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 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 test whether the addition of SpyTag peptide could confer the artificial functions to the biofilm, such as covalent immobilization of SpyCatcher-fusion proteins.

There’s a significant difference between the two groups. This suggests our CsgA-SpyTag could sufficiently connect with SpyCatcher-CotA .

Verification- Using SpyCatcher-CotA

We used ABTS as indicator to measure the laccase activity of the biofilm after the fixing process as a whole.

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

Reference:

“Uranium Reaper.” Uranium Reaper - Team: Peking, 2016.igem.org/Team:Peking.