Part:BBa_K2387032

CpxR-eYFPn[1-154] and CpxR-eYPFc[155-238] + araC/pBAD promoter

The [http://parts.igem.com/Part:BBa_K2387005 N-terminus of eYFP] and the[http://parts.igem.com/Part:BBa_K2387006 C-terminus of eYFP] are fused to Cpx response regulator [http://parts.igem.com/Part:BBa_K2387002 CpxR] in order to visualize the activation of the Cpx pathway. Upon activation of the Cpx pathway, CpxR gets phosphorylated by E. coli endogenous CpxA after which it can homodimerize. This protein-protein interaction can be visualized using BiFC, hence the fusion of eYFPn[1-154] and eYFPc[155-238]. The fusion is put under the control of the L-arabinose inducible araC/pBAD promoter. Strong RBS BBa_B0034 is used to regulate transcription.

CpxR-eYFPn and CpxR-eYFPc fusions were linked together using a [http://parts.igem.com/Part:BBa_K1486004 Flexible Linker] consisting of two times the amino acids GGGGS.

This part is used to visualize the activation of the Cpx pathway via Bimolecular Fluorescence Complementation by using the CpxR-CpxR interaction.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  Illegal XhoI site found at 1283
  Illegal XhoI site found at 2498
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  Illegal AgeI site found at 1694
  Illegal AgeI site found at 2909
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  

Usage and Biology

BBa_K2387032 is created as a means to detect activation of the Cpx pathway of E. coli. This is done using a method called Bimolecular Fluorescence Complementation (BiFC) [1]. To optimize experimental results, wet-lab experience and computer models were used.