Team:Edinburgh/Project/Bioelectric-Interface/Bio-electric-Interface-BioBricks-Cloning

From 2012.igem.org

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Bio-electric interface
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Bio-electric Interface:
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Bio-electric interface BioBricks cloning
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BioBrick Cloning and Characterisation(Methods and Results)
</p>
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Procedure
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Methods
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- 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. <i>S. oneidensis</i> cultures were grown on LB agar at room temperature not exceeding 30&deg;C. Plates were subcultured each week. <i>E. coli</i> cultures were grown on LB agar at room temperature and subcultured by lab staff when needed.
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- Bacteria 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. <i>S. oneidensis</i> cultures were grown on LB agar at room temperature not exceeding 30&deg;C. Plates were subcultured each week. <i>E. coli</i> cultures were grown on LB agar in a 37 degree incubator overnight and then left to grow at room temperature and subcultured by lab staff when needed.
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<br /><br />
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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. <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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- PCR: most PCR reactions were performed using the 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 the range of 50-55&deg;C. <i>S. oneidensis</i> cell suspension in sterile water was used as template for <i>mtrA, MtrCAB, S. oneidensis ccmA-E</i> and <i>ccmF-H</i> genes. <i>E. coli</i> cell suspension in sterile water was used as template for <i>napC </i> and <i>E. coli</i> <i>ccmA-H</i> genes.
<br /><br />
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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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- PolyA tailing: for several genes polyA tailing was performed using Taq polymerase and the 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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<br /><br />
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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 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. Gel staining was done using the SYBR-Safe staining solution.
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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 standardised QIAquick Gel Extraction Kit. For pure PCR products OpenWetWare protocol CFrench: <a href="http://openwetware.org/wiki/Cfrench:DNAPurification1">DNAPurification1</a> was used.
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- Gel purification and DNA purification: for <i>ccm</i> 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 standardised QIAquick Gel Extraction Kits. 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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- Vectors used: For most reaction the standard BioBrick vector pSB1C3 (provided by the Registry) was used, except for samples that were subjected to polyA tailing which were then ligated into the 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. Analytical restriction digests were also performed for miniprep samples using the original protocol.
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- Restriction digestions: Restriction digests were performed for PCR products along with vector digestion following the OpenWetWare <a href="http://openwetware.org/wiki/Cfrench:restriction1">CFrench:restriction1 protocol</a>. For enhanced efficiency, varying ratios of insert to vector were used with the optimum ratio reached at about 3:1 to 5:1 ratio of insert digest to vector digest. 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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- Ligations: Digested samples were mixed with 1 ul T4 ligase buffer and 1 ul T4 ligase and mixed with water to reach the 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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- Fusion PCR: following the ligation the samples were used as template for fusion PCR, following KodPCR protocol using forward primer for the gene and reverse primer for the vector. Extension time was adjusted to suit the length of the 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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- Transformation: Ligation and fusion PCR products were used to transform <i>E coli</i> JM109 competent cells using the OpenWetWare protocol <a href="http://openwetware.org/wiki/Cfrench:compcellprep1">Cfrench:compcellprep1</a> for preparation of competent cells and cell transformation).
