Difference between revisions of "Part:BBa K3185004"
0012massan (Talk | contribs) |
(→References) |
||
Line 28: | Line 28: | ||
<br> | <br> | ||
==References== | ==References== | ||
− | + | 1 Boraston, A.B., Bolam, D.N., Gilbert, H.J., and Davies, G.J. (2004). | |
+ | Carbohydrate-binding modules: Fine-tuning polysaccharide recognition. | ||
+ | <i>Biochem. J. </i>382, 769–781. | ||
<br> | <br> | ||
− | |||
<br> | <br> | ||
− | + | 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. | ||
+ | <i>Proc. Natl. Acad. Sci. U. S. A. </i>113, 1202–1207. | ||
+ | <br> | ||
+ | <br> | ||
+ | 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). | ||
+ | <i>Appl. Microbiol. Biotechnol. </i>103, 4801–4812. | ||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K3185004 parameters</partinfo> | <partinfo>BBa_K3185004 parameters</partinfo> | ||
<!-- --> | <!-- --> |
Revision as of 11:34, 18 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. 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Unknown
- 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.
SDS-PAGE
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.