 µg/mL
- Incubate plates at 37°C overnight
- Count colonies and estimate transformation efficiency
- Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion
Cloning with pUC Plasmid of Lac Operation (CCA_San_Diego Specific)
- Synthetic DNA
- Synthetic flnB, dbfA1, dbfA2: 3219 bp
- Synthetic flnE, flnD1, ORF16, flnC: 3545 bp
- Synthetic phnF, phnE, phnC, phnD: 4227 bp
- Synthetic phnAc, phnAd, phnB: 3174 bp
- HindIII restriction enzyme, Thermo Fisher, Cat No. ER0501
- Restriction digest Buffer R, Thermo Fisher, Cat No. 00267554
- Antarctic Phosphatase, New England Biolabs Cat No. M0289G
- Agarose, 100 g, Fisher, Cat No. BP-164-100
- 50XTAE Electrophoresis Buffer, 1L, (1X: 40 mMTris, 20mM Acetic Acid, 1 mM EDTA), Thermofisher, Cat No. B49
- Sybr Safe DNA Gel Stain, Invitrogen, Cat No. S33102
- 100 bp DNA marker, Invitrogen, Cat No. 15628-019
- 10X Blue Juice DNA loading buffer, Invitrogen, 10816-015
- Reagent grade water
- Gel DNA Recovery Kit, Zymo Research, Cat No. D4007
- 1.5 mL tubes
- Vortex
- Ice bucket and ice
- Synthetic flnB, dbfA1, dbfA2: 3219 bp
- Synthetic flnE, flnD1, ORF16, flnC: 3545 bp
- Synthetic phnF, phnE, phnC, phnD: 4227 bp
- Synthetic phnAc, phnAd, phnB: 3174 bp
Preparation of Recipient Vector (Insert 1)
- This insert contains the vector (origin of replication of pUC and the first part of the catabolic pathway under the control of the T7 promoter + inducible lacI +RBS)
- Digest clone containing synthetic gene containing insert 1 with the restriction enzyme HindIII that cuts only at one site.
- Restriction Digest Set-up (20µl reaction) in a 1.5 mL tube
- Add the reagents as described below.
- Incubate at 37°C for 1 hours
- Add 2 µl of phosphatase buffer
- Add 1 µl of phosphatase
- Incubate at 37°C for 30 minutes
- Add 2.3 µl of loading buffer at the end of the reaction
- Load the reaction on a 1% Agarose gel
- After running the electrophoresis, cut the linearized band.
- Purify the DNA from the gel using the Zymoclean Gel DNA Recovery Kit
- Elute the DNA from the Zymoclean column with 12 µl of sterile filtered H2O
Component Volume (μl) Final condition
Reagent grade water 8.0 μl
10X Buffer R 2.0 μl 1x
Vector-Insert 1 1.0 μl 0.5 µg
HindIII (10U/µl) 1.0 μl 10U
Preparation of Insert (Insert 2)
- This insert contains the second part of the catabolic pathway under the control of the T7 promoter + inducible lacI +RBS)
- Digest plasmid containing synthetic gene designated as insert 2 with the restriction enzyme HindIII that cuts at 2 sites (HindIII sites are flanking the genes)
- Set-up restriction digestion (20µl reaction) in a 1.5 mL tube
- Turn on water bath at 37°C
- Add the reagents in the order and with volume described in the table below.
- Spin the tube briefly for 15 seconds at 10,000 rpm
- Incubate the tube at 37°C for 1 hours
- Add 2 µl of loading buffer in the tube at the end of the reaction
- Load the digestion reaction on a 1% Agarose gel, TAE
- After running the electrophoresis for 2 hours at 80V, cut out with a razor blade the linearized band of the desired size.
- Purify the DNA from the gel using the Zymoclean Gel DNA Recovery Kit.
- Elute the DNA from the Zymoclean column with 12 µl of sterile filtered H2O
Component Volume (μl) Final condition
Reagent grade water 12.5 μl
10X Buffer R 1.5 μl 1x
Vector-Insert 2 1.0 μl ~0.4 µg
HindIII (10U/µl) 1.0 μl 10U
Preparation of Recipient Vector (Insert 1)
Component | Volume (μl) | Final condition |
---|---|---|
Reagent grade water | 8.0 μl | |
10X Buffer R | 2.0 μl | 1x |
Vector-Insert 1 | 1.0 μl | 0.5 µg |
HindIII (10U/µl) | 1.0 μl | 10U |
Preparation of Insert (Insert 2)
- This insert contains the second part of the catabolic pathway under the control of the T7 promoter + inducible lacI +RBS)
- Digest plasmid containing synthetic gene designated as insert 2 with the restriction enzyme HindIII that cuts at 2 sites (HindIII sites are flanking the genes)
- Set-up restriction digestion (20µl reaction) in a 1.5 mL tube
- Turn on water bath at 37°C
- Add the reagents in the order and with volume described in the table below.