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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. 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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- Transformed cell selection: Transformed cells were spread on LB agar with chloramphenicol (when using pSB1C3 vector) or LB agar with carbenicillin, Xgal and IPTG. 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 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 QIAprep Spin MiniPrep Kit.. Minipreps were then restriction digested and run on the gel
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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 the OpenWetWare protocol <a href="http://openwetware.org/wiki/Cfrench:minipreps1">Cfrench:minipreps1</a> or standarised QIAprep Spin MiniPrep Kit. Minipreps were then restriction digested and run on a gel.
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- Sequencing: size confirmed minipreps were then sent for sequencing in the University of Edinburgh GenePool.
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- Sequencing: size confirmed minipreps were then sent for sequencing at the University of Edinburgh GenePool.
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<b>NapC</b><br />
<b>NapC</b><br />
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- Throughout summer we have managed to clone napC gene from <i>E coli</i> (<a href="http://partsregistry.org/Part:BBa_K917003">BBa_K917003</a>). We have inserted the gene into the standard BioBrick vector pSB1C3 and submitted it to the parts registry. We have then linked napC gene to lac promoter (<a href="http://partsregistry.org/Part:BBa_K917012">BBa_K917012</a>) to characterise its functionality.<br />
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- Throughout the summer we have managed to clone the <i>napC</i> gene from <i>E coli</i> (<a href="http://partsregistry.org/Part:BBa_K917003">BBa_K917003</a>). We have inserted the gene into the standard BioBrick vector pSB1C3 and submitted it to the parts Registry. We have then linked the <i>napC</i> gene to the lac promoter (<a href="http://partsregistry.org/Part:BBa_K917012">BBa_K917012</a>) to characterise its functionality.<br/><br />
<img id="fig01" src="https://static.igem.org/mediawiki/2012/b/bf/Bio-el-interface-fig02.JPG"><br />
<img id="fig01" src="https://static.igem.org/mediawiki/2012/b/bf/Bio-el-interface-fig02.JPG"><br />
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Figure 1: napC PCR
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<b>Figure 1:</b> <i>napC</i> PCR
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<img id="fig02" src="https://static.igem.org/mediawiki/2012/9/9b/Bio-el-interface-fig03.JPG"><br />
<img id="fig02" src="https://static.igem.org/mediawiki/2012/9/9b/Bio-el-interface-fig03.JPG"><br />
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Figure 2: napC miniprep
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<b>Figure 2:</b> <i>napC</i> miniprep
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<img id="fig03" src="https://static.igem.org/mediawiki/2012/d/d8/Gelpic010.jpg"><br />
<img id="fig03" src="https://static.igem.org/mediawiki/2012/d/d8/Gelpic010.jpg"><br />
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Figure 3: pLac-napC construct analytical digestion with XbaI and PstI <br />
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<b>Figure 3:</b> pLac-napC construct analytical digestion with XbaI and PstI <br />
Lanes 3, 4 = pSB1C3-Plac-lacZ'-napC, clones 1 and 2, digested with XbaI-PstI. Clone 2 looks as expected, clone 1 has an unexpected band around 0.6 kb.  
Lanes 3, 4 = pSB1C3-Plac-lacZ'-napC, clones 1 and 2, digested with XbaI-PstI. Clone 2 looks as expected, clone 1 has an unexpected band around 0.6 kb.  
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<b>MtrA</b><br />
<b>MtrA</b><br />
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- We also managed to obtain MtrA gene (<a href="http://partsregistry.org/Part:BBa_K917008">BBa_K917008</a>) of S. oneidensis and cloned it into pSB1C3 plasmid.<br />
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- We also managed to obtain the <i>mtrA</i> gene (<a href="http://partsregistry.org/Part:BBa_K917008">BBa_K917008</a>) of <i>S. oneidensis</i> and cloned it into the pSB1C3 plasmid.<br />
<img id="fig04" src="https://static.igem.org/mediawiki/2012/2/29/Bio-el-interface-fig04.JPG"><br />
<img id="fig04" src="https://static.igem.org/mediawiki/2012/2/29/Bio-el-interface-fig04.JPG"><br />
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Figure 4: MtrA PCR
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<b>Figure 4:</b> MtrA PCR
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<img id="fig05" src="https://static.igem.org/mediawiki/2012/6/69/Bio-el-interface-fig05.JPG"><br />
<img id="fig05" src="https://static.igem.org/mediawiki/2012/6/69/Bio-el-interface-fig05.JPG"><br />
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Figure 5: MtrA transformation
