Part:BBa_K4229067

mTurquoise2 with C-terminal SnoopCatcher regulated by tetA/B promotor

This BioBrick was used as a fluorescent reporter fused with a SnnopCatcher, enabling recruitment by the SpyTag.

We used this fusion protein to test the functionality of our compartments: the minimal wiffleball (BBa_K4229047), full wiffleball (BBa_K4229049), and SPD-5 (BBa_K4229078). All compartmentalization proteins have an N-terminal SpyTag.

The SnoopTag/Catcher system was tested with those two reporter genes. A fluorescent signal in the bacteria that expressed either protein could be detected. Upon co-expression with compartmentalization proteins, foci were formed (especially well with the full wiffleball) with both reporter genes:

Fluorescent microscopy of T1 catching the mVenus2 and mTurquoise2 when the minimal or full wiffleball construct is expressed: A. Controls for induction; B. T1 with and without the Spy/Snp tags; scale bar = 5µm

It was later confirmed that in fact mTurqouise was cached by T1 by a western blot comparing the full wiffleball and the minimal wiffleball.

: Western Blot of the BMC minimal wiffleball (pT1spysnp) and full wiffleball (pT1spysnpT2T3) + mTurquoise2

With that, we can say that our mTurquiose2 with the snoopCatcher is successfully expressed and caught by our compartment.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 59
  Illegal PstI site found at 856
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 59
  Illegal PstI site found at 856
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 59
  Illegal BglII site found at 68
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 59
  Illegal PstI site found at 856
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 59
  Illegal PstI site found at 856
  Illegal AgeI site found at 121
  Illegal AgeI site found at 1017
  
• 1000
  COMPATIBLE WITH RFC[1000]