Difference between revisions of "Part:BBa K3185008"

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We wanted to know if hydrophobin can bind to PET by themselves. We put SpyCatcher(''<partinfo>BBa_K1159200</partinfo>'') 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].
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We wanted to know if hydrophobin can bind to PET by themselves. We put SpyCatcher (''<partinfo>BBa_K1159200</partinfo>'') 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].
 
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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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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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==Purification==
 
==Purification==
 
[[File:Hydrophobin.png|300px|thumb|right|Fig.1 SDS-PAGE of imidazole elutes, CBB stained.]]
 
[[File:Hydrophobin.png|300px|thumb|right|Fig.1 SDS-PAGE of imidazole elutes, CBB stained.]]
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<h3><font size="4.5">Expression</font> </h3>
 
<h3><font size="4.5">Expression</font> </h3>
 
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<h3><font size="4.5">Purification </font></h3>
 
<h3><font size="4.5">Purification </font></h3>
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1. <i>E.coli<i/> which expressed this part were lysed with sonification.<br>
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1. <i>E.coli</i> 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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2. Proteins are purified from lysate with GST-sepharose.<br>
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3. Imidazole eluates were visualized and confirmed by SDS-PAGE followed by CBB staining.<br>
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3. Glutathione eluates were visualized and confirmed by SDS-PAGE followed by CBB staining.<br>
 
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This purification method failed. As shown in Fig.1, we didn't see any band in the specific molecular weight of the protein.   
 
This purification method failed. As shown in Fig.1, we didn't see any band in the specific molecular weight of the protein.   
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==Result==
 
==Result==
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We couldn't get any results in our experiment because of failure of protein purification.
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We couldn't get any result in our experiment because we failed the protein purification.
  
 
==Reference==
 
==Reference==

Latest revision as of 02:26, 22 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 SDS-PAGE of imidazole elutes, CBB stained.

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 GST-sepharose.
3. Glutathione eluates were visualized and confirmed by SDS-PAGE followed by CBB staining.

This purification method failed. As shown in Fig.1, we didn't see any band in the specific molecular weight of the protein.

Result

We couldn't get any result in our experiment because we failed the protein purification.

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.