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<b>Figure 5:</b> <i>mtrA</i> transformation
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<br /><br />
<img id="fig06" src="https://static.igem.org/mediawiki/2012/9/90/Bio-el-interface-fig06.JPG"><br />
<img id="fig06" src="https://static.igem.org/mediawiki/2012/9/90/Bio-el-interface-fig06.JPG"><br />
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Figure 6: MtrA miniprep
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<b>Figure 6:</b> <i>mtrA</i> miniprep
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Obtained MtrA gene contains an internal PstI site which needs to be mutagenised prior to submission and use.
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The obtained <i>mtrA</i> gene contains an internal PstI site which needs to be mutated out prior to submission and use.
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<b>cymA</b><br />
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<b>CymA</b><br />
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We have managed to clone cymA gene (<a href="http://partsregistry.org/Part:BBa_K917009">BBa_K917009</a>) from <i>S. oneidensis</i>. We have tested the gene for internal restriction sites and linked cymA gene to lac promoter (<a href="http://partsregistry.org/Part:BBa_K917014">BBa_K917014</a>) to characterise its functionality.<br/>
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We have managed to clone the <i>cymA</i> gene (<a href="http://partsregistry.org/Part:BBa_K917009">BBa_K917009</a>) from <i>S. oneidensis</i>. We have tested the gene for internal restriction sites and linked the <i>cymA</i> gene to the lac promoter (<a href="http://partsregistry.org/Part:BBa_K917014">BBa_K917014</a>) to characterise its functionality.<br/>
<img id="fig07" src="https://static.igem.org/mediawiki/2012/e/ea/Gelpic003.jpg"><br />
<img id="fig07" src="https://static.igem.org/mediawiki/2012/e/ea/Gelpic003.jpg"><br />
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Figure 7: lanes 3, 4 = pSB1C3-cymA clones 1 and 2, analytically digested with EcoRI. Band has size correct for linearised plasmid with the gene(3kb) <br />
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<b>Figure 7:</b> lanes 3, 4 = pSB1C3-cymA clones 1 and 2, analytically digested with EcoRI. Band has size correct for linearised plasmid with the gene(3kb) <br />
lanes 5, 6 = pSB1C3-cymA clones 1 and 2, double digested with EcoRI/SpeI. <br /><br />
lanes 5, 6 = pSB1C3-cymA clones 1 and 2, double digested with EcoRI/SpeI. <br /><br />
<img id="fig08" src="https://static.igem.org/mediawiki/2012/7/71/Gelpic005.jpg"><br />
<img id="fig08" src="https://static.igem.org/mediawiki/2012/7/71/Gelpic005.jpg"><br />
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Figure 8: pSB1C3-cymA clones 1 and 2, testing internal restriction sites. <br />  
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<b>Figure 8:</b> pSB1C3-cymA clones 1 and 2, testing internal restriction sites. <br />  
Lanes 1, 2 = clones 1 and 2, NdeI.<br />
Lanes 1, 2 = clones 1 and 2, NdeI.<br />
Lanes 3, 4 = clone 1, XbaI and XbaI/HindIII. <br />  
Lanes 3, 4 = clone 1, XbaI and XbaI/HindIII. <br />  
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Gel results appear as expected <br /><br />
Gel results appear as expected <br /><br />
<img id="fig09" src="https://static.igem.org/mediawiki/2012/c/c7/Gelpic009.jpg"><br />
<img id="fig09" src="https://static.igem.org/mediawiki/2012/c/c7/Gelpic009.jpg"><br />
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Figure 9: Lanes 4 to 6, pSB1C3-Plac-lacZ'-cymA clones 1-3, analytically digested with XbaI-PStI. <br />
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<b>Figure 9:</b> Lanes 4 to 6, pSB1C3-Plac-lacZ'-cymA clones 1-3, analytically digested with XbaI-PStI. <br />
Clones 1 and 2 show bands of appropriate sizes. <br /><br />
Clones 1 and 2 show bands of appropriate sizes. <br /><br />
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<b>ccm cytochrome maturation cluster of <i>E. coli</i></b><br />
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<b><i>ccm</i> cytochrome maturation cluster of <i>E. coli</i></b><br />
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We have cloned <i>E. coli</i> ccm gene cluster (<a href="http://partsregistry.org/Part:BBa_K917006">BBa_K917006</a>), analysed its internal restriction sites and linked it with lac promoter (<a href="http://partsregistry.org/Part:BBa_K917013">BBa_K917013</a>).<br />
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We have cloned the <i>E. coli</i> <i>ccm</i> gene cluster (<a href="http://partsregistry.org/Part:BBa_K917006">BBa_K917006</a>), analysed its internal restriction sites and linked it with the lac promoter (<a href="http://partsregistry.org/Part:BBa_K917013">BBa_K917013</a>).<br />
<img id="fig10" src="https://static.igem.org/mediawiki/2012/d/d3/Bio-el-interface-fig08.JPG"><br />
<img id="fig10" src="https://static.igem.org/mediawiki/2012/d/d3/Bio-el-interface-fig08.JPG"><br />
