Revision as of 19:06, 30 June 2017

Crude oil is composed of many complex PAHs (approximately one third of crude oil is complex aromatics) that are difficult to degrade and destroy vital components in our marine and terrestrial ecosystems. It is pertinent to find a time and cost effective procedure that will catabolize some of the most prevalent, toxic PAHs in crude oil --fluorene, phenanthrene, and naphthalene--to salicylate, an innocuous compound able to be degraded in metabolic cycles. Certain bacteria utilize intermediates in their degradative pathways to break down such PAHs. These intermediates create simpler pathways that result in lesser complication regarding gene combination and nucleotide sequencing.

We propose a novel methodology of degradation of multiple PAHs through combinational implementation of these bacteria-derived pathways into Escherichia Coli. Thus, this treatment will allow for broad spectrum transformation of multiple PAHs within the same oil environment into safer residues. By incorporating a fluorescent protein within the genetic structure of our engineered bacteria, the monitoring and isolation of the bacteria from the spill site will be easily achievable. These bacteria can then be implemented in the field to clean various oil spills and/or used in bioreactors, achieving efficient detoxification through combinational genetic bioremediation.

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 <h1> Our Project </h1>
-<p>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.</p>
+<p>Crude oil is composed of many complex PAHs (approximately one third of crude oil is complex aromatics) that are difficult to degrade and destroy vital components in our marine and terrestrial ecosystems. It is pertinent to find a time and cost effective procedure that will catabolize some of the most prevalent, toxic PAHs in crude oil --fluorene, phenanthrene, and naphthalene--to salicylate, an innocuous compound able to be degraded in metabolic cycles. Certain bacteria utilize intermediates in their degradative pathways to break down such PAHs. These intermediates create simpler pathways that result in lesser complication regarding gene combination and nucleotide sequencing. </p>
-<p>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.</p>
+<p>We propose a novel methodology of degradation of multiple PAHs through combinational implementation of these bacteria-derived pathways into Escherichia Coli. Thus, this treatment will allow for broad spectrum transformation of multiple PAHs within the same oil environment into safer residues. By incorporating a fluorescent protein within the genetic structure of our engineered bacteria, the monitoring and isolation of the bacteria from the spill site will be easily achievable. These bacteria can then be implemented in the field to clean various oil spills and/or used in bioreactors, achieving efficient detoxification through combinational genetic bioremediation.
-<p>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.
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Difference between revisions of "Team:CCA San Diego"

Revision as of 19:06, 30 June 2017

CCA_San_Diego

Our Project

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