Part:BBa_K3788020

Expression of the timer device fused to the RFP protein

Introduction  :

The Part BBa_K3788020 is a part composed with BBa_K3788017 part (lexA-repressed promoter), BBa_K3788015 and BBa_E1010 (RFP).

This construction allows the fusion of the RFP to the regulatory sequences of cal under the control of the lexA-repressed promoter.

Thus, it is composed of a fragment of the cal gene encoding the CaL lysis protein. It also contains the caa and cai genes coding respectively for the ColA toxin (colicin A) and the CaI immunity protein.

In nature, the caa and cai genes are in operon, and the gene encoding the immunity protein is constitutively expressed and is found on the antisense strand of the operon. The regulation of this system is complex and contains many regulatory elements.

This system is located on a plasmid present in certain populations of wild E. coli. It induces the accumulation of ColA toxin in the host cell and then causes the latter to die by the action of the lysis protein. This releases ColA into the medium and kills cells that do not have the immunity gene present on this plasmid.

The cal gene encodes the CaL protein, a 28 to 41 residue lipopeptide, which is produced as a precursor and then processed. When mature, it forms pores in the membrane, causing the host to near lysis and the leakage of cellular components into the medium.

Design  :

This construct allows the study of the previously reported latency period between induction of the caa-cal operon system, production of ColicinA and production of the CaL lysis protein.

Since the caa and cal genes are both regulated by the same promoter, we concluded that the delay between the synthesis of colicin A and CaL depended on the regulatory elements present in the sequence between the caa and cal genes. The BBa_K3788018 part composed of this regulatory sequence and the cal gene. This was designed as a “timer lysis device” (BBa_K3788018).

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 808
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1128
  Illegal BamHI site found at 801
  Illegal XhoI site found at 677
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 877
  Illegal AgeI site found at 2129
  Illegal AgeI site found at 2241
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 631