- Spin the tube briefly for 15 seconds at 10,000 rpm
- Incubate the tube at 37°C for 1 hours
- Add 2 µl of loading buffer in the tube at the end of the reaction
- Load the digestion reaction on a 1% Agarose gel, TAE
- After running the electrophoresis for 2 hours at 80V, cut out with a razor blade the linearized band of the desired size.
- Purify the DNA from the gel using the Zymoclean Gel DNA Recovery Kit.
- Elute the DNA from the Zymoclean column with 12 µl of sterile filtered H2O
Component | Volume (μl) | Final condition |
---|---|---|
Reagent grade water | 12.5 μl | |
10X Buffer R | 1.5 μl | 1x |
Vector-Insert 2 | 1.0 μl | ~0.4 µg |
HindIII (10U/µl) | 1.0 μl | 10U |
Cloning with Constructive Promoter of Different Strengths (CCA_San_Diego Specific)
- Synthetic DNA [Fluorene F1, F2] and [Phenanthrene P1 and P2]
- Synthetic F1 : flnB, dbfA1, dbfA2: 3219 bp
- Synthetic F2: flnE, flnD1, ORF16, flnC: 3545 bp
- Synthetic P1: phnF, phnE, phnC, phnD: 4227 bp
- Synthetic P2: phnAc, phnAd, phnB: 3174 bp
- Synthetic DNA [Promoter areas 100, 101, 110]
- Synthetic Promoter 100: 88 bp
- Synthetic Promoter 101: 88 bp
- Synthetic Promoter 110: 88 bp
- Agarose, 100 g, Fisher, Cat No. BP-164-100
- 50XTAE Electrophoresis Buffer, 1L, (1X: 40 mMTris, 20mM Acetic Acid, 1 mM EDTA), Thermofisher, Cat No. B49
- Sybr Safe DNA Gel Stain, Invitrogen, Cat No. S33102
- 1kb plus DNA ladder DNA marker, Thermo Scientific, Cat No. SM1334
- 10X Blue Juice DNA loading buffer, Invitrogen, 10816-015
- Reagent grade water
- Gel DNA Recovery Kit, Zymo Research, Cat No. D4007
- 1.5 mL tubes
- Vortex
- Ice bucket and ice
- Cryo box for restriction enzyme
- P1000 and P200 with corresponding tips
- Synthetic F1 : flnB, dbfA1, dbfA2: 3219 bp
- Synthetic F2: flnE, flnD1, ORF16, flnC: 3545 bp
- Synthetic P1: phnF, phnE, phnC, phnD: 4227 bp
- Synthetic P2: phnAc, phnAd, phnB: 3174 bp
- Synthetic Promoter 100: 88 bp
- Synthetic Promoter 101: 88 bp
- Synthetic Promoter 110: 88 bp
Preparation of Promoter Areas with RBS
- The promoters/RBS regions were designed by the CCA_IGEM team and synthetized by IDT.
- For the writing on plates, the designations for the promoters + RBS were as follows:
- BBa_J23100 + RBS =100
- BBa_J23101 + RBS =101
- BBa_J23110 + RBS =110
- Double Digestion of promoter + RBS region with restriction enzymes EcoRI and SpeI
-
- Restriction Digest Set-up (20µl reaction) in a 1.5 mL tube
- Add the reagents as described below.
- Incubate the tube at 37°C for 15 min
- Note: there is no need to add a loading buffer because the digestion buffer already includes it.
- Load the reaction on a 1.5 % Agarose gel, TAE
- After running the electrophoresis for 1 hour at 80V, cut the linearized band.
- Purify the DNA from the gel using the Zymoclean Gel DNA Recovery Kit
- Elute the DNA from the Zymoclean column with 12 µl of sterile filtered H2O
Component Volume (μl) Final condition
Reagent grade water 12.0 μl
10X Buffer R 2.0 μl 1x
Promoter+RBS Region 4.0 μl ~1 µg
EcoRI (10U/µl) 1.0 μl
SpeI (10U/µl) 1.0 μl
Preparation of insert F1, F2, P1, and P2
- The following polycistronic sequences are cloned behind the 3 different promoter regions:
- For fluorene catabolic pathway
- FLUO-Insert 1 or F1 [Synthetic flnB, dbfA1, dbfA2]
- FLUO-Insert 2 or F2 [Synthetic flnE, flnD1, ORF16, flnC]
- For phenanthrene catabolic pathway
- PHE-Insert 1 or P1 [Synthetic phnF, phnE, phnC, phnD]
- PHE-Insert 2 or P2 [Synthetic phnAc, phnAd, phnB]
- The fragments are digested with 2 enzymes: XbaI and Pst I
- Digest plasmid containing synthetic gene designated F1, F2, P1, and P2 with the restriction enzyme XbaI and PstI
- Set-up restriction digestion (15 µl reaction) in a 1.5 mL tube
- Turn on water bath at 37°C
- Add the reagents in the order and with volume described in the table below.
