Coding

Part:BBa_K2132003

Designed by: Shijie Gu   Group: iGEM16_ShanghaitechChina   (2016-10-12)
Revision as of 08:30, 12 October 2016 by LechenQian (Talk | contribs)


mCherry-SpyTag

This is a reporter protein for any protein with SpyCatcher tagged. mCherry-SpyTag is stable under room temperature. It can bind to SpyCatcher within 20 min. In our project, we used it to see if the expression of CsgA-SpyCatcher is successful.


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]

As figure illustrated, his-CsgA-SpyCatcher-his mutant incubated with mcherry-SpyTag show a clear biofilm-associated mcherry fluorescence signal, which indicating the accurate conformation and function of the SpyTag and SpyCatcher linkage system. The third figure is merged by the first and second figures of each sample are snapped respectively under green laser field with 558 nm wavelength and bright field of fluorescence microscopy, Zeiss Axio Imager Z2. As for controls, strains secreted CsgA–histag and ΔCsgA both are unable to specifically attach to SpyTag thus no distinct localization highlight of red fluorescence on E.coli. That to a large extent prove the specificity of our desired linkage between SpyTag and SpyCatcher system.

Functional Parameters

[edit]
Categories
Parameters
None