Team:USTC/Demonstrate/1
Mtr CAB
In our project, Mtr CAB is the most important and fundamental proteins as it plays the role to transfer extracellular electrons into the cytoplasm through the membrane. To examine whether the Mtr protein complex has the function as we expected, we used our engineered strain pMC( strain co-expressed Mtr CAB and Ccm A-H) to construct a bio-cathode. We monitored the current of the bio-cathode to see whether there would be a higher current in the experiment group than the WT strain.
Here, first we did an bacteria PCR to monitor the maintenance of the recombinant plasmids(pM28 contains the mtr CAB’s gene and the pTBC contains the ccm A-H’s gene). As we can see in figure 1, we could confirm that the strain is fine to use.
Figure 1. Electrophoresis result of PCR of Mtr and Ccm
So we started our bio-cathode assay to examine our theory. The protocol of the bio-cathode assay can be found in the notebook part in our wiki, look it up if you want to know more details!! In figure 2, we can easily confirm that the Mtr CAB protein complex was mature, as the pellets were red in pMC group, no matter the strain had been induced or not. When we did it the first time, there was no significant difference of the current of the bio-cathode between WT and our strain pMC(data not shown). We speculated that it was because we did NOT have the starvation step when we first did it, which is to cultivate the bacteria in a minimal salts medium for a certain time, like 4 to 6 hours. Because we did NOT have this starvation step, although we already used PBS to wash the bacteria 2 to 3 times, those nutritions still contained inside of the bacteria, providing another electron source when we were running the bio-cathode. So when the cathode was given a certain voltage, the bacteria still wouldn’t take up the electrons from the electrode.
Figure 2. Color of bacteria sediments
So we performed this bio-cathode assay for a second time, adding this starvation step into the protocol. In addition, after the starvation step, we used 1 mL of minimal salts medium to resuspend the bacteria and dropped it onto the graphite electrode to form a bio-film, which could help to make a better connection between the bacteria and the electrode, especially when we were using the Mtr pathway to transfer electrons. Here, in figure 3, you can see how we made this biofilm. 2 to 3 hours later, with a sufficient airflow in the laminar flow hood, the graphite electrode would dry up and form a great biofilm. With this biofilm, electrons could be transferred to the Mtr C protein directly from the electrode which can increase the efficiency of electron transferring. Then what we need to do was to construct this bio-cathode, put every part of this “toy” together and get the oxygen out of this container. Here in figure 4 is how we clear the oxygen out of the bio-cathode to create an anaerobic environment. Lastly, we connected the bio-cathode to the electric-chemical station to give a certain voltage to the cathode and monitor the current of the cathode as time went by as how figure 5 shows.
Figure 3. Electrode coated with bacteri
Figure 4. Preparation for reaction system
Figure 4. Preparation for reaction system
Figure 5. Device for bio-cathode
Figure 6 is the result of this experiment. From the figure, we can easily notice that the red line, which is the Mtr-induced group, had a 50% higher current than the other two group after the bio-cathode turned into a stable state . This could strongly prove that the engineered strain pMC can transfer electrons into the cytoplasm, which led to the increasing of the cathode-current. But there would be a chance that this difference between these 3 groups was just the background noise between this three cathode, resulting from the hardware’s varieties. So we added fumarate into the system to see whether there would be a cathode catalyzed current happened in the pMC group. That’s why there was a sharp increasing in the figure. When we added fumarate into the system, the electrons on the electrode finally found a way to leak to—— the fumarate. So there would be a strong electron flow when we added fumarate into the system. But after a short time we introduced this sudden change into the system, the current will become stable again, slowly climbing back to the current it was. However, the time it took to get back to stable state can be a strong evident to prove our assumption——our engineered E.coli can transfer extracellular electrons into the cytoplasm!! The red line’s curve happened after we added fumarate into the system is kind of a typical curve of cathode-catalyze-current!! So, with this result, the cathode’s current to time under a certain voltage, we can confidently say that the Mtr CAB system work!!
