Difference between revisions of "Part:BBa K3185007"
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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. | + | <li>Cells were grown in 200ml LB media (100μg/ml Ampicillin) at 37<sup>o</sup>C shaking at 140 rpm to an OD600 <br> of 0.5, verifying via a spectrophotometer. |
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Revision as of 09:29, 21 October 2019
SPYCatcher -> sfGFP -> TA2
Usage and Biology
Tachystation A2(TA2) is a protein from Tachypleus tridentatus[1]. The paper shows it binds to polyurethane (PU)[2].
We used it as the PU binding protein. We also inserted Superfolder GFP (sfGFP, BBa_I746916) which folding interval is shortened by improving natural GFP 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 SpyCatcher/SpyTag system to bind it to other parts.
This part has four tags. First is 6×His-tag inserted in the N-terminus of SpyCatcher(BBa_K1159200) 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 put it between BamHI site and Ndel site on pET11-a. The expression plasmids were introduced into BL21(DE3) and expressed by T7 promoter/ T7 RNAP system. Ni-NTA agarose was used for the purification.
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.
Purification
1. E.coli which expressed this part were lysed with sonification.
2. Proteins are purified from lysate with Ni-NTA agarose(QIAGEN).
3. Imidazole eluates were visualized and confirmed by SDS-PAGE followed by CBB staining.
This purification method works. As shown in Fig.1, the protein successfully purified.
Result
![](/wiki/images/f/f1/191008_Dot_Blot.png)
A 3µL of protein solution dropped on PET film, then left for 20min. Then the film was washed in TBST for 5min x3, then placed with Anti-His-tag-HRP conjugated for 1h. ECL substrate was added, then chemiluminescence was imaged by LAS-3000. The exposure time is 6min.
![](/wiki/images/6/6e/Fiber_CBB.png)
References
1 Osaki, T., Omotezako, M., Nagayama, R., Hirata, M., Iwanaga, S., Kasahara, J., Hattori, J., Ito, I., Sugiyama, H., and Kawabata, S.I. (1999).
Horseshoe crab hemocyte-derived antimicrobial polypeptides, tachystatins, with sequence similarity to spider neurotoxins.
J. Biol. Chem. 274, 26172–26178.
2 Islam, S., Apitius, L., Jakob, F., and Schwaneberg, U. (2019).
Targeting microplastic particles in the void of diluted suspensions.
Environ. Int. 123, 428–435.