Difference between revisions of "Team:USTC/Improve"

This year, we improve the function of part BBa . In former iGEM teams, this part, or specifically this protein, are used to transfer intracellular electrons out to the electrode. In another word, it is a useful component if we want to build up a bio-anode. However, few iGEM teams, or even other researchers, have noticed that this protein, Mtr CAB can transfer electrons bidirectionally!! In our project, we successfully proved that Mtr CAB can enable our engineered bacteria to transfer extracellular electrons into the cytoplasm!

1.Plasmid construction and co-transformation of ccm A-H and mtr CAB

We amplified gene ccm A-H from the genome of E.coli(BL21) by PCR and inserted this gene to pSB1C3 with promoter pTet upstream successfully. The sequence of ccm A-H was validated with DNA sequencing by Sangon. Besides, we constructed another plasmid pM28 with promoter T7 and gene mar CAB downstream. After the construction, We co-transformed these two plasmids into strain BL21. Then we picked some colonies for cultivation and confirmed the co-transformation of these two plasmids (shown in Figure 1). We inoculated confirmed colonies to 2x YT media and cultivate it for 12 hours at 30˚C, 250 rpm. 2 mL of overnight culture was used to inoculate 200 mL 2xYT media and were grown for 16 hours at 30 ˚C. After cultivation, we confirmed the maintenance of 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 successfully expressed mature MtrA and MtrC

After cultivation, we collected our bacteria from 1 mL media by centrifugation. Obviously, bacteria with ccm A-H turned red compared with wild type, which proved that ccm A-H was expressed successfully as heme are attached to MtrA&C properly.(shown in Figure 3).

We lysed the bacteria and extracted the membrane and periplasmic fractions, respectively. Then we ran 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 Mtr CAB with the band of approximate molecular weight, but the expression of CcmA-H is not sure (shown in Figure 4). We attached a His-tag to Mtr C so the expression of MtrC can be confirmed by the result of Western blot (shown in Figure 5).

To insure the function of Ccm A-H directly, we employed heme staining which is a common chemical analysis method for heme covalently bonding to peptides to confirm whether Mtr CAB protein was mature or not. According to the principle of heme staining, if Ccm A-H have catalyzed the attachment of heme to MtrA&C, there will be visible blue bond at corresponding position on the gel. By comparing the position of blue bond with protein marker, we made sure that our MtrA and MtrC are mature. These results proved that Ccm A-H functions well directly and our Ccm is expressed successfully indirectly (shown in Figure 6).

In a word, as the Mtr CAB protein complex have been matured, we can proved the expression of Ccm A-H indirectly and the function of Ccm A-H directly.

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. Western for strain expressing Mtr CAB only and WT

Figure 10. Heme staining for stain expressing Mtr CAB only and WT

From these two experiments, we can reach the conclusion that MtrA&C get mature because of the function of CcmA-H which proved the successful expression of Ccm A-H. Moreover, we also confirmed the expression of Mtr CAB. In a word, we successfully constructed a mature Mtr CAB system with the co-expression of CcmA-H

3. Function of 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 11, we could confirm that the strain is fine to use.

Figure 11. 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 12, 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 12. Bacteria sediments
(from left to right, WT, pMC not induced, pMC induced)

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 13, 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 14 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 15 shows.

Figure 16 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 16. 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!

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