- Spin the tube briefly for 15 seconds at 10,000 rpm
- Incubate the tube at 37°C for 15 min
- Note: there is no need to add a loading buffer because the digestion buffer already has it.
- Load the digestion reaction on a 1% Agarose gel, TAE
- After running the electrophoresis for 2 hours at 80V, cut out with a razor blade the linearized band of the desired size.
- Purify the DNA from the gel using the Zymoclean Gel DNA Recovery Kit.
- Elute the DNA from the Zymoclean column with 12 µl of sterile filtered H2O
Component Volume (μl) Final condition
Reagent grade water 10.0 μl
10X Buffer 1.5 μl 1X
Preparation of Insert
F1, F2, P1 and P2
1.5 μl ~0.4 µg
Xbal 1.0 μl
Pstl 1.0 μl
- For fluorene catabolic pathway
FLUO-Insert 1 or F1 [Synthetic flnB, dbfA1, dbfA2]
FLUO-Insert 2 or F2 [Synthetic flnE, flnD1, ORF16, flnC]
For phenanthrene catabolic pathway
PHE-Insert 1 or P1 [Synthetic phnF, phnE, phnC, phnD]
PHE-Insert 2 or P2 [Synthetic phnAc, phnAd, phnB]
The fragments are digested with 2 enzymes: XbaI and Pst I
Digest plasmid containing synthetic gene designated F1, F2, P1, and P2 with the restriction enzyme XbaI and PstI
Set-up restriction digestion (15 µl reaction) in a 1.5 mL tube
Turn on water bath at 37°C
Add the reagents in the order and with volume described in the table below.
Spin the tube briefly for 15 seconds at 10,000 rpm
Incubate the tube at 37°C for 15 min
Note: there is no need to add a loading buffer because the digestion buffer already has it.
Load the digestion reaction on a 1% Agarose gel, TAE
After running the electrophoresis for 2 hours at 80V, cut out with a razor blade the linearized band of the desired size.
Purify the DNA from the gel using the Zymoclean Gel DNA Recovery Kit.
Elute the DNA from the Zymoclean column with 12 µl of sterile filtered H2O
Preparation of Promoter Areas with RBS
- BBa_J23100 + RBS =100
- BBa_J23101 + RBS =101
- BBa_J23110 + RBS =110
Component | Volume (μl) | Final condition |
---|---|---|
Reagent grade water | 12.0 μl | |
10X Buffer R | 2.0 μl | 1x |
Promoter+RBS Region | 4.0 μl | ~1 µg |
EcoRI (10U/µl) | 1.0 μl | |
SpeI (10U/µl) | 1.0 μl |
- For fluorene catabolic pathway
- FLUO-Insert 1 or F1 [Synthetic flnB, dbfA1, dbfA2]
- FLUO-Insert 2 or F2 [Synthetic flnE, flnD1, ORF16, flnC]
- For phenanthrene catabolic pathway
- PHE-Insert 1 or P1 [Synthetic phnF, phnE, phnC, phnD]
- PHE-Insert 2 or P2 [Synthetic phnAc, phnAd, phnB]
- The fragments are digested with 2 enzymes: XbaI and Pst I
- Digest plasmid containing synthetic gene designated F1, F2, P1, and P2 with the restriction enzyme XbaI and PstI
- Set-up restriction digestion (15 µl reaction) in a 1.5 mL tube
- Turn on water bath at 37°C
- Add the reagents in the order and with volume described in the table below.
- Spin the tube briefly for 15 seconds at 10,000 rpm
- Incubate the tube at 37°C for 15 min
- Note: there is no need to add a loading buffer because the digestion buffer already has it.
- Load the digestion reaction on a 1% Agarose gel, TAE
- After running the electrophoresis for 2 hours at 80V, cut out with a razor blade the linearized band of the desired size.
- Purify the DNA from the gel using the Zymoclean Gel DNA Recovery Kit.
- Elute the DNA from the Zymoclean column with 12 µl of sterile filtered H2O
Component Volume (μl) Final condition Reagent grade water 10.0 μl 10X Buffer 1.5 μl 1X Preparation of Insert
F1, F2, P1 and P21.5 μl ~0.4 µg Xbal 1.0 μl Pstl 1.0 μl