Edinburgh/Project/Bioelectric-Interface/Methods-and-Bibliography

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Materials and Methods <a href="#" onclick="expand('all-methods')">(expand)</a>
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<span class="intense-emphasis">cloning BioBricks</span>
- microorganisms used: <i>Escherichia coli</i> JM109 and <i>Shewanella oneidensis</i> MR-1. Both organisms were obtained from cultures in Chris French’s lab at the University of Edinburgh.
<br /><i>S. oneidensis</i> cultures were grown on LB agar at room temperature not exceeding 30&deg;C. Plates were subcultured each week.
<br /><i>E. coli</i> cultures were grown on LB agar at room temperature and subcultured by lab staff when needed.
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- PCR: most PCR reactions were performed following OpenWetWare protocol CFrench: <a href="http://openwetware.org/wiki/Cfrench:KodPCR">KodPCR</a>. Optimal annealing temperature for <i>S. oneidensis</i> genes was found to be around 50-52&deg;C while <i>E. coli</i> genes showed good results with annealing temperatures in range of 50-55&deg;C.
<br /><i>S. oneidensis</i> cell suspension in sterile water was used as template for MtrA, MtrCAB, <i>S. oneidensis</i> ccmA-E and ccmF-H genes
<i>E. coli</i> cell suspension in sterile water was used as template for napC and <i>E. coli</i> ccmA-H genes.
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- polyA tailing: for several genes polyA tailing was performed using Taq polymerase and following protocol: 20 minutes denaturation at 95&deg;C, followed by addition of Taq polymerase, followed by 15 minutes extension at 72&deg;C. 
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- gel electrophoresis: gel analysis was used following OpenWetWare CFrench: <a href="http://openwetware.org/wiki/Cfrench:AGE">AGE protocol</a> except 0,5 TAE buffer was used rather that 1x TAE. Staining procedure involved SYBR-Safe.
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- gel purification and DNA purification: for ccm and several ligation attempts for other genes the PCR samples were run on the gel then the appropriate bands were cut out and purified using standarised [company name][product name]  gel purififcation kit. For pure PCR products OpenWetWare protocol CFrench: <a href="http://openwetware.org/wiki/Cfrench:DNAPurification1">DNAPurification1</a> was used. 
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- Vectors used: For most reaction standard BioBrick vector pSB1C3 (provided by the registry) was used, except for samples that were subjected to polyA tailing which were then ligated into pGEM vector (Promega)
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- Restriction digestion: Restriction digests were performed for PCR products along with vector digestion following OpenWetWare <a href="http://openwetware.org/wiki/Cfrench:restriction1">CFrench:restriction1 protocol</a>. For enhanced efficiency varying ratio of insert to vector were used with optimum reached at about 3:1 to 5:1 ratio of insert digest to vector digest.
<br />Analytical restriction digests were also performed for miniprep samples using the original protocol.
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- Ligation: Digested samples were mixed with 1 ul T4 ligase buffer and 1 ul T4 ligase and mixed with water to reach final volume of 20 ul if necessary.  
Alternatively, polyA tailed PCR sampels were mixed with pGEM vector and used directly for ligation.
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- Fusion PCR: following the ligation the samples were used as template for fusion PCR, following KodPCR protocol using forward primer of the gene and reverse primer for the vector. Extension time was adjusted to the length of vector with insert.
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- Transformation: Ligation and fusion PCR products were used to transform <i>E coli</i> JM109 competent cells using OpenWetWare protocol <a href="http://openwetware.org/wiki/Cfrench:compcellprep1">Cfrench:compcellprep1</a>, protocol for preparation of competent cells and cell tansformation).
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-  Transformed cell selection: Transformed cells were spread on LB agar with chloramphenicol (for pSB1C3 vector) or LB agar with Carbenicillin, Xgal and IPTG.
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Following overnight incubation at 37&deg;C white colonies were chosen (rather than red colonies from pSB1C3-RFP vector or blue colonies from pGEM vector) and subcultured on the plates containing the same medium.
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- Miniprepping: Subcultures were used to set up overnight liquid cultures in 2,5 ml of LB. Miniprepping was performed using either OpenWetWare protocol <a href="http://openwetware.org/wiki/Cfrench:minipreps1">Cfrench:minipreps1</a> or standarised miniprep kit [name].
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Minipreps were then restriction digested and run on the gel
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- Sequencing: size confirmed minipreps were then sent for sequencing in the University of Edinburgh GenePool.
<span class="intense-emphasis">Fuel cell</span>
- Fuel cells were constructed using carbon weave electrodes and reference electrodes provided by Matthew Knighton from Dr Bruce Ward’s lab. 
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- Fuel cells were assembled by inserting bottle cap with attached carbon weave electrode into 500 or 250 ml standard glass bottles. The electrodes were attached to the caps using silicone sealant. Bottles were then autoclaved. In sterile conditions, reference electrodes were dipped in alcohol, inserted into the cap of the bottles and sealed with silicon sealant. Half fuel cells were then filled with media and inoculated with bacteria and sealed with parafilm in order to ensure anaerobic growth. They were then grown in room temperature
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- media used: standard LB, M9 (<a href="http://openwetware.org/wiki/M9_medium/minimal">minimal growth medium</a>) supplemented with 0,4% glycerol or sodium acetate. 
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- Measurements were obtained using a digital multimeter.
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<a name="bibliography">Bibliography</a> <a href="#" onclick="expand('works-cited')">(expand)</a>
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<b>1. Jensen, H. M., Albers, A. E., Malley, K. R., Londer, Y. Y. , Cohen, B. E., Helms, B. A., Weigele, P., Groves, J. T. & Ajo-Franklin, C. M.</a> (2010). <i>Engineering of a synthetic electron conduit in living cells.</i> PNAS  107, 19213-19218
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<b>2. Stewart, V., Lu, Y. & Darwin, A. J.</b> (2002). <i>Periplasmic Nitrate Reductase (NapABC Enzyme) supports Anaerobic Respiration by Escherichia coli K-12.</i> Journal of Bacteriology 184, 1314-1323
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<b>3. Marritt, S. J., Lowe, T. G., Bye, J., McMillan, D.G.G., Shi, L., Frederickson, J., Zachara, J., Richardson, D. J., Cheesman, M. R., Jeuken L.J.C. & Butt, J. N.</b> (2012). <i>A functional description of CymA, an electron-transfer hub supporting anaerobic respiratory flexibility in Shewanella.</i> Biochemical Journal 444, 465-474
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<b>4. Richter, K., Schicklberger, M., Gescher, J.</b> (2011). <i>Dissimilatory reduction of extracellular electron acceptors in anaerobic respiration.</i> Applied and Environmental Microbiology 78, 913-921
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