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Project Results

Click here to see details of our lab results

Our initial aim was to express the MtrCAB operon found in Shewanella oneidensis MR-1 in Escherichia coli. Another objective was to investigate the potential benefits of the proteins CymA and OmcA within the microbial fuel cell. From reviewing the literature we quickly discovered that cloning these genes into E.coli would be a challenge as they can be potentially toxic to the cell. Due to this we decided to try to express each gene individually with His-10 tags to show that they can be expressed in E.coli.

Another potential hurdle for our project is the fact that E.coli does not have the advantage of nanowires in order to transport their electrons as S. oneidensis. Therefore, in order to overcome this the K12 derivative, DH5α was used.

Westminster team took advantage on the generous offer from IDT. In doing so, the decision was made to acquire the five genes (MtrA, MtrB, MtrC, CymA, OmcA), that were the focus of this project, synthesised as gBlocks. We were fortunate enough to be selected to use a novel cloning technique designed by, RDP (Rapid DNA Prototyping). 20 primers were ordered in order to convert the gBlocks into BioBrick and RDP formats. These were designed in SnapGene to include a GC clamp and annealing temperature of 60-70°C.





After ordering these primers we ran a PCR reaction to amplify the gBlocks and add the Prefix and suffix or X and Z ends to the five genes of interest. The gels below show the results:







RDP constructs to test individual genes:

To build the constructs we planned to use rapid DNA prototyping cloning method supplied to us by Synbiota. Our first constructs were made as verification to show that each individual gene can be expressed in E.coli

.

dA18-ChlR – Chloramphenicol resistance anchor
Pr.2 – Medium strength promoter
Rbs.3.1 – Medium strength ribosomal binding site
PlKr.Xa – Cleavable (factor Xa) protein linker
His10 – His10 tag
Ori.3 dT18 – High copy number cap



RDP construct to test electrical output:

To test the genes we have been working on in a microbial fuel cell we decided to make two plasmid constructs. MtrCAB as a whole operon was designed along with OmcA-CymA, these constructs were built with different antibiotic resistance so when cloning into an expression strain we could use a duel antibiotic selection marker system.





Converting RDP to BioBrick standard:

To convert the RDP constructs we made into BioBrick format we decided to order custom primers. These primers were designed to anneal to the anchor and cap region of the construct, thus not losing any of the composite part. In total four primers were ordered, two forward for the X and Z anchors and two reverse for the X and Z end caps.





Digestion and ligation into pSB1C3

To conform to iGEM competition rules, before freeze drying and sending the BioBricks to the iGEM headquarters we needed to ligate our parts to the digested plasmid backbone pSB1C3. Our experiments thus far had been going according to plan, building the RDP constructs and converting them to BioBrick format had been relatively successful. Unfortunately we had mixed results when trying to ligate these parts into the plasmid backbone and due to time constraints we were not able to troubleshoot in time for the deadline.





Results:




Power density tests were conducted on the second day when all the MFCs were in their pseudo-steady-state conditions. As shown in Figure 1, the MtrCAB-CymA system generated the highest maximum power density of 90 ± 5mW m-2; MtrC study generated 87±6mWm-2, MtrA produced 73±4mWm-2 while MtrCAB produced the least maximum power density of 41± 3mWm-2.

Discussion:
The experiment aimed to enhance electricity recovery in MFCs by heterologously expression of synthetic electron conduit from Shewanella oneidensis into E. coli as this model organism is ideal for growth. The results indicated that MtrCAB-cymA modified E. coli TOP10 gave the maximum power generation. The power produced in correlation with MtrCAB E. coli suggest that cymA is very important and enhanced MtrCAB electron transfer potential more than two times on power generation. The result supported the proposed pathway of electron transfer from CymA to MtrA and from MtrA to MtrC within the MtrCAB complex. MtrC the outer membrane cytochrome modified E. coli result which produced the second highest maximum power generation suggested the protein is responsible majorly for extracellular electron transfer processes utilize by Shewnaella oneidensis as the result is significantly similar to MtrCAB-CymA modified E. coli.