Part:BBa_K2242419
pTET+ccmA-H
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. For those proteins in this family, they all need to get through a process before they become a mature one, which means they can function as they are meant to, the covalent heme ligation. First, those apo-cytochrome c will be secreted to the periplasm, then a protein complex——ccm A-H will catalyze the process of covalent heme ligation, to attach the heme onto the apo-cytochrom c at the right position, after which our cytochrome c proteins can become mature and function as we expected. In our project, we co-express this ccm A-H protein complex with cytochrome c Mtr CAB to mature Mtr CAB to shuttle electrons from electrode into cytoplasm of our engineered E.coli to increase the synthesis efficiency. We use a constitutively on promoter pTET to control gene ccmA-H’s expression.
1.Transformation and Expression
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).
Figure 3.The bacteria sediments
Figure 4. SDS-PAGE for membrane and periplasm fraction
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).
Figure 5. Western blot for membrane and periplasm fraction
Figure 6. Heme staining for membrane and periplasm fraction
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.
Figure 7. Bacteria PCR for strain expressing Mtr CAB only and WT
Figure 8. SDS-PAGE for strain expressing Mtr CAB only and WT
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
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 534
Illegal BamHI site found at 3323
Illegal BamHI site found at 3551
Illegal BamHI site found at 5895 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 188
Illegal AgeI site found at 1015 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 4602
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