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Bio-electric interface
Bio-electric interface BioBricks cloning
Procedure
- Microorganisms 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 at room temperature and subcultured by lab staff when needed.
- PCR: most PCR reactions were performed 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 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 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. Staining procedure involved SYBR-Safe.
- 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: DNAPurification1 was used.
- 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)
- Restriction digestion: Restriction digests were performed for PCR products along with vector digestion following OpenWetWare CFrench:restriction1 protocol. 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.
- 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.
- 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.
- Transformation: Ligation and fusion PCR products were used to transform E coli JM109 competent cells using OpenWetWare protocol < a href="http://openwetware.org/wiki/Cfrench:compcellprep1">Cfrench:compcellprep1, protocol for preparation of competent cells and cell tansformation).
- 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°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.
- Miniprepping: Subcultures were used to set up overnight liquid cultures in 2,5 ml of LB. Miniprepping was performed using either OpenWetWare protocol Cfrench:minipreps1 or standarised QIAprep Spin MiniPrep Kit.. Minipreps were then restriction digested and run on the gel
- Sequencing: size confirmed minipreps were then sent for sequencing in the University of Edinburgh GenePool.
Results
NapC
- Throughout summer we have managed to clone napC gene from E coli. We have inserted the gene into the standard BioBrick vector pSB1C3 and submitted it to the parts registry.
Figure 1: napC PCR
Figure 2: napC miniprep
https://static.igem.org/mediawiki/2012/d/d8/Gelpic010.jpg
Figure 3: 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.
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 MtrA gene of S. oneidensis and cloned it into pSB1C3 plasmid.
Figure 4: MtrA PCR
Figure 5: MtrA transformation
Figure 6: MtrA miniprep
Submitted MtrA gene contains a PstI site which needs to be mutagenised prior to use.
- We have also obtained good quality pure PCR products of MtrCAB and ccm genes from S. oneidensis and ccm genes from E. coli.
Figure 7: MtrCAB PCR
Figure 8: ccm genes from S. oneidensis and E. coli
Discussion and conclusions
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).
We managed to obtain napC and MtrA genes which are now ready for testing, using haem staining and half fuel cells. MtrA gene still contains and internal PstI site which has to be mutated out prior to submission. We intend to insert a promoted in front of both genes and test these new BioBricks using haem staining and half fuel cells using our current results as reference. However it is possible that the transformed cells will require functional copy of ccm genes in order to function properly. Ccm genes are responsible for cytochrome maturation which is necessary for proper folding of multihaem cytochromes.
We have also had some success in cloning ccm and cymA which are the remaining components of the inspected system. We intend to combine these genes and test them together in order to assess the efficiency of the system.
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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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.
