Coding

Part:BBa_K3185007

Designed by: Masahiro Sakono   Group: iGEM19_Kyoto   (2019-10-04)
Revision as of 16:30, 21 October 2019 by Daidai (Talk | contribs) (→‎Result)


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 TA2 (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


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

Fig.1 SDS-PAGE of imidazole elutes, CBB stained.

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

Fig.2 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.3 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.4 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.5 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.6a 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.
Fig.6b BaCBM2 bind most to PET fiber
SDS-PAGE’s gel band intensity quantified with ImageJ. The y-axis shows amounts of protein which bind to 20cm PET fiber.

PET film assay

We tried to compare our proteins with each other by the film dot blotting.

As shown in Fig.2, the negative control protein, SpyCatcher (SPYC), did not stain PET film at all. In contrast, the plastic-binding proteins tested here strongly stained the PET film. As stains spread, we could not quantify their signals. This blot spreading might be due to the plastic-binding proteins’ fast binding rate. The proteins in excess liquid could have bound to the neighbor area of the film in the first wash step.

Although this experiment suggested our plastic-binding proteins can quickly bind to PET’s smooth surface, we could not compare binding affinity quantitatively.

PET cloth assay

We quantified how much protein binds to PET fiber with fluorescence. TA2 proteins can be seen with fluorescent signals because it is fused with sfGFP.

We bought a white PET T-shirt and cut it into pieces. The lysate of E.coli which expresses protein is used for this experiment. The concentrations of fluorescent proteins were measured by SDS-PAGE and CBB-staining, and an equal amount of proteins were used for each assay. Diluted proteins were spotted on a piece of PET cloth. The cloth was incubated for 20 min at room temperature, then washed by TBST for 5 min 3 times. Finally, fluorescent proteins were photographed.
Fig.3a shows the cloth before wash, and 3b shows the same cloth after wash. As shown in figures, sfGFP was completely washed out. In sharp contrast, TA2 strongly stuck to PET cloth. The intensity of dot was quantified with ImageJ.
As shown in Fig.4, about 75% of TA2 were still observed on PET cloth after wash, showing these proteins are really strong PET cloth binders.

PET fiber assay

We showed two fluorescent plastic-binding proteins bind to PET cloth very tightly. Next, we demonstrated proteins’ binding in a more realistic target: PET fiber. In cloth, fibers are close to each other, so they might create a hydrophobic environment between them. In the fiber form, they are surrounded by water, so plastic-binding proteins might behave in a different way.

In this experiment, PET fibers were soaked in water or protein solutions, then washed in TBST for 5 mins three times. The concentrations of protein solutions were 2000 ng/µL. sfGFP and other sfGFP-fused protein’s fluorescence were observed by a fluorescence microscope in 460 nm exciting light.

Clearly, when TA2 were fused to sfGFP, the PET fiber was brightly stained by fluorescence. Water control did not show any fluorescence, indicating that no autofluorescence was observed with these fibers. sfGFP control also showed no signals, meaning that sfGFP binding was mediated by the plastic-binding domain in the sfGFP fusion proteins.

We next compared with other plastic-binding proteins quantitatively. The equal length of PET fibers ware soaked in protein solutions and proteins bound were visualized in SDS-PAGE and CBB stain.

As shown in Fig.6a and 6b, BaCBM2 bind the most to PET fiber. According to the references, BaCBM2 and CenA are polyethylene terephthalate (PET)-binding proteins, LCI KR-2 is a polypropylene (PP) binding protein, and TA2 is a polyurethane (PU) binding protein. Therefore, this result is consistent with the reported observation. It is of interest that LCI KR-2 (PP binding) and TA2 (PU binding) proteins also showed a moderate but measurable amount of PET-binding.

Protein conjugation thorough SpyCatcher/SpyTag system

We conjugated SpyC->TA2 with SpyTag inserted TmEncapsulin (BBa_K3185000) through SpyCatcher/SpyTag system. SpyCatcher and SpyTag form an isopeptide bond between them when they are mixed. SpyTag inserted TmEncapsulin has SpyTags inserted on its surface.

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.


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