Team:TU Delft-Leiden/Project/Life science/EET/characterisation

Module Electron Transport - Characterization

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Our BioBrick BBa_K1316012 encodes the mtrCAB genes under control of an adjusted T7 lac promoter. The MtrC, MtrA and MtrB proteins form a conduit to transfer electrons across the membrane. To complete the implementation of the extracellular electron pathway we constructed BBa_K1316011 as well , a BioBrick encoding the ccm genes under control of the pFAB640 promoter. The ccm proteins help to mature the MtrCAB conduit. The elaborated combination of promoters and coding sequences that we used was found to generate the largest maximal current [1]. To test if this is true, we characterized BBa_K1316012 and BBa_K1316011 . We made use of SDS-page and UV-vis to see if the proteins are expressed. In addition, we made use of a potentiostat to do advanced current measurements. In this way we were able to visualize expression of ccm genes, and showed that induction of our BioBricks indeed results in a current flow.

Characterization of BBa_K1316011

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 translocate 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 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 has been done. E. coli (C43) cultures were transformed with BBa_K1316011 and then cultivated aerobically. E. coli (C43) CcmA-H+NdeI from the Ajo-Franklin lab and E. coli (C43) without plasmid were used as a positive and negative control, respectively. E. coli (C43) CcmA-H+NdeI already have the Ccm system and the MtrCAB operon, so is expected to have expression of MtrA, MtrB and MtrC proteins bacause of the Ccm system. BBa_K1316011 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) CcmA-H+NdeI 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.

Before membrane protein purification, BBa_K1316011 and the controls where induced with IPTG. Analysis of the pellets shown that there was a difference in color in BBa_K1316011 and E. coli (C43) CcmA-H+NdeI compared with E. coli (C43). The BBa_K1316011 and E. coli (C43) CcmA-H+NdeI 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.

According to Reedy, C. J. & Gibney, B. R. et al (2004)[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) CcmA-H+NdeI offer enhanced cytochrome c expression compared to the E. coli (C43) strain without the Ccm or MtrCAB plasmids.

SDS-PAGE

A SDS-PAGE has been done with the membrane fractions for E. coli (C43), E. coli (C43) CcmA-H+NdeI 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) CcmA-H+NdeI. When the MtrCAB operon is included, the membrane fractions of E. coli (C43) CcmA-H+NdeI 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]

There’re 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) CcmA-H+NdeI. But becasue of the weak MtrCAB promotor, the expression of MtrCAB proteins is at a low level and that may be the reason why the MtrCAB proteins are not 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): 617–49. doi:10.1021/cr0206115. PMID 14871137 (2004)