Part:BBa_K3185008
GST -> SPYCatcher -> Hydrophobin
Usage and Biology
Hydrophobin is a protein from Bacillus subtilis. In this paper (BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm), it shows that they self-assemble and get hydrophobic region. 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 on N-terminus of hydrophobin because we used SpyTag/SpyCatcher system to bind hydrophobin to other parts. Also, according to the paper (BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm) which shows how to purify hydrophobins, they add GST, so we used the pGEX-6P-1whose backbone is GST as a vector too.
This part has three tags. First is 6×His-tag inserted in the N-terminus of SpyC 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 (Programmable polyproteins built using twin peptide superglues),
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 688
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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
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