Our Project

We are engineering two strains of E. coli in order to develop a circuit that allows researchers to culture both strains together in a fixed ratio. We will be using two orthogonal quorum sensing systems, likely Rhl and Las (or perhaps Lux and Cin). One strain of E. coli will express the gene for RhlI, which synthesizes the AHL, of which activates RhlR. The other strain of E. coli will express the gene for LuxI, which synthesises the AHL, hence activating LuxR. This gives a direct correlation between the amount of each AHL present and the corresponding population's size.

Each strain will express both transcriptional activators, RhlR and LuxR, and activate transcription of genes downstream of promoters pRhl and pLus, respectively. These promoters will be placed upstream of their respective STAR and Anti-STAR coding sequences. If the two populations are out of balance, the STAR RNA logic system will induce the expression of a growth inhibiting protein, T7 phage protein GP0.4 or GP0.2, in the larger population.

We are engineering a strain of E. coli (K12) that will degrade some of the most prevalent PAHs in crude oil—fluorene and phenanthrene—to salicylate and phthalate, the latter of which are much safer for the environment and can be utilized in various metabolic cycles. The process of engineering the bacteria begins with finding various genomic sequences from other bacteria that can degrade such PAHs to an extent, and it is continued by piecing together these genomes to find the correct sequence for one degradation pathway. For the bacterial pathways to be novel and efficient, the nucleotide sequence must undergo codon optimization, and the RBS sequence that sits between every ORF will be chosen specifically to create a new sequence. Since we will be using a low copy plasmid, a strong promoter and a regular terminator will be placed at the beginning and end of the sequence, as well as a promoter that will respond to concentrations of phthalate and salicylate to promote a gene that will fluoresce with the use of fluorescent protein, thus indicating that the process is approaching completion (or working). There may be some steps taken to regulate the enzymes produced in order to not only systematically and effectively use the energy and resources of the bacteria, but also to make sure the enzymes do not reach toxic concentrations.

Information

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