Part:BBa_K4468004

PmrB(FP)

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 884
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 884
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 403
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 884
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 884
  Illegal AgeI site found at 1078
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

The PmrCAB of Salmonella is a system that can be stimulated and induced by exogenous metal ions. The main functions are executed by three parts, the transmembrane protein PmrB, the intracellular protein PmrA, and the promoter PmrC. In wild-type Salmonella, it can adsorbs extracellular Fe3+, and phosphorylates protein PmrA. Then the phosphorylated PmrA will bind on the promoter PmrC to initiate expression.
The expression product of PmrB is a single pass transmembrane protein whose 34to 64 amino acids are located outside the membrane to adsorb Fe3+. Near the C-terminus is a protein kinase that can phosphorylate PmrA. On the basis of protein PmrB(LanM), we further reformed its protein sequence of the extracellular domain. Thanks to HUST China 2017, we already knew that the peptide dLBT also has similar function, except that dLBT is worse than LanM and only Tb3+ could be adsorbed. So we added a linker between LanM and dLBT in order to join the bipartite sequences together and reintegrated at the adsorption domain of PmrB. We believed that the new fusion protein will highly improve the absorption ability of lanthanides. We named this protein PmrB(FP).

Molecular cloning

First of all, we need to amplificated all the commercially synthesized plasmid to acquire enough amount for further study. After transformation, colony PCR is applied for confirmation. Then we go for plasmid extraction.
Using E. coli to extraction. Through designed primers, we have obtained different high copies linearized fragments from our plasmids by PCR. These fragments are then connected together by homologous recombination to form a complete plasmid. After transformed into E. coli, colony PCR was applied for confirmation. Then we go for extracting plasmids again.
Finally we transformed our recombinant plasmids into E. coli BL21(DE3) competent cells. Correct as checked by colony PCR.

File:HUST-China-0-.png

Fig.1 Colony PCR result of Oprf-Sitag-FP and PmrB(FP)-PmrC transformed E.coli

The band of Oprf-Sitag-FP from colony PCR is about 2000bp, identical to the theoretical length of 1944bp estimated by the designed primer location and The band of PmrB(FP)-PmrC from colony PCR is about 1800bp, identical to the theoretical length of 1801bp estimated by the designed primer location, which could demonstrate that this target plasmid had successfully transformed into E.coli