Difference between revisions of "Part:BBa K3185006"
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We used LCI-KR2 for binding protein to PP. We inserted superfolder GFP (sfGFP) whose folding interval is shortened by improving natural GFP on the N-terminus of LCI (''<partinfo>BBa_I746916</partinfo>''). By doing so we wanted to do the binding assay with fluorescence. Moreover, we put SpyCatcher(SpyCatcher:''<partinfo>BBa_K1159200</Partinfo>'')[ on N-terminus of sfGFP because we used SpyTag/SpyCatcher system to bind it to other parts.
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We used LCI-KR2 for binding protein to PP. We inserted superfolder GFP (sfGFP) whose folding interval is shortened by improving natural GFP on the N-terminus of LCI (''<partinfo>BBa_I746916</partinfo>''). By doing so we wanted to do the binding assay with fluorescence. Moreover, we put SpyCatcher(''<partinfo>BBa_K1159200</Partinfo>'')[ on N-terminus of sfGFP because we used SpyTag/SpyCatcher system to bind it to other parts.
 
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Revision as of 12:32, 18 October 2019


SPYCatcher -> sfGFP -> LCI KR-2

Usage and Biology

LCI is a protein from Bacillus subtili. The paper shows that it can bind to polypropylene(PP)[1]. The paper shows the improved variant, LCI-KR2(Y29R and G35R; variant KR-2)[2]. Its affinity is 5.4±0.5 times stronger than natural LCI.

We used LCI-KR2 for binding protein to PP. We inserted superfolder GFP (sfGFP) whose folding interval is shortened by improving natural GFP on the N-terminus of LCI (BBa_I746916). By doing so we wanted to do the binding assay with fluorescence. Moreover, we put SpyCatcher(BBa_K1159200)[ on N-terminus of sfGFP because we used SpyTag/SpyCatcher system to bind it to other parts.

This part has four tags. First is 6×His-tag inserted on the N-terminus of SpyC for protein purification. Second is MYC-tag inserted between sfGFP and Spy-Catcher to detect it by using the antibody. The third is a TEV protease site and we put it into two regions because it was used for protein purification in the paper[3].

We inserted it into the site between the BamHI site on the pGEX11-a and Ndel site. We used BL21 (DE3) for gene expression. We used Ni-NTA Agarose for purification. After that, we confirmed the molecular weight of LCI by using SDS-PAGE. The result is shown below.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 1174
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 460

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



References

[1](Anchor peptides: A green and versatile method for polypropylene functionalization)
[2](KnowVolution of the Polymer-Binding Peptide LCI for Improved Polypropylene Binding)
[3](Directed evolution of polypropylene and polystyrene binding peptides/Programmable polyproteams built using twin peptide superglues)