Coding

Part:BBa_K3185003

Designed by: Masahiro Sakono   Group: iGEM19_Kyoto   (2019-10-04)
Revision as of 05:57, 19 October 2019 by Daidai (Talk | contribs) (Purification)


SPYCatcher

Usage and Biology

SpyCatcher is a protein that came from the CnaB2 domain of FbaB, Streptococcus pyogenes(SpyCatcher:BBa_K1159200). In a natural environment, CnaB2 domain is used for attaching to host cells. In a paper, it is partially changed and divided into two domains.[1] Nowadays, these two protein domains are known as SpyCatcher/SpyTag system because they bind irreversibly with a covalent bond.

In our experiment, we used the SpyCatcher/SpyTag system and designed only SpyCatcher part for assay(SpyCatcher:BBa_K1159200, SpyTag:BBa_K1159201). In addition, this has two tag or cleavage sites. First is 6×His-tag inserted in the N-terminus of SpyC for protein purification. Second is a TEV protease site because, in the paper, it was used for protein purification[2]. However, we didn’t use it in our experiment.

We put it between the BamHI site and the Ndel site on pET11-a. We used BL21 (DE3) for gene expression. We used the Ni-NTA agarose for purification. After that, we confirmed the molecular weight of SpyCatcher 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

penguin



References

1 Zakeri, B., Fierer, J.O., Celik, E., Chittock, E.C., Schwarz-Linek, U., Moy, V.T., and Howarth, M. (2012).
Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.
Proc. Natl. Acad. Sci. U. S. A. <i>109.

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.

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