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Figure 10: <i>E coli</i> ccm genes PCR (right lanes)<br /><br />
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<b>Figure 10:</b> <i>E coli</i> <i>ccm</i> genes PCR (right lanes)<br /><br />
<img id="fig11" src="https://static.igem.org/mediawiki/2012/6/62/Gelpic004.jpg"><br />  
<img id="fig11" src="https://static.igem.org/mediawiki/2012/6/62/Gelpic004.jpg"><br />  
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Figure 11: pSB1C3-ccm clones 1-6, digested with EcoRI. <br />
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<b>Figure 11:</b> pSB1C3-ccm clones 1-6, digested with EcoRI. <br />
Clones 1 and 5 show bands of expected size, the other clones are too small <br /><br />
Clones 1 and 5 show bands of expected size, the other clones are too small <br /><br />
<img id="fig12" src="https://static.igem.org/mediawiki/2012/f/fc/Gelpic006.jpg"><br />
<img id="fig12" src="https://static.igem.org/mediawiki/2012/f/fc/Gelpic006.jpg"><br />
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Figure 12: pSB1C3-ccm clones 1 and 5, testing internal restriction sites. <br />
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<b>Figure 12:</b> pSB1C3-ccm clones 1 and 5, testing internal restriction sites. <br />
Lanes 1, 2 = EcoRI/SpeI digestion. <br />
Lanes 1, 2 = EcoRI/SpeI digestion. <br />
Lanes 3, 4 = BamHI digestion. <br />
Lanes 3, 4 = BamHI digestion. <br />
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Results appear as expected assuming 2 of the 3 ClaI sites are uncuttable due to overlapping dam methylation <br /><br />
Results appear as expected assuming 2 of the 3 ClaI sites are uncuttable due to overlapping dam methylation <br /><br />
<img id="fig13" src="https://static.igem.org/mediawiki/2012/d/d8/Gelpic010.jpg"><br />
<img id="fig13" src="https://static.igem.org/mediawiki/2012/d/d8/Gelpic010.jpg"><br />
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Figure 13: Lanes 1, 2 = pSB1C3-Plac-lacZ'-ccm, clones 1 and 2, digested with XbaI-PstI.  <br />
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<b>Figure 13:</b> Lanes 1, 2 = pSB1C3-Plac-lacZ'-ccm, clones 1 and 2, digested with XbaI-PstI.  <br />
Clone 2 looks as expected, clone 1 has an unexpected band around 0.6 kb. <br /><br />
Clone 2 looks as expected, clone 1 has an unexpected band around 0.6 kb. <br /><br />
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<b>MtrCAB and <i>S. oneidensis</i> ccm</b><br />
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<b>MtrCAB and <i>S. oneidensis ccm</i></b><br />
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- We have also obtained good quality pure PCR products of MtrCAB and ccm genes from <i>S. oneidensis</i>
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We have also obtained good quality pure PCR products of <i>mtrCAB</i> and <i>ccm</i> genes from <i>S. oneidensis</i><br/>
<img id="fig14" src="https://static.igem.org/mediawiki/2012/6/64/Bio-el-interface-fig07.JPG" width="350"><br />
<img id="fig14" src="https://static.igem.org/mediawiki/2012/6/64/Bio-el-interface-fig07.JPG" width="350"><br />
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Figure 14: MtrCAB PCR
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<b>Figure 14:</b> MtrCAB PCR
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<img id="fig15" src="https://static.igem.org/mediawiki/2012/d/d3/Bio-el-interface-fig08.JPG" ><br />
<img id="fig15" src="https://static.igem.org/mediawiki/2012/d/d3/Bio-el-interface-fig08.JPG" ><br />
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Figure 15: ccm genes from <i>S. oneidensis</i> and <i>E. coli</i>
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<b>Figure 15:</b> <i>ccm</i> genes from <i>S. oneidensis</i> and <i>E. coli</i>
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</p>
</p>
<p class="h2">
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Discussion and conclusions
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BioBrick characterisation <br />
</p>
</p>
<p class="normal-text">
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<b>We managed to obtain napC, cymA, ccm and mtrA genes which are now ready for testing, using haem staining and half fuel cells.</b> MtrA gene still contains and internal PstI site which has to be mutated out prior to submission. <b>We have linked napC, ccm and cymA to lac promoter to test these new BioBricks using haem staining and half fuel cells with our current results as reference.</b> However it is possible that the transformed cells will require multiple genes  to function properly. Ccm genes are responsible for cytochrome maturation which is necessary for proper folding of multihaem cytochromes such as NapC, CymA and especially decahaem cytochrome MtrA.
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<i>ccm, napC and cymA</i><br />
