Part:BBa_K4468010
T7-PmrA-T7-PmrB(LanM)-T7 Terminator-PmrC-Oprf-Sitag-LanM-T7 Terminator
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1523
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2762
Illegal PstI site found at 1523 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 834
Illegal BamHI site found at 3692
Illegal XhoI site found at 1324
Illegal XhoI site found at 3104 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1523
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1523
Illegal AgeI site found at 1717
Illegal AgeI site found at 2546 - 1000COMPATIBLE 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(LanM)-T7 Terminator-PmrC-oprf-Sitag-LanM-T7 Terminator. It can express PmrA and PmrB protein without any induction. Besides, lanthanide ions in the external solution can initiate the expression of oprf-Sitag-LanM protein that can achieve a large amount of lanthanide adsorption and attachment on the surface of silica column.
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(LanM)
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).
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-LanM
Oprf-Sitag-LanM is a protein composed of oprf, Sitag 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. 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 them in a whole, we have created a new protein that can stick on our E.coli membrane and fix to silica column with its engineered bacteria together. 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.
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.
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.
Silica absorbability
Immunofluorescence labeling
None |