Part:BBa_K4229066

mVenus with N-terminal spyCatcher regulated by tetA/B promotor

mVenus2 (together with mTurquoise2,[BBa_K4229064]) was used as a reporter by fusing it with a SpyCatcher, enabling recruitment by the SpyTag. We used this fusion protein to test the functionality of our compartments: the minimal (BBa_K4229047) and full wiffleball (BBa_K4229049), and SPD5 (BBa_K4229078). All compartmentalisation proteins have an N-terminal SpyTag. For experimental data please refer to the data listed in Biobrick BBa_K4229067.

The Snoop/SpyTag Snoop/SpyCatcher was tested with those two reporter genes. A fluorescent signal in the bacteria that expressed either protein could be detected.Upon co-expression with compartmentalisation 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 BMC construct is expressed: A Controls for induction; B T1 with and without the Spy/Snp tags; scalebar 5µm

It was later confirmed that in fact mVenus was cached by T1 by a western blot comparing the full wiffleball with and without the necessary tags.

Western Blot comparison of the BMC minimal wiffleball with and w/o tags (pHT1) + mVenus2

With that, we can say that our mVenus tagged with the SpyCatcher is successfully expressed and caught by our compartment.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

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

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

  Illegal EcoRI site found at 59
  Illegal BglII site found at 68
  Illegal XhoI site found at 1075
  
• 23
  INCOMPATIBLE WITH RFC[23]

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

  Illegal EcoRI site found at 59
  Illegal AgeI site found at 508
  
• 1000
  COMPATIBLE WITH RFC[1000]