Figure 6. The current result of the bio-cathode.
In conclusion, the Mtr CAB system can really function as an electron pathway to transfer extracellular electrons into the cytoplasm, even though it’s expressed in E.coli, but not it’s origin host Shewanella.!! In another word, our conduction system can function as we expected, transferring those electrons from the electrode into the cytoplasm, which means our E.coli can transform itself like transformer from a normal form to a special form that can “eat” electrons!
Ccm A-H
1.Construction of ccm on pSB1C3 and co-transformation of ccm and mtr
We get ccm gene from the genome of E.coli by PCR and insert this sequence to pSB1C3 with pTet upstream successfully. The sequence of our ccm is validated by DNA sequencing from SangoTech. We co-transform the plasmids containing ccm and the plasmids containing mtr in BL21. Then we pick some colonies for cultivation and confirm the co-transformation of two plasmids (shown in Figure 1). We inoculate confirmed colony to 2x YT media and grown with 250 rpm shaking for 12 hours at 30˚C. 5 mL of overnight culture is used to inoculate 1L 2xYT media and were grown for 16 hours at 30 ˚C. After cultivation, we confirm the existence of our two plasmids in BL21 by bacteria PCR (shown in Figure 2).
Figure 1. Bacteria PCR for strain pMC co-expressing Mtr CAB & Ccm A-H
2.We express mature MtrA and MtrC successfully
After cultivation, we collect our bacteria from 1 mL media by centrifugation. Obviously, our bacteria with ccm become red compared with wild type which shows our Ccm is expressed successfully because heme are attached to MtrA&C expressed on the outer membrane (shown in Figure 3).
Figure 3.
Figure 4.
We lyse the bacteria and extract the membrane and periplasmic fractions, respectively. Then we run SDS-PAGE of sample of each fraction. The molecular weight of MtrC, MtrB and MtrA is 72kDa, 77kDa and 36kDa respectively. We can confirm the expression of MtrABC from the bond of approximate molecular weight, but the expression of CcmA-H is not sure (shown in Figure 4). We attach a His-tag to MtrC so the expression of MtrC is confirmed from the result of Western blot (shown in Figure 5).
Figure 5.
Figure 6.
To insure the function of Ccm directly, we employ the method of TMBZ stain which is a common chemical analysis method for heme covalently bond to peptides. According to the theory of TMBZ stain, if Ccm A-H have catalyzed the attachment of heme to MtrA&C, there will be visible blue bond at corresponding position. By comparing the position of blue bond with protein marker, we make sure that our MtrA and MtrC are mature. These results prove that our Ccm functions well directly and our Ccm is expressed successfully indirectly (shown in Figure 6).
Figure 7.
Figure 8.
Besides, we design an experiment as a negative control. We transform the plasmids containing mtr (shown in Figure 7). Then we induce the expression of mtr without ccm under aerobic condition. We run SDS-PAGE and western blot of our samples (shown in Figure 8, Figure 9) and detect the heme via TMBZ stain (shown in Figure 10). It’s obvious that our MtrCAB is expressed compared with wild type from SDS-PAGE result. But there is no blue bond after TMBZ stain so we conclude that our Mtr is immature. These results also reveal the fact that Ccm A-H have no impact on the expression of MtrCAB but play a vital role in catalyzing the maturation of MtrA&C.
Figure 9.
Figure 10.
From these two experiments, we can reach the conclusion that MtrA&C get mature because of the function of CcmA-H which prove the successful expression of Ccm A-H. We construct mature MtrCBA system with the expression of CcmA-H and the first part of our project conduction system is fulfilled.
Reference:
[1] Thomas, P. E., Ryan, D., & Levin, W. (1976). An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Analytical biochemistry, 75(1), 168-176.[2] Jensen, H. M. (2013). Engineering Escherichia coli for molecularly defined electron transfer to metal oxides and electrodes. University of California, Berkeley
