Part:BBa_K3788021

Expression of GFP and of the timer device fused to the RFP protein

Introduction  :

The Part BBa_K3788021 is a part composed from the parts BBa_K3788017 (lexA-repressed promoter), BBa_K3788015, BBa_E1010 (RFP) fused to cal gene and BBa_E0040 (GFP) fused to caa gene.

With this design, this part integrates a part of the cal gene, coding for the lysis protein CaL. It is also composed from caa and cai genes, respectively for the Colicin A toxin and the immunity protein Cal.

In normal conditions, caa and cai genes are positioned as an operon, and the cai gene is located on the antisense strand and expressed in a constitutive manner.

This system’s regulation is constituted from many regulation elements, which permits it to obtain a fine-tuned regulation.

This system is located on a plasmid present in some E. Coli populations. It allows Colicin A accumulation on the host cell, and then induces its death due to CaL action, which provokes Colicin A liberation on the extracellular medium, which induces the death of bacteria that don’t produce Cal protein.

cal gene codes for CaL protein, a lipopeptide with a length between 28 and 48 amino acid residues. This protein is produced as a precursor and then processed. Once the protein is processed, it can form pores in the bacterial membrane, which leads to the quasi-lysis of the host cell, and provokes a leak from the cellular component into the medium.

Design :

This construction is used to study the time-shift between ColA and Cal production after induction of the promoter, by measuring the fluorescence kinetics.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1528
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1848
  Illegal BamHI site found at 1521
  Illegal XhoI site found at 1397
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1597
  Illegal AgeI site found at 2849
  Illegal AgeI site found at 2961
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 867
  Illegal SapI.rc site found at 1351