Difference between revisions of "Part:BBa K3185008"
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This part has three tags. First is 6×His-tag inserted in the N-terminus of SpyCatcher for protein purification. Second is MYC-tag inserted between sfGFP and SpyCatcher to detect it by using the antibody. Third is a TEV protease site and we put it between Spy-C and 6xHis-tag because it was used for protein purification in the paper[2].
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This part has three tags. First is 6×His-tag inserted in the N-terminus of SpyCatcher for protein purification. Second is MYC-tag inserted between sfGFP and SpyCatcher to detect it by using the antibody. Third is a TEV protease site and we put it between SpyCatcher and 6xHis-tag because it was used for protein purification in the paper[2].
 
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Revision as of 12:39, 18 October 2019


GST -> SPYCatcher -> Hydrophobin

Usage and Biology

Hydrophobin is a protein from Bacillus subtilis. In this paper, it shows that they self-assemble and get hydrophobic region[1]. OLS_Canmore_Canada team from iGEM tried to improve efficiency of the to degrade plastic (improve the ability to degrade plastic which PETase has.)

We wanted to know if hydrophobin can bind to PET by themselves. We put SpyCatcher(BBa_K1159200) on N-terminus of hydrophobin because we used SpyTag/SpyCatcher system to bind hydrophobin to other parts. Also, according to the paper which shows how to purify hydrophobins, they add GST, so we used the pGEX-6P-1whose backbone is GST as a vector too[1].

This part has three tags. First is 6×His-tag inserted in the N-terminus of SpyCatcher for protein purification. Second is MYC-tag inserted between sfGFP and SpyCatcher to detect it by using the antibody. Third is a TEV protease site and we put it between SpyCatcher and 6xHis-tag because it was used for protein purification in the paper[2].

We inserted it in the C-terminal of GST on the pGEX-6P-1.We used BL21 (DE3) for gene expression. We used Ni-NTA Agarose for purification. After that, we confirmed the molecular weight of hydrophobin by using SDS-PAGE. The result is shown below.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 688
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1444
    Illegal SapI.rc site found at 85

Purification


Expression

  • Cells were grown in 200ml LB media (100μg/ml Ampicillin) at 37oC shaking at 140 rpm to an OD600 of 0.5, verifying via a spectrophotometer.
  • Protein was expressed in 0.1mM IPTG for 2hours.

SDS-PAGE



Reference

1 Al, L. H. and N. R. S.-W. et. (2019).
BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm.
Journal of Chemical Information and Modeling, 53(9), 1689–1699.

2 Veggiani, G., Nakamura, T., Brenner, M. D., Gayet, R. V., Yan, J., Robinson, C. V., & Howarth, M. (2016).
Programmable polyproteams built using twin peptide superglues.
Proceedings of the National Academy of Sciences of the United States of America, 113(5), 1202–1207.