Difference between revisions of "Part:BBa K2242363"
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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> | <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> | ||
<p><img src="https://static.igem.org/mediawiki/2017/b/b2/USTC-result-ccm-2.png" width="30%" style="margin:0 40%;"></p> | <p><img src="https://static.igem.org/mediawiki/2017/b/b2/USTC-result-ccm-2.png" width="30%" style="margin:0 40%;"></p> | ||
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 5205
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 247