Difference between revisions of "Part:BBa K3185008"
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==Purification==
 
==Purification==
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[[File:Hydrophobin.png|300px|thumb|right|Fig.1 hydrophobin]]
 
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<h3><font size="4.5">Expression</font> </h3>
 
<h3><font size="4.5">Expression</font> </h3>
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<li>Protein was expressed in 0.1mM IPTG for 2hours.
 
<li>Protein was expressed in 0.1mM IPTG for 2hours.
 
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<h3><font size="4.5">SDS-PAGE</font></h3>
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<h3><font size="4.5">Purification </font></h3>
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[[File:Hydrophobin.png|800px|thumb|left|hydrophobin!]]
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1. E.coli which expressed this part were lysed with sonification.<br>
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2. Proteins are purified from lysate with Ni-NTA agarose(QIAGEN).<br>
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3. Imidazole eluates were visualized and confirmed by SDS-PAGE followed by CBB staining.<br>
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<br>
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This purification method failed. As shown in Fig.1, the protein successfully purified.
 
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Revision as of 09:34, 21 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]. iGEM OLS_Canmore_Canada 2018 team used this protein as PET binding protein.(BBa_K2650003) Because PET is hydrophobic, hydrophobin might have hydrophilic interaction with PET.

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 SpyCatcher/SpyTag 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.The expression plasmids were introduced into BL21(DE3) and expressed by T7 promoter/ T7 RNAP system. Glutathione sepharose beads was used for the purification.

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

Fig.1 hydrophobin


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.

Purification

1. E.coli which expressed this part were lysed with sonification.
2. Proteins are purified from lysate with Ni-NTA agarose(QIAGEN).
3. Imidazole eluates were visualized and confirmed by SDS-PAGE followed by CBB staining.

This purification method failed. As shown in Fig.1, the protein successfully purified.

Result

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.