Difference between revisions of "Part:BBa K3185007"

Revision as of 09:24, 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

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.

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Result

Fig.1 Plastic-binding protein binding to PET film
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.

Fig.2 Cloth dot blot by fluorescent plastic-binding protein before washing.
The dilution collection of each protein was dropped on PET cloth, then left for 20min. The protein fluorescent was imaged by LAS-3000. The exposure time is 10sec.

Fig.3 Percentage of protein retention on PET cloth
As shown above, our fluorescent plastic-binding proteins bind to PET cloth. and stay even they are washed. In order to demonstrate in a more outstanding way, we took the movie that is shown below.

Fig.4 Plastic-binding proteins also bind to PET fiber
PET fibers were soaked in each protein solution, then washed in TBST for 5min x3. Fluorescent was observed in 460nm exciting light and imaged with 0.25sec exposure time. Magnification is 10x.

Fig.5 SDS-PAGE gel for quantification of amounts of proteins bind to PET fiber 20cm of PET fibers were soaked in protein solutions, then washed in TBST for 5min three times. Washed fibers were soaked in 50µL of 2x SDS sample buffer. Bounded proteins were eluted with boiling. SDS-PAGE for 40min in 200V. CBB stained.

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.