Oprf-Sitag-LanM

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 463
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1393
Illegal XhoI site found at 805
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 247
1000
COMPATIBLE WITH RFC[1000]

Usage and Biology

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.

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-LanM 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, 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.3 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.

Fig.4 SDS-PAGE result of membrane protein oprf-Sitag-LanM(GolS).

The band of oprf-Sitag-LanM(GolS) is about 60kDa, identical to the theoretical length of 62.82kDa and still within explainable and acceptable range of glycosylation or phosphorylation modification. Oprf-Sitag-LanM(GolS) 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.

Absorption of REE

Fig.8 PmrL’s absorption rate of REE.

PmrL is short for BBa_K4468010. The abscissa is 7 kinds of REE and the ordinate is the absorption rate of REE. On average, the absorption rate reached the maximum when pH=5.22 and minimum when pH=5.22. When pH is higher than 5.22, the adsorption rate decreases sharply at first and then increases with pH increasing. The highest adsorption rate of Samarium element even reached 93%.