Part:BBa_K3185004

SPYCatcher -> BaCBM2

Usage and Biology

BaCBM2 is a Carbonhydrate-binding Module family 2(CBM2) derived from Bacillus anthracis. CBMs are protein domains mainly found in carbohydrate-active enzymes. According to an article[1], BaCBM2 is proved to have the highest binding ability to PET film, so we introduce this protain to the part as PET-binding protain.

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. When this part connected with SpyTag inserted Tm Encapsulin(りんく), the compound does complete work as flocculant.

This part has three tags or binding sites;6xHis-tag,MYC-tag and TEV-binding site.6xHis-tag is inserted at the N-terminal of the SpyCatcher and used for affinity purification of this part. MYC-tag is also inserted between SpyCatcher and BaCBM2 in order to be detected with antiboby in Western brotting. TEV-binding site is inserted in the original article[2], and we left it as it was so as not to change the function, although the site is not used in our experiment.

We lay this part on pET-11a plasmid between BamHI and NdeI restriction sites, and using the T7 promoter to express the resulting protein in BL21(DE3).

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 37^oC shaking at 140 rpm to an OD600 of 0.5, verifying via a spectrophotometer.

• Protein was expressed in 0.1mM IPTG for 2hours.