Part:BBa_K4468003

PmrB(LanM)

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 788
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 788
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 99
  Illegal XhoI site found at 589
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 788
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 788
  Illegal AgeI site found at 982
  
• 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. We luckily found that the absorption capacity is only related to its 34-64 amino acids. In that case, we turned them into LanM, a peptide with the ability to adsorb lanthanides, thus achieving the function of specifically adsorbing lanthanides. Besides, its kinase activity still remains intact. Now the PmrCAB system convert to absorb lanthanides to induce downstream genes’ expression. We named this protein PmrB(LanM).

Molecular cloning

Using E. coli to extract our plasmids. 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.

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

The band of Oprf-Sitag-LanM from colony PCR is about 1800bp, identical to the theoretical length of 1797bp estimated by the designed primer location and the band of PmrB(LanM)-PmrC from colony PCR is about 1750bp also identical to the theoretical length of 1735bp, which could demonstrate that this target plasmid had successfully transformed into E.coli

SDS-PAGE

Fig.2 SDS-PAGE result of membrane protein PmrB(LanM).

The band of PmrB(LanM) is about 40kDa, identical to the theoretical length of 38.38kDa and still within explainable and acceptable range of glycosylation or phosphorylation modification. PmrB(LanM) could be confirmed as successfully expressed.