Difference between revisions of "Part:BBa K3185004"
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3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature. Then 6µL of 2x SDS sample buffer was added. 10µL of each sample was loaded. SDS-PAGE for 30min in 200V. The gel was CBB stained. | 3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature. Then 6µL of 2x SDS sample buffer was added. 10µL of each sample was loaded. SDS-PAGE for 30min in 200V. The gel was CBB stained. | ||
]] | ]] | ||
+ | <h3><font size="4.5">PET film assay</font> </h3> | ||
+ | We tried to compare our proteins with each other by the film dot blotting.<br><br> | ||
+ | |||
+ | 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.<br><br> | ||
+ | |||
+ | Although this experiment suggested our plastic-binding proteins can quickly bind to PET’s smooth surface, we could not compare binding affinity quantitatively.<br><br> | ||
==References== | ==References== |
Revision as of 11:43, 21 October 2019
SPYCatcher -> BaCBM2
Usage and Biology
BaCBM2 is a Carbohydrate-Binding Module (CBM) from Bacillus anthracis. CBM often found in Carbohydrate related enzymes. It can bind to not only highly crystallized cellulose but also PET because it has a binding site formed by aromatic amino acids [1].In this paper, they research binding affinity of some kinds of CBM and PET. As a result, it is found that BaCBM2 has the most strong binding affinity to PET [2].
We used BaCBM2 as PET binding domain. We put SpyCatcher on N-terminus of BaCBM2 because we used SpyCatcher/SpyTag system to bind it to other parts(SpyCatcher:BBa_K1159200, SpyTag:BBa_K1159201). Also, this has three tag and cleavage sites. First is 6×His-tag inserted in the N-terminus of SpyC for protein purification. Second is MYC-tag inserted between SpyC and CBM to detect it by using the antibody. Third is a TEV protease site because, in the paper, it was used for protein purification [3]. However, we didn’t use it in our experiment.
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]
- 1000COMPATIBLE WITH RFC[1000]
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)
![](/wiki/images/f/f6/191001_SPYt-SPYC_2.png)
3µL of SpyCatcher-Plastic-binding protein (SpyC-PBP) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for 16h at room temperature. Then 6µL of 2x SDS sample buffer was added. 10µL of each sample was loaded. SDS-PAGE for 30min in 200V. The gel was CBB stained.
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.
References
1 Boraston, A.B., Bolam, D.N., Gilbert, H.J., and Davies, G.J. (2004).
Carbohydrate-binding modules: Fine-tuning polysaccharide recognition.
Biochem. J. 382, 769–781.
2 Veggiani, G., Nakamura, T., Brenner, M.D., Gayet, R. V., Yan, J., Robinson, C. V., and Howarth, M. (2016).
Programmable polyproteams built using twin peptide superglues.
Proc. Natl. Acad. Sci. U. S. A. 113, 1202–1207.
3 Weber, J., Petrović, D., Strodel, B., Smits, S.H.J., Kolkenbrock, S., Leggewie, C., and Jaeger, K.E. (2019).
Interaction of carbohydrate-binding modules with poly(ethylene terephthalate).
Appl. Microbiol. Biotechnol. 103, 4801–4812.