Part:BBa_K4468011

T7-PmrA- T7-PmrB(FP)-T7 Terminator-PmrC-Oprf-Sitag-FP- T7 Terminator

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1619
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2858
  Illegal PstI site found at 1619
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1138
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1619
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1619
  Illegal NgoMIV site found at 3293
  Illegal NgoMIV site found at 4127
  Illegal AgeI site found at 1813
  Illegal AgeI site found at 2642
  
• 1000
  COMPATIBLE WITH RFC[1000]

Description

This is a composite component for the absorption and recovery of rare earth elements, especially for lanthanides. It consists of T7-PmrA- T7-PmrB(FP)-T7 Terminator-PmrC-oprf-Sitag-FP- T7 Terminator. It can express PmrA and PmrB protein with IPTG induction. Besides, lanthanide ions in the external solution can initiate the expression of oprf-Sitag-FP protein that can achieve a large amount of lanthanide adsorption and attachment on the surface of silica column. FP protein fuses dLBT and LanM proteins together. Its adsorption efficiency and amount of lanthanides are both improved compared with ordinary LanM protein.

Usage and Biology

PmrA

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 PmrA is an intracellular protein which can be actived during phosphorylation by protein kinase at the C-terminus of PmrB. Phosphorylated PmrA is able to bind on the promoter PmrC and initiate its downstream genes’ expression.

PmrB(FP)

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).

PmrC

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.
PmrC is a promoter that can bind to phosphorylated PmrA and initiate expression.

Oprf-Sitag-FP

Oprf-Sitag-FP is a protein composed of oprf, Sitag, dLBT and LanM peptides. It is the main element for adsorption and recovery of lanthanides. Oprf has a membrane-binding domain, which helps the protein binding on the cell membrane of our engineered bacteria. Sitag is a tag that can connect with silicon element. It allows us to easily fix our protein just using a silica column. dLBT can highly absorb Tb³⁺, the most representative element of lanthanides. Thanks to HUST-China 2017, we successfully obtained the dLBT with the best adsorption effect from 12 different LBT domains. LanM has great characteristics of efficient and specific absorbing lanthanides which can effectively absorb the free lanthanides in the environment. Through GS linker to combine the first three peptides and rigid linker to combine dLBT and LanM, we have connected them as a whole and created a new protein that can stick on our E. coli membrane and fix to silica column with its engineered bacteria together. We named dLBT-LanM as FP for short. When the mining wastewater flows through the column, the lanthanides can be effectively adsorbed, so as to achieve the purpose of rare earth element recovery.

Fig.1 The absorption principle of Sitag.

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.2 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

Fig.3 Colony PCR result of our four pathways transformed E. coli

The band of PmrCAB-LanM from colony PCR is about 5000bp, identical to the theoretical length of 5156bp estimated by the designed primer location, the band of PmrCAB-FP from colony PCR is about 5000bp also identical to the theoretical length of 5399bp. Besides, the band of GolS-LanM from colony PCR is about 4900bp identical to the theoretical length of 5049bp and he band of GolS-LanM from colony PCR is about 5000bp identical to the theoretical length of 5196bp, which could demonstrate that the target plasmids had successfully transformed into E. coli.

SDS-PAGE

After confirming through colony PCR and sequencing, we used the successfully transformed E. coli BL21 (DE3) for expression. We induced with IPTG and Tb³⁺ or IPTG and Cu²⁺ then followed by cell disruption to detect membrane proteins, as our fusion proteins would be expressed on the cell membrane.

Fig.4 SDS-PAGE result of PmrA after purification of total protein extraction product through Nickel-affinity chromatography column

The target protein located around 26-30kDa, similar the theoretical 26.87kDa. PmrA could be confirmed as successfully expressed. The concentration of E. coli total protein is so high that huge amount of impure protein is included during elution. But due to difference from impure or permeate bands, its consistency among several times of elution, this band could be verified as our target PmrA.

Fig.5 SDS-PAGE result of membrane protein PmrB(FP).

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

Fig.6 SDS-PAGE result of membrane protein oprf-Sitag-FP(PmrCAB) induced by different lanthanides.

After induction using different lanthanide ions, we obtained several strains that successfully expressed the oprf-Sitag-FP(PmrCAB). All their membrane proteins were detected by SDS-PAGE. The band of oprf-Sitag-FP(PmrCAB) is about 70kDa, identical to the theoretical length of 68.03kDa and still within explainable and acceptable range of glycosylation or phosphorylation modification. Oprf-Sitag-FP(PmrCAB) could be confirmed as successfully expressed.

Immunofluorescence labeling

Fig.7 Image of immunofluorescence labeling of the fusion protein from E. coli.

It is obvious to see that our target proteins are successfully expressed and immobilized on the cell membrane through the fluorescence on the bacteria cell.

Absorption of REE

Fig.8 Absorption rate of Tb.

This is the REE absorption of Tb. The abscissa is 4 kinds of pathways: PmrL is short for BBa_K4468010. PmrFP is short for BBa_K4468011. GolSL is short for BBa_K4468012. GolSFP is short for BBa_K4468013. And the ordinate is the absorption of Tb. For our fusion proteins, the adsorption of Tb was great. The lowest adsorption rate is about 60% and the highest rate is more than 80%.