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We have tested the expression of NapC and CymA proteins in <i>ccm</i> transformed cells (<i>ccm</i> gene cluster BioBrick was transferred into pSB4K5 vector to allow for double epxression of <i>ccm</i> and <i>cymA/napC</i> genes). Cell pellets were scanned prior to sonication and <i>cymA</i> and <i>napC</i> transformed cells show slightly more intense colour, indicating higher concentration of haem in cells. <br /><br />
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<img src="https://static.igem.org/mediawiki/2012/6/69/Cytochrome_compare.jpg" width="700"><br /><br />
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Following the sonication protein samples were run on a gel and were stained for haem using the following protocol: <br /> <br />
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The NuPAGE® gel was first soaked in 100 ml solution-I
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(30 ml methanol, 70 ml 250 mM NaAc, pH=5.2) for 10 min, and then was moved into 50 ml
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solution-II (15 mg 3,3',5,5'-Tetramethylbenzidine (TMBZ), 15 ml methanol and 35 ml 250 mM NaAc, pH=5.2; TMBZ was prepared
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by dissolving the powder in methanol and placed in the dark) in an opaque box to prevent
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the light. The box was incubated for 25 min at room temperature. 150 ul 30% (w/w) H2O2
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solution was then added into the box which was further incubated in the dark for 3~5 min
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after gentle mixing. The stained bands were fixed by washing the gel with dH2O.
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The longer products (MtrCAB and ccm genes) seem to be more problematic to clone, with digestion/ligation step being the limiting factor, despite using several alternative techniques (polyA tailing, fusion PCR).
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Haem staining resulted in clear bands forming in all samples, probably representing one of the Ccm proteins, most likely CcmE (haem chaperone protein). In <i>napC</i> and <i>cymA</i> samples additional bands were present, indicated by the arrows.<br /><br />
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<img src="https://static.igem.org/mediawiki/2012/2/2c/Gel_haem_compare_.jpg" width="700"><br />
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We had some success in cloning mtrCAB and <i> S. oneidensis</i> ccm genes which may enhance the efficiency of the system. We intend to clone these genes into the pSB1C3 vector (<a href="http://partsregistry.org/Part:BBa_K917007">BBa_K917007</a>), link them to a promoter and test them together in order to assess the efficiency of the system.
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Haem staining: lane 1: pure cytochrome c <b>control</b>, <br />
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lane 2: protein extract from <i><b>ccm</b></i> transformed cells, membrane fraction; <br />
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lane 3: protein extract from <i><b>ccm</i> and <i>cymA</i></b> transformed cells, membrane fraction; <br />
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lane 4: protein extract from <i><b>ccm</i> and <i>napC</i></b> transformed cells, membrane fraction; <br />
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lane 5: protein extract from <i><b>ccm</b></i> transformed cells, cytosol fraction; <br />
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lane 6: protein extract from <i><b>ccm</i> and <i>cymA</i></b> transformed cells, cytosol fraction; <br />
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lane 7: protein extract from <i><b>ccm</i> and <i>napC</i></b> transformed cells, cytosol fraction
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These results show that expression of CymA and NapC is successful. Interestingly NapC predominates in the soluble fraction and CymA appears in the membrane fraction. This is an unexpected result as both proteins are intermembrane proteins. We hope to inspect this fact closer to determine its significance in our system.
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<a href="https://2012.igem.org/Team:Edinburgh/Project/Bioelectric-Interface"><span class="intense-emphasis">&lt;&lt;Prev</span></a><span style="color:white;">__</span>2/4</span><span style="color:white;">__</span><a href="https://2012.igem.org/Team:Edinburgh/Project/Bioelectric-Interface/Microbial-Half-Fuel-Cells"><span class="intense-emphasis">Next&gt;&gt;</span></a></span>
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The complete electron export conduit should be able to reliably export electrons in response to an external stimulus. This system can be used to enhance the current biosensor systems. One possible application would be to link our system to arsenic promoter and construct a reliable, cheap arsenic biosensor which would generate easy to interpret data that can be stored on a computer.
 
