Difference between revisions of "Part:BBa K3185007"
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<partinfo>BBa_K3185007 short</partinfo> | <partinfo>BBa_K3185007 short</partinfo> | ||
==Usage and Biology== | ==Usage and Biology== | ||
| − | Tachystation A2(TA2) is a protein from <i>Tachypleus tridentatus</i>. | + | Tachystation A2(TA2) is a protein from <i>Tachypleus tridentatus</i>[1]. The paper (Directed evolution of polypropylene and polystyrene binding peptides) shows it binds to the polystyrene (PS). |
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| − | 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 | + | 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]. |
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==References== | ==References== | ||
| + | [1]Horseshoe Crab Hemocyte-derived Antimicrobial Polypeptides, Tachystatins, with Sequence Similarity to Spider Neurotoxins | ||
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| + | [2] Directed evolution of polypropylene and polystyrene binding peptides/Programmable polyproteams built using twin peptide superglues | ||
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===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K3185007 parameters</partinfo> | <partinfo>BBa_K3185007 parameters</partinfo> | ||
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Revision as of 08:00, 18 October 2019
SPYCatcher -> sfGFP -> TA2
Usage and Biology
Tachystation A2(TA2) is a protein from Tachypleus tridentatus[1]. 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[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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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]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