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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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