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Latest revision as of 19:52, 26 October 2012

Bio-electric Interface:

BioBrick Cloning and Characterisation(Methods and Results)

Methods

- Bacteria used: Escherichia coli JM109 and Shewanella oneidensis MR-1. Both organisms were obtained from cultures in Chris French’s lab at the University of Edinburgh. S. oneidensis cultures were grown on LB agar at room temperature not exceeding 30°C. Plates were subcultured each week. E. coli cultures were grown on LB agar in a 37 degree incubator overnight and then left to grow at room temperature and subcultured by lab staff when needed.

- PCR: most PCR reactions were performed using the following OpenWetWare protocol CFrench: KodPCR. Optimal annealing temperature for S. oneidensis genes was found to be around 50-52°C while E. coli genes showed good results with annealing temperatures in the range of 50-55°C. S. oneidensis cell suspension in sterile water was used as template for mtrA, MtrCAB, S. oneidensis ccmA-E and ccmF-H genes. E. coli cell suspension in sterile water was used as template for napC and E. coli ccmA-H genes.

- PolyA tailing: for several genes polyA tailing was performed using Taq polymerase and the following protocol: 20 minutes denaturation at 95°C, followed by addition of Taq polymerase, followed by 15 minutes extension at 72°C.

- Gel electrophoresis: gel analysis was used following OpenWetWare CFrench: AGE protocol except 0,5 TAE buffer was used rather that 1x TAE. Gel staining was done using the SYBR-Safe staining solution.

- 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 standardised QIAquick Gel Extraction Kits. For pure PCR products OpenWetWare protocol CFrench: DNAPurification1 was used.

- Vectors used: For most reaction the standard BioBrick vector pSB1C3 (provided by the Registry) was used, except for samples that were subjected to polyA tailing which were then ligated into the pGEM vector (Promega)

- Restriction digestions: Restriction digests were performed for PCR products along with vector digestion following the OpenWetWare CFrench:restriction1 protocol. For enhanced efficiency, varying ratios of insert to vector were used with the optimum ratio reached at about 3:1 to 5:1 ratio of insert digest to vector digest. Analytical restriction digests were also performed for miniprep samples using the original protocol.

