Team:Ionis Paris/E.coli
We chose to use E. coli strain DH5α as chassis for amplified our plasmid and to use E. coli strain BL21 to express our recombinant protein. All our parts are optimized for E. coli and this is an extensively characterized model organism. E. coli DH5a is a non-pathogenic strain of E.coli that was developed for laboratory cloning use. The genomic structure of this strain is a singular circular chromosome consisting of 4,686,137 nucleotides, 4359 genes, of which 4128 are protein encoding genes.
E. coli DH5a is an excellent strain for laboratory cloning procedures. It has the ability to accept plasmid insertion exceptionally well and its several mutations enable high-efficiency transformations. Among those mutation, the endA1 mutation allows for lower endonuclease degradation which ensures higher plasmid transfer rates and the recA1 mutation reduces homologous recombination for a more stable insert. A deoR mutation enables the bacteria to grow on minimal media with only one carbon source (iosine) but also to efficiently integrate large inserts. The GyrA mutation avoid deletions between direct repeats that could occur in the insert on a plamid. [1][2] E. coli DH5a reproduces by successive binary fission with a generation time of approximately 30 minutes with optimum growth occurring at 37 degrees centigrade. E. coli are facultative aerobic bacteria and are capable of ATP synthesis via both aerobic respiration and anaerobic fermentation. This particular strain can be identified and distinguished from other E. coli strains by examining the genetic sequence of its 16s small ribosomal subunit, which has been fully sequenced. [3] E.coli BL21 is a non-pathogenic strain of E.coli that was created by F. William Studier and Barbara A. Moffatt. This strain was developed especially for recombinant protein synthesis as it lacks lon and ompT proteases. [4] It is important to study the effect of pollutants on our chassis to better understand our results and predict the possible outcomes of those effects on the sensitivity of our biosensor. Toluene has been employed for many years by microbiologists to sterilize cultures and to maintain solutions in a sterile condition. Therefore, toluene has a deleterious effect on bacteria[5]. At low concentration, toluene does not directly disrupt the cells nor denature its components. However, material lost from the cell treated with toluene was analyzed. Release of this material, largely RNA (especially ribosomal RNA) and protein, was due to the complete and rapid destruction of galactosidase permease as well as the disturbance of tryptophan permease activity. Others permease activities seem also to be affected [5][6]. Moreover, toluene seems to elicit the enzymatic degradation of ribosomes followed by degradation of ribosomal RNA. Toluene treated cells also lose the ability to concentrate substrates and their macromolecule synthesis ability was affected. The toluene-treated cells were shown to exclusively synthesize membrane proteins [7]. As the respiratory chain of toluene treated cells seems to be intact, prior work has established that toluene does not trigger the total destruction of the membrane and that its lethal effects were mainly due to the loss of the ability to concentrate substrate, the loss of selective permeability and the loss of ribosomes. Toluene treated cells retain the machinery to provide themselves with energy [5]. References (Links are provided when available):E.Coli
Strain choice & quick description
V.O.Cs & Toluene
Effects on E.coli
