Team:Calgary/Project

From 2011.igem.org

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<h2>Engineering the Biosensor</h2>
<h2>Engineering the Biosensor</h2>
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<p>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.</p>
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<p>Engineering the biosensor involved three main components: finding an appropriate sensory element, characterizing a novel reporter and selecting and designing tools for an appropriate chassis for our system. Information on the sensory element can be found on our Promoter Project page. More details on our reporter can be found on the Reporter Project page.  More details on our chassis can be found on our Chassis Project page.</p>
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A diagram similar to the ismos poster one showing the two gene elements i.e. promoter and reporter might be nice
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<p>In 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.</p>
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<p>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.</p>
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Revision as of 05:00, 28 September 2011


A Biosensor for Naphthenic Acids

Naphthenic Acids in the Oil Sands

Figure 1. Structure of Naphthenic Acids as discussed in Whitby, C. et al. 2010.

Naphthenic Acids (NAs) are a group of recalcitrant, hydrophobic, compounds which may contain a variety of structures. All napthenic acids contain a conserved carboxylic acid group followed by a hydrocarbon chain. Attached to this hydrphobic chain can be between one and four hydrogenated ring systems. The classification of what defines a napthenic acid has been of great debate in the scientific community, but there diversity has made them difficult to better understand them. 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.

Figure 1. Courtesy of Pembina

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.



Figure 1. LC50 values for Adult kutum, sturgeon, and roach when exposed to Napthenic Acids. Adapted from Dokholyan VK., Magomedov AK. (1983).


Monitoring Naphthenic Acids

Having the ability to monitor the levels of NAs is mandated by Canadian law and would be useful in assessing whether or not any future detoxification or remediation efforts were effective. Other applications including examining the surrounding areas for seepage of NAs into ground water, and examine . 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.

Figure 1. LC50 values for Adult kutum, sturgeon, and roach when exposed to Napthenic Acids. Adapted from Dokholyan VK., Magomedov AK. (1983).


Engineering the Biosensor

Engineering the biosensor involved three main components: finding an appropriate sensory element, characterizing a novel reporter and selecting and designing tools for an appropriate chassis for our system. Information on the sensory element can be found on our Promoter Project page. More details on our reporter can be found on the Reporter Project page. More details on our chassis can be found on our Chassis Project page.

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