Latest revision as of 13:52, 30 October 2017

pFAB640 regulating the ccmAH cluster

Cytochrome c maturation (ccm) cluster regulated by the best performing promoter generated by Goldbeck et al.[1]

Characterization of the Ccm genes

The CCM cluster is a cluster consisting of genes encoding for several (parts of) proteins. The cytochrome C maturation (Ccm) system consists of heme delivery proteins that help the conduit proteins, such as the proteins in the MtrCAB operon, to mature properly by translocating heme in the periplasm and catalyzes the formation of thioether bonds that link heme to two cysteine residues. The axial ligands are then coordinated to the heme iron and the holocytochrom C is folded. In these strains, no conduit proteins are inserted, but the heme delivery proteins should be more highly expressed. When there is an increased expression of heme delivery proteins, that can already be seen by the redness of the pellets (because of the heme) after the membrane purification.

Membrane purification and UV-VIS

To look into the expression of the cytochrome c maturation (Ccm) proteins, an UV-VIS spectrum has been recorded. E. coli (C43) cultures were transformed with BBa_K1316011 and then cultivated aerobically. E. coli (C43) Ccm+MtrCAB and E. coli (C43) without plasmid were used as a positive and negative control, respectively. E. coli (C43) Ccm+MtrCAB already has the Ccm system and the MtrCAB operon, so is expected to have expression of MtrA, MtrB and MtrC proteins because of the Ccm system. BBa_K1316011 is expected to have more expression of the Ccm proteins than the normal E. coli (C43) strain and more equal expressions of Ccm proteins compared with E. coli (C43) Ccm+MtrCAB because of the inserted Ccm operon. When there is an increased expression of heme delivery proteins, that can already be seen by the redness of the pellets (because of the heme) after induction with IPTG.

Figure 1: pictures of the pellets shown that there was a difference in color in BBa_K1316011 and E. coli (C43) Ccm+MtrCAB compared to E. coli (C43).

Before membrane protein purification, BBa_K1316011 and the controls where induced with IPTG. Visual analysis of the pellets shown that there was a difference in color in BBa_K1316011 and E. coli (C43) Ccm+MtrCAB compared with E. coli (C43). The BBa_K1316011 and E. coli (C43) Ccm+MtrCAB had a red color, which could be indicating the increased production of cytochrome c proteins because of the heme delivery proteins. Membrane protein purifications where done by low-speed and high-speed centrifugation.

Figure 2: Image of a UV-VIS with membrane fractions of BBa_K1316011 (green) and E. coli (C43) Ccm+MtrCAB (positive control) (pink) and E. coli (C43) without plasmid (negative control) (blue). y-as: wavelength (nm)

According to [2], there should be a peak around 550nm for cytochrome c proteins. Using the UV-VIS results, there is a peak for all membrane fractions around 550nm, so it is possible to confirm the expression of cytochrome c proteins in all the samples. There is a difference between E. coli (C43) without plasmid and BBa_K1316011, as shown in figure 1. These observations confirm that BBa_K1316011 and E. coli (C43) Ccm+MtrCAB offer enhanced cytochrome c expression compared to the E. coli (C43) strain without the Ccm or MtrCAB plasmids.

SDS-PAGE

An SDS-PAGE has been done with the membrane fractions for E. coli (C43), E. coli (C43) Ccm+MtrCAB and BBa_K1316011. As mentioned before, no conduit proteins, such as the proteins in the MtrCAB operon, are included in BBa_K1316011, but they are included in E. coli (C43) Ccm+MtrCAB. When the MtrCAB operon is included, the membrane fractions of E. coli (C43) Ccm+MtrCAB should contain MtrA, a 32-kD periplasmic decaheme cytochrome c, MtrC is a 69-kD cell-surface-exposed lipid-anchored decaheme cytochrome c and MtrB is a 72-kD predicted twenty-eight strand β-barrel outer membrane protein.[1]

Figure 3: Image of a SDS page with membrane fractions of colonies transformed with BBa_K1316011. Colonies transformed with E. coli (C43) Ccm+MtrCAB and E. coli (C43) were used as positive and negative control, respectively.

There are no significant differences between BBa_K1316011 and both positive and negative controls observed using SDS-PAGE. There are no MtrCAB proteins observed when looking into the SDS-PAGE lane for E. coli (C43) Ccm+MtrCAB. Due to the the weak MtrCAB promotor, the expression of MtrCAB proteins might be too low to be visible on the gel.

References

1. C.P. Goldbeck et al., Tuning promoter strengths for improved synthesis and function of electron conduits in E. coli, ACS Synth. Biol. 2 (3), pp 150–159 (2013)

2. Reedy, C.J. & Gibney, B.R. et al., Heme protein assemblies, Chem Rev 104 (2), pp 617–49 (2004)

Contribution from team iGEM2017_USTC

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.

Introduction

The new part we have submitted is part BBa_K2242666. Although we all know this protein complex, Ccm A-H, can mature cytochrome c. However, there are only a few of characterization and proof about it. So this year, we team USTC, confirmed the function of this part BBa_K1316011 from team iGEM14_TU_Delft-Leiden and added more proof about it. Besides, we also add a constitutively on promoter, pTet, to regulate the expression of this gene.

The reason we chose this part for contribution is because there was no sample for this part in iGEM HQ. Besides, there is only a few characterization for this protein complex. However, this protein complex Ccm A-H is an essential component for cytochrome c. Any iGEM team or researchers want to mature cytochrome c aerobically needs this part! For its great importance, we chose this part for contribution!

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