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.
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).
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).
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