Part:BBa_K1316011
pFAB640 regulating the ccmAH cluster
Cytochrome c maturation (ccm) cluster regulated by the best performing promoter generated by Goldbeck et al.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 730
Illegal BamHI site found at 3519
Illegal BamHI site found at 3747
Illegal BamHI site found at 6091 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 384
Illegal AgeI site found at 1211 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 4798
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