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| − | <div class="panel-heading" role="tab" id="P1">
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| − | <h4 class="panel-title">
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| − | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P1-collapse" aria-expanded="false" aria-controls="P1-collapse">
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| − | <div class="col-md-11">Competent Cell Preparation</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div>
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| − | <br>
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| − | 1. Grow Paracoccus cultures in liquid media
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| − | 2. Dilute 500mL of Paracoccus denitrificans culture in 500mL SGM17 medium
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| − | ***COLD ROOM FROM HERE***
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| − | 3.Harvest by centrifuge 250mL samples each in 2 four centrifuge bottles at 5000xg at 4 C. Carefully dump out supernatant.
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| − | 4.Completely resuspend (shake vigorously without up & down motion to avoid liquid loss) the pellets from the previous step in all centrifuge bottles each with 10050mL of 0.5 M sucrose solution with 10% glycerol.
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| − | 5. Combine the liquid(containing resuspended pellets) into one centrifuge bottle, add 200mL water to the other empty centrifuge bottle for counterbalance.
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| − | 6. Centrifuge the two bottles at 5000xg at 4 C.
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| − | 7. Dump out supernatant and resuspend the pellets with 100mL of 0.5 M sucrose solution with 10% glycerol. Counter-balance bottle should have 100mL of water.
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| − | 8. Centrifuge the two bottles at 5000xg at 4 C.
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| − | 9. Dump out supernatant and resuspend the resulting pellets in 10mL of 0.5 M sucrose solution with 10% glycerol.
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| − | 10. Aliquot the final 10mL of liquid into 0.25mL(250uL) with 40 microcentrifuge tubes.
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| − | 11. Flash freeze with liquid nitrogen and store at -80 C.
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| − | <br>
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| − | <button data-toggle="collapse" data-target="#pdtransformation">Transformation via Electroporation</button>
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| − | <div id="pdtransformation" class="collapse"><br>
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| − | <li>1. Thaw and mix 50 uL portions cells with 5 uL DNA
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| − | <li>2. Transfer to ice cold electroporation cuvette
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| − | <li>3. Electroporate with single pulse set at 25 uF and 2.0 kV for 4.5 to 5 ms
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| − | <li>4. The electroporator as a Gene-Pulser by BioRad connected to a 200-(-) resistor also by biorad
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| − | <li>5. IMMEDIATELY following electroporation: carefully mix suspension with 0.95 mL ice cold SGM17 containing 20 mM MgCl2, 2 mM CaCl2 and incubate on ice for 5 minutes
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| − | <li>6. Dilutions were made in the SGM17 media
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| − | <li>7. Incubate cells at 30 C for 2 hours
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| − | <li>8. Take 100 uL portions and plate.
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| − | <h2>Measurement protocols</h2>
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| − | <button data-toggle="collapse" data-target="#promotercharacterization">Mn-Sod, VhB and T7 Promoter Characterization</button>
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| − | <b>Isolate Single colonies</b>
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| − | <li>1. Select glycerol stock cultures from -80 C storage to begin
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| − | <li>2. Thaw on ice for 15 minutes and observe when the cell stock begins to melt
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| − | <li>3. Take a plastic p-loop or other spreading instrument and spread on a selective media agar plate containing the appropriate antibiotics
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| − | <li>If the culture is P. denitrificans, culture on PD media or standard LB
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| − | <li>If the culture is E. coli use LB media
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| − | <li>If using untransformed bacteria, equivalent non-selective media should be used
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| − | <li>4. Incubate both P. denitrificans and E. coli overnight at 37 C
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| − | <li>5. Isolate single colonies after overnight incubation and resuspend in 60 mL volumes of liquid selective LB media with appropriate antibiotics
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| − | <br><br>
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| − | <b>Preculture for Device Testing</b>
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| − | <button data-toggle="collapse" data-target="#ammoniumconversion">Measuring Efficiency of Ammonium Conversion and Nitrate Production Over Time
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| − | textextextextextextextext
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