Difference between revisions of "Part:BBa K2232012:Experience"
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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). | 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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− | <center><html><img src='https://static.igem.org/mediawiki/parts/thumb/b/be/Nhac3.jpeg/570px-Nhac3.jpeg' style="width: | + | <center><html><img src='https://static.igem.org/mediawiki/parts/thumb/b/be/Nhac3.jpeg/570px-Nhac3.jpeg' style="width:30%;margin:0 auto"> |
<center>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). | <center>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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Latest revision as of 20:14, 1 November 2017
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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