- Ligations: Digested samples were mixed with 1 ul T4 ligase buffer and 1 ul T4 ligase and mixed with water to reach the final volume of 20 ul if necessary. Alternatively, polyA tailed PCR sampels were mixed with pGEM vector and used directly for ligation.

- Fusion PCR: following the ligation the samples were used as template for fusion PCR, following KodPCR protocol using forward primer for the gene and reverse primer for the vector. Extension time was adjusted to suit the length of the vector with insert.

- Transformation: Ligation and fusion PCR products were used to transform E coli JM109 competent cells using the OpenWetWare protocol Cfrench:compcellprep1 for preparation of competent cells and cell transformation).

- Transformed cell selection: Transformed cells were spread on LB agar with chloramphenicol (when using pSB1C3 vector) or LB agar with carbenicillin, Xgal and IPTG. Following overnight incubation at 37°C white colonies were chosen (rather than red colonies from pSB1C3-RFP vector or blue colonies from pGEM vector) and subcultured on plates containing the same medium.

- Miniprepping: Subcultures were used to set up overnight liquid cultures in 2,5 ml of LB. Miniprepping was performed using either the OpenWetWare protocol Cfrench:minipreps1 or standarised QIAprep Spin MiniPrep Kit. Minipreps were then restriction digested and run on a gel.

- Sequencing: size confirmed minipreps were then sent for sequencing at the University of Edinburgh GenePool.

Results

NapC
- Throughout the summer we have managed to clone the napC gene from E coli (BBa_K917003). We have inserted the gene into the standard BioBrick vector pSB1C3 and submitted it to the parts Registry. We have then linked the napC gene to the lac promoter (BBa_K917012) to characterise its functionality.


Figure 1: napC PCR


Figure 2: napC miniprep


Figure 3: pLac-napC construct analytical digestion with XbaI and PstI
Lanes 3, 4 = pSB1C3-Plac-lacZ'-napC, clones 1 and 2, digested with XbaI-PstI. Clone 2 looks as expected, clone 1 has an unexpected band around 0.6 kb.

MtrA
- We also managed to obtain the mtrA gene (BBa_K917008) of S. oneidensis and cloned it into the pSB1C3 plasmid.

Figure 4: MtrA PCR


Figure 5: mtrA transformation


Figure 6: mtrA miniprep

The obtained mtrA gene contains an internal PstI site which needs to be mutated out prior to submission and use.

CymA
We have managed to clone the cymA gene (BBa_K917009) from S. oneidensis. We have tested the gene for internal restriction sites and linked the cymA gene to the lac promoter (BBa_K917014) to characterise its functionality.

Figure 7: lanes 3, 4 = pSB1C3-cymA clones 1 and 2, analytically digested with EcoRI. Band has size correct for linearised plasmid with the gene(3kb)
lanes 5, 6 = pSB1C3-cymA clones 1 and 2, double digested with EcoRI/SpeI.


Figure 8: pSB1C3-cymA clones 1 and 2, testing internal restriction sites.
Lanes 1, 2 = clones 1 and 2, NdeI.
Lanes 3, 4 = clone 1, XbaI and XbaI/HindIII.
Lanes 5, 6 = clone 2, XbaI and XbaI/HindIII.
Gel results appear as expected


Figure 9: Lanes 4 to 6, pSB1C3-Plac-lacZ'-cymA clones 1-3, analytically digested with XbaI-PStI.
Clones 1 and 2 show bands of appropriate sizes.

ccm cytochrome maturation cluster of E. coli
We have cloned the E. coli ccm gene cluster (BBa_K917006), analysed its internal restriction sites and linked it with the lac promoter (BBa_K917013).

