Difference between revisions of "Part:BBa K3185007"

Revision as of 11:39, 18 October 2019

SPYCatcher -> sfGFP -> TA2

Usage and Biology

Tachystation A2(TA2) is a protein from Tachypleus tridentatus[1]. The paper shows it binds to the polystyrene (PS)[2].

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 (BBa_I746916). 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[2].

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 37^oC 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]Horseshoe Crab Hemocyte-derived Antimicrobial Polypeptides, Tachystatins, with Sequence Similarity to Spider Neurotoxins
[2]Directed evolution of polypropylene and polystyrene binding peptides/Programmable polyproteams built using twin peptide superglues

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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 (''<partinfo>BBa_1746916</partinfo>''). By doing so, we wanted to do the binding assay with fluorescence.
+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 (''<partinfo>BBa_I746916</partinfo>''). 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.
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