Difference between revisions of "Part:BBa K2242363"
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===Usage and Biology===
 
===Usage and Biology===
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                                                        <p id="first" class="scrollspy label label-pink">1.Plasmid construction and co-transformation of ccm A-H and mtr CAB</p>
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        <p class="indent_word">We amplified gene ccm A-H from the genome of <span class="italic">E.coli</span>(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). </p>
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        <p><img src="https://static.igem.org/mediawiki/2017/b/b2/USTC-result-ccm-2.png" width="30%" style="margin:0 40%;"></p>
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        <p style="text-align:center!important">Figure 1. Bacteria PCR for strain pMC co-expressing Mtr CAB & Ccm A-H</p>
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        <p id="second" class="scrollspy label label-pink">2.We successfully expressed mature MtrA and MtrC</p>
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        <p class="indent_word">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). </p>
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        <img src="https://static.igem.org/mediawiki/2017/f/f7/USTC-result-ccm-10.jpeg" width="50%" style="margin:0 30%;">
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        <p style="text-align:center!important">Figure 3.The bacteria sediments</p>
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        <img src="https://static.igem.org/mediawiki/2017/6/6a/USTC-result-ccm-9.jpeg" width="130%" style="margin:0 -5%;">
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        <p style="text-align:center!important">Figure 4. SDS-PAGE for membrane and periplasm fraction</p>
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        <p class="indent_word"> 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). </p>
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        <img src="https://static.igem.org/mediawiki/2017/8/87/USTC-result-ccm-3.png" width="80%" style="margin:0 10%;">
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        <p style="text-align:center!important">Figure 5. Western blot for membrane and periplasm fraction</p>
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        <img src="https://static.igem.org/mediawiki/2017/1/1e/USTC-result-ccm-4.png" width=120%" style="margin:0 %;">
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        <p style="text-align:center!important">Figure 6. Heme staining for membrane and periplasm fraction</p>
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                                                        </div>
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        <p class="indent_word">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). </p>
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        <p class="indent_word">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.
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        <img src="https://static.igem.org/mediawiki/2017/4/4c/USTC-result-ccm-5.png" width="55%" style="margin:0 40%;">
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        <p style="text-align:center!important">Figure 7. Bacteria PCR for strain expressing Mtr CAB only and WT</p>
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        <img src="https://static.igem.org/mediawiki/2017/9/94/USTC-result-ccm-6.png" width="73%" style="margin:0 15%;">
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        <p style="text-align:center!important">Figure 8. SDS-PAGE for strain expressing Mtr CAB only and WT</p>
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                                                        </div>
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        <p class="indent_word">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. </p>
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        <img src="https://static.igem.org/mediawiki/2017/5/53/USTC-result-ccm-8.png" width="70%" style="margin:0 20%;">
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        <p style="text-align:center!important">Figure 9. Western for strain expressing Mtr CAB only and WT</p>
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        <img src="https://static.igem.org/mediawiki/2017/9/9b/USTC-result-ccm-7.png" width="30%" style="margin:0 30%;">
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        <p style="text-align:center!important">Figure 10. Heme staining for stain expressing Mtr CAB only and WT</p>
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        <p class="indent_word">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 </p>
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        <p id="third" class="scrollspy label label-pink">3. Function of Mtr CAB</p>
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        <p class="indent_word">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.</p>
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        <p class="indent_word">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. </p>
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        <img src="https://static.igem.org/mediawiki/2017/9/94/USTC-result-Mtr-1.png" width="30%" style="margin:0 40%;">
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        <p style="text-align:center!important">Figure 11. Electrophoresis result of PCR of Mtr and Ccm</p>
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        <p class="indent_word">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.</p>
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        <img src="https://static.igem.org/mediawiki/2017/6/6f/USTC-result-Mtr-2.jpeg" width="40%" style="margin:0 30%;">
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        <p style="text-align:center!important">Figure 12. Bacteria sediments<br>(from left to right, WT, pMC not induced, pMC induced)</p>
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        <p class="indent_word">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. </p>
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                                                        <img src="https://static.igem.org/mediawiki/2017/thumb/5/50/USTC-demo-3.jpeg/800px-USTC-demo-3.jpeg" width="80%" style="margin:0 4%;">
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                                                        <p style="text-align:center!important">Figure 13. Preparation for bio-film</p>
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                                                        <img src="https://static.igem.org/mediawiki/2017/thumb/a/a3/USTC-demo-2.jpeg/800px-USTC-demo-2.jpeg" width="80%" style="margin:0 4%;">
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                                                        <p style="text-align:center!important">Figure 14. Preparation for reaction system<br>(to exclude oxygen out of the container)</p>
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        <img src="https://static.igem.org/mediawiki/2017/thumb/f/f0/USTC-result-Mtr-5.jpeg/800px-USTC-result-Mtr-5.jpeg" width="40%" style="margin:0 30%;">
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        <p style="text-align:center!important">Figure 15. Bio-cathode device</p>
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        <p class="indent_word">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!!</p>
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        <img src="https://static.igem.org/mediawiki/2017/9/91/USTC-result-Mtr-10.jpeg" width="80%" style="margin:0 10%;">
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        <p style="text-align:center!important">Figure 16. The current result of the bio-cathode.</p>
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        <p class="indent_word">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!</p>
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        <p class="get_bold1">Reference: </p>
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        [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.
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        [2] Jensen, H. M. (2013). Engineering Escherichia coli for molecularly defined electron transfer to metal oxides and electrodes. University of California, Berkeley
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Revision as of 13:49, 29 October 2017


Metal Reductase CAB

Mtr CAB is a protein complex located on the outer membrane of Shewanella originally, transferring electrons from the cityplasm to the outside of the bacteria. However, according to the lately research, we found that this Mtr CAB protein complex can also transport electrons into the cytoplasm from the electrode or other electron donors from the outside. This is the role Mtr CAB plays in our project. We introduce this protein complex into our engineered E.coli to transport electrons into the cytoplasm. Mtr CAB consists of three proteins, Mtr A, Mtr B, Mtr C. Mtr B is anchored onto the outer membrane. With its ß barrel conformation, it can help to locate the Mtr A and Mtr C and increase the whole complex’s stability. Mtr A and Mtr C are the two protein that can actually transfer electrons with the heme attached into these two protein at the right position. MtrA is a 32-kD periplasmic decaheme cytochrome c, and MtrC is a 69-kD cell-surface-exposed. Electrons are collected by Mtr C then it will shuttle through this electron tunnel and go to Mtr A, then these electrons will be transferred to CymA on the inner membrane and final get to the NADH dehydrogenase through the electron transfer chain. With this protein complex, we can utilize the electrons from the electrode or some chemical compound outside of the bacteria can turn them into NADH when they get into the cytoplasm of our engineered bacteria, increasing the NADH’s concentration inside of the cytoplasm, which means their the reduction power ——the ability to synthesize, will be pump up. As we have mentioned, it’s the heme attached to the Mtr A and Mtr C that enable them to shuttle electrons. So these two protein belong to a protein family called cytochrome c.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 5205
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 247