Figure 10: E coli ccm genes PCR (right lanes)


Figure 11: pSB1C3-ccm clones 1-6, digested with EcoRI.
Clones 1 and 5 show bands of expected size, the other clones are too small


Figure 12: pSB1C3-ccm clones 1 and 5, testing internal restriction sites.
Lanes 1, 2 = EcoRI/SpeI digestion.
Lanes 3, 4 = BamHI digestion.
Lanes 5, 6 = ClaI digestion.
Results appear as expected assuming 2 of the 3 ClaI sites are uncuttable due to overlapping dam methylation


Figure 13: Lanes 1, 2 = pSB1C3-Plac-lacZ'-ccm, clones 1 and 2, digested with XbaI-PstI.
Clone 2 looks as expected, clone 1 has an unexpected band around 0.6 kb.

MtrCAB and S. oneidensis ccm
We have also obtained good quality pure PCR products of mtrCAB and ccm genes from S. oneidensis

Figure 14: MtrCAB PCR


Figure 15: ccm genes from S. oneidensis and E. coli

BioBrick characterisation

ccm, napC and cymA
We have tested the expression of NapC and CymA proteins in ccm transformed cells (ccm gene cluster BioBrick was transferred into pSB4K5 vector to allow for double epxression of ccm and cymA/napC genes). Cell pellets were scanned prior to sonication and cymA and napC transformed cells show slightly more intense colour, indicating higher concentration of haem in cells.



Following the sonication protein samples were run on a gel and were stained for haem using the following protocol:

The NuPAGE® gel was first soaked in 100 ml solution-I (30 ml methanol, 70 ml 250 mM NaAc, pH=5.2) for 10 min, and then was moved into 50 ml solution-II (15 mg 3,3',5,5'-Tetramethylbenzidine (TMBZ), 15 ml methanol and 35 ml 250 mM NaAc, pH=5.2; TMBZ was prepared by dissolving the powder in methanol and placed in the dark) in an opaque box to prevent the light. The box was incubated for 25 min at room temperature. 150 ul 30% (w/w) H2O2 solution was then added into the box which was further incubated in the dark for 3~5 min after gentle mixing. The stained bands were fixed by washing the gel with dH2O.

Haem staining resulted in clear bands forming in all samples, probably representing one of the Ccm proteins, most likely CcmE (haem chaperone protein). In napC and cymA samples additional bands were present, indicated by the arrows.


Haem staining: lane 1: pure cytochrome c control,
lane 2: protein extract from ccm transformed cells, membrane fraction;
lane 3: protein extract from ccm and cymA transformed cells, membrane fraction;
lane 4: protein extract from ccm and napC transformed cells, membrane fraction;
lane 5: protein extract from ccm transformed cells, cytosol fraction;
lane 6: protein extract from ccm and cymA transformed cells, cytosol fraction;
lane 7: protein extract from ccm and napC transformed cells, cytosol fraction

These results show that expression of CymA and NapC is successful. Interestingly NapC predominates in the soluble fraction and CymA appears in the membrane fraction. This is an unexpected result as both proteins are intermembrane proteins. We hope to inspect this fact closer to determine its significance in our system.



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Bibliography (expand)

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. (2010). Engineering of a synthetic electron conduit in living cells. PNAS 107, 19213-19218

2. Stewart, V., Lu, Y. & Darwin, A. J. (2002). Periplasmic Nitrate Reductase (NapABC Enzyme) supports Anaerobic Respiration by Escherichia coli K-12. Journal of Bacteriology 184, 1314-1323

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. (2012). A functional description of CymA, an electron-transfer hub supporting anaerobic respiratory flexibility in Shewanella. Biochemical Journal 444, 465-474

4. Richter, K., Schicklberger, M., Gescher, J. (2011). Dissimilatory reduction of extracellular electron acceptors in anaerobic respiration. Applied and Environmental Microbiology 78, 913-921

Close bibliography.