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

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.

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.

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.