Team:SHSBNU China/Improve
Improve
I. Overview
Fig1.1: CsgA-SpyTag
CsgA is a amyloid protein which is a dominant component of the biofilms of E. coli. We fused a functional peptide domain SpyTag with gene CsgA (previous part: BBa_K1583000), forming CsgA-SpyTag (Improved Part: BBa_K2684006). By utilizing the steady isopeptide bond combination ability of the SpyTag-SpyCatcher system , it is possible to fix different SpyChatcher-fusing proteins, like enzymes or receptors, to the biofilm to introduce a variety of artificial functions.
(Team: Peking, 2016)
Fig1.2: SpyTag and SpyCatcher System
II. Verification - Using sfGFP-SpyCatcher
We used sfGFP-SpyCatcher as indicator to test whether the addition of SpyTag peptide could confer the artificial functions to the biofilm, such as covalent immobilization of SpyCatcher-fusion proteins.
Protocol for SpyTag-SpyCatcher system verification
We tested our improved part BBa_K2684006 and the previous part BBa_K1583000 with the same protein expression system settings (E. coli BL21, pET28a vector, and the same induction and expression protocol).
The decrease of florescence of the supernatant indicates the immobilization of sfGFP-SpyCatcher, and there’s a significant difference between the two groups.
III. Verification - Using SpyCatcher-CotA
The immobilization of SpyCatcher-CotA fusion protein to the CsgA-SpyTag biofilm was tested, and a commercial laccase activity assay kits was used to measure the laccase activity.
Protocol for Biofilm x Laccase
The difference between our improved part and the previous part is significant. Thus, we proved that SpyCatcher-CotA can be attached to the biofilm containing functional peptide SpyTag.
IV. Conclusion
To conclude, we introduce a new ability to the previous CsgA part, which is capturing all fusion protein with SpyCatcher domain, by fusing SpyTag peptide to CsgA coding sequence. We verified the protein immobilization function of our improved part using sfGFP-SpyCatcher and SpyCatcher-CotA, and proved that SpyCatcher fusion protein is still functional after fixed to the biofilm.
Reference:
“Uranium Reaper.” Uranium Reaper - Team: Peking, 2016.igem.org/Team:Peking.