Difference between revisions of "Part:BBa K3185007"
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We used it as the PS binding protein. We also inserted Superfolder GFP (sfGFP) which we improved GFP to make the folding time shorter in the N-terminal of LCI (Part:　BBa_1746916). By doing so, we wanted to do the binding assay with fluorescence.
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We used it as the PS binding protein. We also inserted Superfolder GFP (sfGFP) which we improved GFP to make the folding time shorter in the N-terminal of LCI (Part:BBa_1746916). By doing so, we wanted to do the binding assay with fluorescence.
 
Moreover, we put SpyCatcher on N-terminus of sfGFP because we used the SpyTag/SpyCatcher system to bind it to other parts.
 
Moreover, we put SpyCatcher on N-terminus of sfGFP because we used the SpyTag/SpyCatcher system to bind it to other parts.
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3185007 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3185007 SequenceAndFeatures</partinfo>
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==Purification==
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<h3><font size="4.5">Expression</font> </h3>
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<li>Cells were grown in 200ml LB media (100μg/ml Ampicillin) at 37<sup>o</sup>C shaking at 140 rpm to an OD600 of 0.5, verifying via a spectrophotometer.
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<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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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 13:53, 17 October 2019


SPYCatcher -> sfGFP -> TA2

Usage and Biology

Tachystation A2(TA2) is a protein from Tachypleus tridentatus. (Horseshoe Crab Hemocyte-derived Antimicrobial Polypeptides, Tachystatins, with Sequence Similarity to Spider Neurotoxins) The paper (Directed evolution of polypropylene and polystyrene binding peptides) shows it binds to the polystyrene (PS).

We used it as the PS binding protein. We also inserted Superfolder GFP (sfGFP) which we improved GFP to make the folding time shorter in the N-terminal of LCI (Part:BBa_1746916). By doing so, we wanted to do the binding assay with fluorescence. Moreover, we put SpyCatcher on N-terminus of sfGFP because we used the SpyTag/SpyCatcher system to bind it to other parts.

This part has four tags. First is 6×His-tag inserted in the N-terminus of SpyCatcher for protein purification. Second is MYC-tag inserted between sfGFP and Spy-Catcher to detect it by using the antibody. Third is a TEV protease site and we put it into two regions because it was used for protein purification in the paper (Directed evolution of polypropylene and polystyrene binding peptides/Programmable polyproteams built using twin peptide superglues).

We inserted it in between the BamHI site and the Ndel site on the pET11-a. We used BL21(DE3) for gene expression.We used Ni-NTA agarose for purification. After that, we confirmed the molecular weight of TA2 by using SDS-PAGE.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 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