Team:UTDallas/Project ProjectOverview

Project Overview

Millions of gallons of toxic petroleum are released into the ocean each year as a result of accidents such as oil spills or natural geological seepage.

Crude oil is a complex mixture of hydrocarbons consisting largely of n-alkane and aromatic hydrocarbons. Saturated hydrocarbons with linear or branched chains comprise the alkane series, which includes paraffinic hydrocarbons. Unsaturated hydrocarbons with benzene rings comprise the aromatic series, which includes the BTEX compounds.

The World Water Assessment Program, the UN’s flagship water protection initiative, describes a host of health risks associated with these chemicals, some of which are enumerated in the table below (adapted from WWAP brochure)

Table

While petroleum constituents are generally found around industry, nitrates are a common ingredient in fertilizers, whose use is widespread in agricultural practice. Nutrient-rich runoff enriches water sources such as lakes, rivers and aquifers with nitrates in a process called eutrophication. This facilitates the onset of algal blooms that deprive the native organisms of oxygen and essential nutrients. Afflicted water sources are difficult and expensive to restore and the process could further harm the wildlife.

Due to stable, unreactive chemical structures, many of the aforementioned chemicals are persistent contaminants that circulate through the environment, thus polluting usable water supplies and marine ecosystems for extended periods of time. In fact, crude residues from the 2002 Prestige spill are still, 8 years later, encountered along Glacian shores. The need to control and mitigate the circulation of such chemicals is therefore both eminent and urgent. To that end, the UT Dallas iGEM team is developing novel, modular biosensors that enable cheap, on-site detection of aromatics, nitrates/nitrites and alkanes in low concentrations using an Escherichia coli chassis.

The aromatics sensor will build on the previous work of the Michigan 2009 and Glasgow 2007 teams. Team Glasgow produced a successfully characterized fusion of the pR constitutive promoter and XylR transcription factor of the pu promoter. Team Michigan produced a fusion of the pu promoter and GFP, but failed to characterize the construct. We successfully characterized the function of the Michigan 2009 construct and assess its sensing capabilities in the presence of aromatics.

The nitrate/nitrite sensor will build on parts submitted by the Edinburgh 2009 team. This includes a fusion of the nitrate/nitrite-inducible pYeaR promoter and GFP. We successfully characterized the function construct with a GFP reporter system and with a variety of nitrate/nitrite compounds, which was not included in the work by Edinburgh.

The alkane sensor will include components of the alkane metabolic pathway of Pseudomonas putida OCT. The promoter region pAlkB drives the transcription of the alk gene cluster. AlkS, its transcription factor, is encoded by the alkS gene. We will perform a fusion of pAlkB to a fluorescent protein part to make a reporter construct. A plasmid containing alkS was kindly provided by Dr. Jan Roelof van der Meer of the University of Lausanne in Switzerland. AlkS binds to n-alkanes to form a dimer that positively regulates the pAlkB promoter to drive the expression of fluorescent protein. The two plasmids containing the promoter/fluorescent protein fusion and the AlkS transcription factor will be co-transformed into DH5a to characterize sensing capabilities.