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

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)

Demonstration

CsgA and CsgA-SpyTag were both cloned into the plasmid of pET28a for further improvement assay. We transferred both part in to ΔCsgA-MG1655 cells as two groups and incubate them till OD≈0.6. Both groups were centrifuged. We used a reaction stock containing SpyCatcher-sfGFP protein, of which the florescence was measured, to react with the palette of the two groups. We allow the reaction undergo 1 hours. After that, we centrifuged both groups again and measure the florescence of the supernatant. The amount of florescence the supernatant decreased indicates how much SpyCatcher-sfGFP protein the palette fixed.

There’s a significant difference between the two groups. Thus, our CgsA-SpyTag is functional whenconbining with proetin fused wity SpyCatcher domain.

Reference:

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