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

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 1523
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 2762
Illegal PstI site found at 1523
21
INCOMPATIBLE 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
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 1523
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 1523
Illegal AgeI site found at 1717
Illegal AgeI site found at 2546
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(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.

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

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.2 SDS-PAGE result of PmrA from composite component BBa_K4468010 and BBa_K4468010 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 FMO.

Fig.4 SDS-PAGE result of membrane protein oprf-Sitag-LanM(PmrCAB) induced by different lanthanides

After induction using different lanthanide ions, we obtained several strains that successfully expressed the oprf-Sitag-LanM(PmrCAB). All their membrane proteins were detected by SDS-PAGE. The band of oprf-Sitag-LanM(PmrCAB) is about 65kDa, identical to the theoretical length of 62.82kDa and still within explainable and acceptable range of glycosylation or phosphorylation modification. Oprf-Sitag-LanM(PmrCAB) could be confirmed as successfully expressed. Besides, following elution result also could verify it.

Silica absorbability

Fig.5 Validation of silica absorbability.

Picture A shows the silica observed under the electron microscope after mixing with our target strain for 5h. Picture B shows the untreated silica. Comparing A and B, the silica in Picture A binds a large number of protein molecules, indicating that our fusion protein can bind to silica successfully.

Fig.6 Silica adsorption curve. Red line: control group. Black line: experimental group.

Measuring the absorbance of medium solution and drawing the relation curve between OD and time. With the increase of silica adsorption time, the OD value of the solution has decreased obviously. It proved that the fusion protein has good absorbability indeed, which is consistent with the results obtained by electron microscopy.

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.

Part:BBa_K4468010