Part:BBa_K3788023

Expression of GFP and of an optimized design of the timer device

Introduction  :

The Part BBa_K3788023 is a part composed with BBa_K3788017 part (lexA-repressed promoter), BBa_K3788016, BBa_E0040 (GFP coding sequence) fused to caa gene.

With this design, this part integrates a part of the caa gene, coding for the Colicin A toxin. It is also composed from cal and cai genes, respectively for the lysis protein CaL 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 : </p>BBa_K3788016 is a BBa_K3788015 improvement. It contains a bigger 5’ extremity of the caa coding sequences, which permit to keep some regulation elements that could be involved in the lysis protein regulation.</p>

This caa contains an alternative LexA binding box identified in silico using virtual footprint (https://www.prodoric.de/vfp).This corresponds to an alternative promoter.

This part design allows the construction of the composite part BBa_K3788023 that corresponds to BBa_K3788019 that had been engineered.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1568
  Illegal PstI site found at 1671
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2266
  Illegal PstI site found at 1568
  Illegal PstI site found at 1671
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2586
  Illegal BamHI site found at 2259
  Illegal XhoI site found at 2135
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1568
  Illegal PstI site found at 1671
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1568
  Illegal PstI site found at 1671
  Illegal NgoMIV site found at 2335
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 867
  Illegal SapI.rc site found at 2089