Part:BBa_K2684006

CsgA-SpyTag

CsgA fused with SpyTag by 2xGGGGS linker
This part was improved from BBa_K1583000: https://parts.igem.org/Part:BBa_K1583000

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]

Demonstration

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.

Fig1.2: SpyTag and SpyCatcher System （Source From Peking 2016 wiki）

Verification- Using sfGFP-SpyCatche

We used sfGFP-SpyCatcher as indicator to measure the amount of sfGFP-SpyCatcher protein our CsgA-SpyTag biofilm could capture. 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 CsgA-SpyTag successfully fixed sfGFP-CatCher protein.

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.

As the **** shows, there is significant difference between CsgA and CsgA-Spytag in enzyme activity when combining with SpyCatcher-CotA
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.