Coding

Part:BBa_K3185004

Designed by: Masahiro Sakono   Group: iGEM19_Kyoto   (2019-10-04)
Revision as of 10:59, 8 October 2019 by 0012massan (Talk | contribs) (→‎SDS Page)


SPYCatcher -> BaCBM2

Usage and Biology

BaCBM2 is a Carbonhydrate-binding Module family 2(CBM2) derived from Bacillus anthracis[1]. CBMs are protein domains mainly found in carbohydrate-active enzymes.

As our project involves connecting different proteins, we decided to use the SpyTag/SpyCatcher system and have inserted the SpyCatcher at the N-terminal of the BaCBM2.

We insert this part in pET-11a plasmid between BamHI and NdeI restriction sites.


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]

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

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