Coding

Part:BBa_K2788020

Designed by: Mingyue Luo   Group: iGEM18_SZU-China   (2018-10-05)


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

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 969
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE 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.

We added a histine-tag at the C terminal of the TupA protein so that histidine-tagged affinity column chromatography can be performed. Besides the histine-tag could be an antibody recognition site, and there are many prepared antibody on sale. Some protein like TupA has slim studies on it. and there is no prepared antibody on sale. Once with his-tag, we can do Western Blot and other technique based on antigen-antibody reaction. The research on TupA’s expression is more smooth.