Difference between revisions of "Part:BBa K2282013"

Revision as of 12:43, 25 October 2017

mRFP under heat inducible system pL/cI857

Usage and Biology

This part codes for the full sequence of mRFP, a red fluorescent protein, above 37°C. The construct is composed of a cI857 coding gene behind the strong constitutive promoter J23100 and RBS and an mRFP behind a pL promoter that is regulated by the gene cI857 and RBS. Both cI857 and mRFP coding genes are in front of a terminator. Leftward promoter (pL) derived from phage ƛ is fully repressed at low temperature by the thermolabile cI857 protein. cI857 loses repression activity on the pL promoter when the temperature rise above 30°C until 42°C, when it can no longer bind to pL. The aim of this construct is to code for mRFP when the temperature rises above 30°C, while the transcription is repressed under 30°C.

Source of this part

BBa_K2282013 part has been assembled to the pL promoter - RBS - Double terminator (E.coli his operon terminator). This double terminator was extracted from the strain E.coli K12 MG1655 complete genome, size length 4,641,652 pb in order to be different from the one in the part BBa_K22820012 and avoid concatemers problems.

Design consideration

This part sums up our final heat sensitive construction. The overall strategy we chose is complex and we were not sure it would work. Our bibliographic review and analysis of the Paris-Saclay iGEM 2017 project (using DNA thermometers) we decided to use strong promoters (Anderson BBa_J23100 with its respective RBS and pL promoter) to obtain higher expression levels. We had a slight problem in sequence design with IDT because their system detected our double use of double terminators as concatemers and did not let us introduce the same double terminator BBa_B0015 so we change the double terminator of the new part by an E.coli terminator operon.

References

Norma A Valdez-Cruz, Luis Caspeta, Néstor O Pérez, Octavio T Ramírez, Mauricio A Trujillo-Roldán - Production of recombinant proteins in E.Coli by the heat inducible expression system based on the phage lambda pL and/or pR promoters - Microbial Cell Factories 2010, 9:18.

Sequence and Features