Part:BBa_K2232012:Experience

iGEM2017 SZU-China

In order to strengthen the alkali tolerance of B.subtilis, which is essential for our chassis to live in the concrete, we constructed an expression vector containing part OF4-nhaC(Fig.1) ,which plays a role as shelter protecting B.subtilis from Alkaline environment.

Fig.1 Construction of the expression vector_P43-nhaC-T1. The P43 and T1 represent strong promoter P43 from B.subtilis and terminator T1 from E. coli rrnB. The part OF4-nhaC was inserted by the restriction site KpanI at 4430 bp and HindIII at 5194 bp.

We transformed the expression vectors into Bacillus subtilis WB800 by optimization of Spizizen method, and the real positive clones was confirmed by kanamycin screening and nucleic acid electrophoresis (Fig.2).

Fig.2 1% Agarose Gel Electrophoresis of DNA extracted from the positive clones and its identification by restriction digestion. The product of plasmid digested showed two signal bands at 733 bp and 6703 bp respectively, which correspond to the length of OF4-nhaC and the blank plasmid. Lane 1: Complete plasmid; Lane 2: Plasmid digested by KpnI and HindIII; Lane M: DL marker.

After transformation of this part, The recombinant B.subtilis WB800_nhaC was grown in LB culture for 24h and the cells were disrupted by sonication in 20 mM Tris-HCl(PH 8.0) buffer. The lysate was then centrifuged and the precipitate were electrophoresed on a sodium dodecyl sulfate(SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining(Fig.3).

Fig.3 SDS-PAGE analysis of membrane protein of original B.subtilis and the transformant of OF4-nhaC. Lane M: Marker ladder; Lane 1 & 2: The recombinant strain WB800_nhaC; Lane 3: Original strain WB800. Lane 1 & 2 showed the same band(in red box) corresponded with the molecular weight of NhaC(36kDa).

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