Coding

Part:BBa_K2684006

Designed by: Yihao Zhang   Group: iGEM18_SHSBNU_China   (2018-10-08)

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.

[edit]
Categories
Parameters
None