Difference between revisions of "Part:BBa K3185007"

Revision as of 13:45, 17 October 2019

SPYCatcher -> sfGFP -> TA2 Sequence and Features

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 __NOTOC__
 <partinfo>BBa_K3185007 short</partinfo>
-meow
 <!-- Add more about the biology of this part here
 ===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).
+<br>
+<br>
+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.
+<br>
+<br>
+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).
+<br>
+<br>
+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.
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