Team:SHSBNU China/Improve
Improve
I. Demonstration
Fig1.1: CsgA-SpyTag
CsgA is a kind of amyloid protein that could form biofilm in E. coli cells. We fused SpyTag domain after 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 and the biofilm forming amyloid protein, it is possible to fix any SpyChatcher fused protein domains to the biofilm.
(Team: Peking, 2016)
Fig1.2: SpyTag and SpyCatcher System
II. Verification - Using sfGFP-SpyCatcher
CsgA Improvement
We used sfGFP-SpyCatcher as indicater to measure the amount of sfGFP-SpyCatcher protein our CsgA-SpyTag biofilm could capture.
Protocol for SpyTag-SpyCatcher system verification
The decrease of florescence of the supernatant indicates how much sfGFP-SpyCatcher protein the biofilm gain.
There’s a significant difference between the two groups. Thus, our CgsA-SpyTag successfully fixed sfGFP-CatCher proetin.
III. Verification - Using SpyCatcher-CotA
We used SpyCatcher-CotA Protein, laccase with SpyCatcher, to fix on to our CsgA-SpyTag biofilm. We used ABTS as indicator to measure the laccase activity of the biofilm after the fixing process as a whole. If our CsgA-SpyTag biofilm has laccase activity, that means the fixing process of SpyCatcher and SpyTag is successful.
Protocol for Biofilm x Laccase
There were significance of difference was indicated as **** which was pretty high. So we proved that SpyCatcher-CotA is successfully combined to the biofilm.
IV. Conclusion
In our experiments, we give CsgA a new ability, which is capturing all fusion protein with SpyCatcher, by adding SpyTag domain after CsgA. We test SpyTag using sfGFP-SpyCatcher and SpyCatcher-CotA. We also proved that fusion protein is still functional after fixed to the biofilm.
Reference:
“Uranium Reaper.” Uranium Reaper - Team: Peking, 2016.igem.org/Team:Peking.