Difference between revisions of "Part:BBa K2788020"
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<partinfo>BBa_K2788020 short</partinfo> | <partinfo>BBa_K2788020 short</partinfo> | ||
| − | This part is the coding sequence (CDS) of a cytoplasmic protein TupA (GenBank: BAB07375.1), which catalyzes the conversion of glucuronic acid and L-glutamic acid to polyglucuronic acid and poly-γ-L-glutamic acid. | + | This part is the coding sequence (CDS) of a cytoplasmic protein TupA (GenBank: BAB07375.1) with hisdine-tag at the C terminal, which catalyzes the conversion of glucuronic acid and L-glutamic acid to polyglucuronic acid and poly-γ-L-glutamic acid. |
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===iGEM2018 SZU-China=== | ===iGEM2018 SZU-China=== | ||
| − | This part C125-TupA-6His is developed from part C125-TupA | + | This part C125-TupA-6His is developed from part C125-TupA,which add a his-tag at the C-terminus of TupA so that we can gain this cytoplasmic protein by histidine-labeled affinity column chromatography, and analysis at the protein level is easier. |
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<p> | <p> | ||
We transfered C125-TupA-6His and C125-TupA into Bacillus subtilis SCK6 respectively, then screened by kanamycin. Transformants were grown in LB broth. Then obtained total protein by ultrasonication. performed histidine-tagged affinity column chromatography to isolate fusion protein, then verified by SDS-PAGE and Coomassie blue staining. (Fig.1) | We transfered C125-TupA-6His and C125-TupA into Bacillus subtilis SCK6 respectively, then screened by kanamycin. Transformants were grown in LB broth. Then obtained total protein by ultrasonication. performed histidine-tagged affinity column chromatography to isolate fusion protein, then verified by SDS-PAGE and Coomassie blue staining. (Fig.1) | ||
Revision as of 10:50, 17 October 2018
C125-TupA-6His
This part is the coding sequence (CDS) of a cytoplasmic protein TupA (GenBank: BAB07375.1) with hisdine-tag at the C terminal, which catalyzes the conversion of glucuronic acid and L-glutamic acid to polyglucuronic acid and poly-γ-L-glutamic acid.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 969
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 789
Illegal BsaI.rc site found at 1181
iGEM2018 SZU-China
This part C125-TupA-6His is developed from part C125-TupA,which add a his-tag at the C-terminus of TupA so that we can gain this cytoplasmic protein by histidine-labeled affinity column chromatography, and analysis at the protein level is easier.
We transfered C125-TupA-6His and C125-TupA into Bacillus subtilis SCK6 respectively, then screened by kanamycin. Transformants were grown in LB broth. Then obtained total protein by ultrasonication. performed histidine-tagged affinity column chromatography to isolate fusion protein, then verified by SDS-PAGE and Coomassie blue staining. (Fig.1)
Fig.1. SDS-PAGE analysis of total protein and the product of protein purification.M: marker ladder; Lane 1: total protein of C125-TupA-6His transformant; Lane2: the product of protein purification of C125-TupA-6His transformant; Lane3: total protein of C125-TupA transformant; Lane4: the product of protein purification of C125-TupA transformant. It can be seen that the histidine-tagged fusion protein was successfully expressed. Lane 2 showed the band corresponded with the molecular weight of TupA(57.3kDa), while lane 4 had no band.