Part:BBa_K4468007

Oprf-Sitag-FP

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 463
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 898
  Illegal NgoMIV site found at 1732
  Illegal AgeI site found at 247
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

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. 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-FP 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, 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 Fig2. 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. Besides, following elution result also could verify it.

Fig.3 SDS-PAGE result of membrane protein oprf-Sitag-FP(GolS).

The band of oprf-Sitag-FP(GolS) 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(GolS) could be confirmed as successfully expressed. Besides, following elution result also could verify it.

Immunofluorescence labeling

Fig.4 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.