Part:BBa_K3099002

PLac+omcA+Mtr

Mtr CAB is a protein complex located on the outer membrane of Shewanella, transferring electrons from the cityplasm to the outside of the bacteria. Mtr CAB consists of three proteins, Mtr A, Mtr B, Mtr C. Mtr B is anchored onto the outer membrane. With its β 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. We improve the existing parts by changing the promoter and make the parts can express in Shewanella Oneidensis MR-1

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 2322
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 384
  Illegal NgoMIV site found at 410
  Illegal NgoMIV site found at 1095
  Illegal NgoMIV site found at 4523
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2709
  

USTC

Mtr CAB is a protein complex located on the outer membrane of Shewanella, transferring electrons from the cytoplasm to the outside of the bacteria. Mtr CAB consists of three proteins, Mtr A, Mtr B, and Mtr C. Mtr B is anchored onto the outer membrane. and it can help to locate the Mtr A and Mtr C as well as increase the stability of the whole complex. Mtr A and Mtr C are the two proteins that can actually transfer electrons with heme attached into these two proteins at the right position.
We improve the existing parts by changing the promoter and make these parts be able to be expressed in Shewanella.
This part was inserted into plasmid, and the correct construction of this recombinant plasmid was confirmed by PCR identification and sequencing of the PCR products.

Figure1. Electrophoresis result PCR of Mtr

The Shewanella Oneidensis MR-1 was grown in LB liquid medium, and extract the protein by heating them. The sample was electrophoresed on a SDS-PAGE, followed by heme-staining.

Figure2. SDS-PAGE for strain expressing Mtr

Besides, we designed the experiment of the reduction of methyl orange (MO) to measure the effect of the part.
The result is pretty good. From Figure 3., we can see that, qualitatively, our engineered strain with the electron transfer system is much stronger than wild type. And then, we quantitatively analyzed how much we improved the reduction ability of S. oneidensis MR-1. We made the exponential fitting curve of MO concentration (Figure 4.): WT y=49.44e-0.0014x; Exp. y=49.64e-0.0136x.
The efficiency of our engineered stain to degrade MO is 10 times higher!

Figure3. Samples after incubation of about 5 hours

Figure4. SDS-PAGE for strain expressing Mtr

Figure5. Quantitatively analysis (a) and (b)

References

[1] Thomas, P. E., Ryan, D., & Levin, W. (1976). An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Analytical biochemistry, 75(1), 168-176.