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Applications of BBa_K346034
Materials and Method:
To test the expression of the fusion protein and the binding function of our bacterial, it is essential to construct both the standard plasmid and commercial plasmid. Firstly, we use PCR to get the lpp-ompa gene with the template of PSD-MBD, which is provided by Summers, containing lpp-ompa sequence. The primer is designed according to the paper. Besides the complementary sequence of the original primer, the enzyme cut site are introduced to the primer with standard prefix(or NdeI restriction site) and the SalI restriction site. And the mbp gene is also got by PCR from the plasmid provided by Pro.Chuan He, with the primers which replace the enzyme cut site with SalI restriction site and standard suffix (or XhoI restriction site). Then the two products of PCR are cut with EcolI(or NdeI), SalI and SalI, PstI (or XhoI)separately as inserts and the standard plasmid PSB1A2 is cut with EcolI and PstI(for the commercial plasmid PET21a, the enzyme is NdeI and XhoI) as vector. Ultimately, the procession of ligation with three fragments(lpp-ompa, mbp and vector) is carried out to get the standard and commercial plasmid with the gene coding for Lpp-OmpA-MBP for lead.
After the construction of the plasmid with the fusion protein gene lpp-ompa-mbp, we add the element which is essential for the expression of protein to the standard plasmid with T7promoter and RBS. Both the sequence and the results of enzyme digestion show that the construction is successful.The size of the expressed proteins have been verified with SDS-page and Western blot.
Results
The lpp-ompa-mbp has been inserted into the commercial plasmid PET21A. Then the plasmid is transferred to E.coli strain BL21, which can generate T7polyerase when induced with IPTG. Both induced cells and uninduced cells(as control) are centrifuged to get the cytosol, the periplasm and the membrane separated. The SDS-page and Western blot show that induced cells expressed an identical IPTG-inducible protein in the membrane with the size of ~27kD for LPP-OMPA-MBP, which is consist with the predicted size of 26.8kD, indicating that Lpp-OmpA-MBP can be expressed normally on the membrane.The results of SDS-PAGE and Western is shown below(SDS lane3&4 Western lane 3).
The function test has also been carried out and the results show that the surface display system has a high efficiency of mercury binding.
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