Difference between revisions of "Part:BBa K3185004"

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==Result==
 
==Result==
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[[File:191001 SPYt-SPYC 2.png|200px|thumb|left|Fig. 1 SDS-PAGE gel for quantification of protein concentration]]
 
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[[File:191008 Dot Blot.png|300px|thumb|left|Fig. Plastic-binding protein binding to PET film
 
[[File:191008 Dot Blot.png|300px|thumb|left|Fig. Plastic-binding protein binding to PET film
 
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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.
 
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.
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[[File:191001 SPYt-SPYC 2.png|200px|thumb|left|Fig. 10 Isopeptide bond formation between Plastic binding proteins and Encapsulin.
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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.
 
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Revision as of 09:38, 19 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 SpyTag/SpyCatcher 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. We used BL21 (DE3) for gene expression. We used Ni-NTA Agarose for purification. After that, we confirmed molecular weight of BaCBM2 by using SDS-PAGE.

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
    COMPATIBLE 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.

SDS-PAGE

konkon







































Result

Fig. 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. 10 Isopeptide bond formation between Plastic binding proteins and Encapsulin.
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.

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.