Part:BBa_K346033:Experience

Applications of BBa_K346033

As to construct the fusion of DsbA-MBP, a commercial plasmid, pET-39b(+), which contains the gene encoding DsbA, was used as the backbone. The entire coding region of the MBP was amplified by PCR from full length MerR with two pairs of primers. The two PCR products were digested with Xba I / BamH I, or BamH I / Xho I, cloned into Spe I / Xho I digested pET-39b(+) in one step (fig. 1) to make pET-39b(+)-DsbA-MBP.

DsbA and MBD gene was amplified by PCR from pET-39b(+)-DsbA-MBP, with the primer containing T7 polymerase and RBS. The PCR product was digested with EcoR I / Pst I and then cloned into EcoR I / Pst I double digested pSB1K3, to achieve the goal of the standardization of the fusion protein (fig. 2).

As to construct the fusion of DsbA-MerR, a commercial plasmid, pET-39b(+), which contains the gene encoding DsbA, was used as the backbone. The entire coding region of the MerR was amplified by PCR from full length MerR with a pair of primer. The two PCR products were digested with Xba I / Xho I, cloned into Spe I / Xho I digested pET-39b(+) in one step (fig. 3) to make pET-39b(+)-DsbA-MerR.

DsbA and MerR gene was amplified by PCR from pET-39b(+)-DsbA-MerR, with the primer containing T7 polymerase and RBS. The PCR product was digested with EcoR I / Pst I and then cloned into EcoR I / Pst I double digested pSB1K3, to achieve the goal of the standardization of the fusion protein (fig. 4).

Results

Figure 2: The expression of Dsba+MBP. We can see that the 40 kd band both in the SDS-PAGE and Western blotting result.

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