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.
Figure 1 shows the variation in voltage-time profiles for all the tested inserts. The figure clearly indicates that MtrCAB-CymA recombinant E. coli gave the highest potential difference of 423±5mV throughout the study. This suggested it had the highest energy gain. Although there was a shift on the second day in voltage production between MtrCAB and MtrC. The voltage was stable throughout the remaining period of the study, clearly indicating that the highest production was obtained when MtrCAB and MtrC-CymA were present, meaning their presence is a key element in the process of electron transfer to obtain the highest voltage.
The results represented in Figure 2B represents the maximum power generation that can be obtained from the various recombinant E. coli. This is the maximum point on each curve as clearly shown. The MtrCAB-CymA produced the maximum power generation of 98±4mWm-2. The recombinant MtrCAB gave 89±2mWm-2 which was more than two fold of the maximum power produced by the MtrA recombinant E. coli. The wild type E. coli gave insignificantly power production (curve not plotted). After the maximum power generation was produced, the gradual drop in power generation was as a result of increasingly voltage drop as the current generation increases (Figure 2A). The ability to maintain voltage production as the current increases differs between the recombinant systems and hence the performances.
Table 1 showed the percentage substrate consumed as COD (chemical oxygen demand) with amount of electron recovery on day 8 of the investigation. The MtrA gave the highest COD degradation of 40±3. However, with regard to coulombic efficiency (CE) MtrCAB gave more CE of 17±1 than other tests. The result showed percentage of substrate consumed and the fraction of the substrate consumed utilised for electricity production in this case the CE. Hence, little substrate was consumed by MtrCAB and suggested to be ineffective on substrate utilization. However, suggest to be very effective on substrate utilization to electricity generation than the other recombinant systems. On the other hand, MtrA suggested to be very effective in substrate consumption.
The experiment aimed to enhance electricity recovery in MFCs by heterologously expression of synthetic electron conduits from S. oneidensis into E. coli as this model organism has proven record of genetic manipulation. The results indicated that MtrCAB-cymA modified E. coli TOP10 gave the maximum 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 significantly high maximum power generation suggested the protein is responsible majorly for extracellular electron transfer processes and is suggested to play substantial influence on electricity produced in both MtrCAB and MtrCAB-CymA complexes.