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Team:Calgary/Project
From 2011.igem.org
The Project
A Biosensor for Naphthenic Acids
Naphthenic Acids in the Oil Sands
Naphthenic Acids (NAs) are a grouping of different carboxylic acids which may contain multiple hydrocarbon rings. Containing both a large hydrocarbon segment and a carboxylic acid group makes them a slightly amphipathic compound. NAs are natively found in oil sands deposits and their surfactant quality contributes to a higher efficiency of oil sands recovery in the hot water extraction process. The majority of the NAs end up in large tailings ponds with the water used in the bitumen extraction process. The NAs continuously accumulate in these large tailings ponds along with other wastes generated in the bitumen extraction process which are left to settle to the bottom of the pond.
The Toxicity of Naphthenic Acids
Unfortunately NAs are a contributor to corrosion in equipment and pipelines. In addition, their surfactant nature also makes them toxic as it allows them to pass through cell membranes and adversely affect a plethora of local organisms. The higher the concentration of the NAs in the environment the greater the potential harm to the local ecosystem.
Monitoring Naphthenic Acids
Having the ability to monitor the levels of NAs in an area would allow for the monitoring of levels of NAs in tailings ponds, and would be useful in assessing whether or not any future detoxification or remediation efforts were working. In addition, the surrounding areas could also be tested for seepage of NAs into ground water. Currently, the only ways to test for the presence of NAs are mass spectrometry and gas chromatography. These methods are both costly and inconvenient since samples must be taken off site for processing and interpreting results would take time. The University of Calgary’s iGEM team is working on developing a novel electrochemical biosensor which would allow for convenient on site monitoring of NAs. Certain components of the system would be re-useable while the actual bacteria involved in the sensing would be disposed after use.
Perhaps a pic of the system? Similar to the one on the ismos poster?Engineering the Biosensor
The electrochemical system would consist of two parts: an NA inducible promoter and a suitable reporter. For the reporter we chose the LacZ gene which produces β -galactosidase which can cleave certain substrates. When β -galactosidase cleaves the substrate chlorophenol red β -D-galactopyranoside (CPRG) it produces chlorophenol red (CPR). CPR can be oxidized when exposed to a current and the voltage can be measured which give a value corresponding to the CPR molecules being oxidized. In this way the quantity of CPR being produced could be monitored, which in turn would be based on how much β-galactosidase was present. You can read more about this system under Project Electro.
A diagram similar to the ismos poster one showing the two gene elements i.e. promoter and reporter might be niceIn order to find the promoter a couple of different approaches were employed. Firstly we attempted to create a genomic library of pseudomonas species known to degrade NAs and then screen it for NA degrading elements which would likely contain a promoter. We also created RT-qPCR experiments to search for genes of interest in those pseudomonas species which were upregulated in response to NAs. The genes selected for the RT-qPCR experiment (to observe upregulation) were selected through an intense bioinformatics search. A CHIP-SEQ experiment was also conducted to search for any genes or proteins (possible gene regulatory elements) which would bind NAs in the pseudomonas species. Information on these approaches can be found under Project Pseudomonas.
In addition, a microalgae species which was known to have some ability to degrade NAs was also examined for a possible promoter. However, this project only involved making a library (for screening purposes) of the chloroplast DNA only. Read more on this project by reading up Project Microalgae.
