Part:BBa_M36252:Experience

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Applications of BBa_M36252

Gasoline contamination of groundwater presents a significant danger to humans and the environment. According to the MSDS of Shell Unleaded Petrol, various components in gasoline are carcinogenic and mutagenic and can lead to systemic organ damage. Gasoline can also easily contaminate groundwater. The EPA estimates that as many as 25% of underground petroleum tanks may be leaking and that even a few quarts of contaminant can devastate a farmstead’s water supply. Furthermore, the presence of harmful levels of contaminant is undetectable by human taste or smell. Methods used to detect gasoline contaminants require relatively bulky and expensive equipment, such as the Penetrometer/Fluorometer System developed by the US Army. To date, it seems that the majority of research into hydrocarbon contaminant detection has been focused on chemical and mechanical methods. Our project aims to design a biological hydrocarbon sensor which, when fused to GFP, could provide a cheap and easily implementable method for hydrocarbon contamination detection. Our group also posits that this technology could be applied to search for new hydrocarbon deposits as well.

We tested our construct in a high copy vector with amp resistance. Our construct was also upstream of the Gemini actuator designed by Martin, et al. From here, we measured GFP expression with varying concentrations of different alkanes. These are the results: [http://a6.sphotos.ak.fbcdn.net/hphotos-ak-snc7/375201_2803945735023_1150011077_3246609_344426867_n.jpg]

Testing of our construct revealed that it does not work. It appears to be constitutively generating a PoPS signal. We hypothesize that the problem lies in the design of PalkB (BBa_M36250), in that there are missing nucleotides upstream of the start of BBa_M36250 which serve to clamp the region shut to RNAP.

References:

Chrestman, A.M., G.D. Comes, S.S. Cooper, and P.G. Malone. "Rapid Detection of Hydrocarbon Contamination in Ground Water and Soil." Hydraulic Engineering: Saving a Threatened Resource—In Search of Solutions: Proceedings of the Hydraulic Engineering Sessions at Water Forum ’92. (1992): 1165-170.

Kok, M., R. Oldenhuis, M P Van Der Linden, P. Raatjes, J. Kingma, P H Van Lelyveld, and B. Witholt. "The Pseudomonas Oleovorans Alkane Hydroxylase Gene Sequence and Expression." Journal of Biological Chemistry 264.10 (1989): 5435-441.

Makart, Stefan, Matthias Heinemann, and Sven Panke. "Characterization of the AlkS/PalkB-expression System as an Efficient Tool for the Production of Recombinant Proteins InEscherichia Coli Fed-batch Fermentations." Biotechnology and Bioengineering 96.2 (2007): 326-36.

Puigbo, P. "OPTIMIZER." Evolutionary Genomics Group. Universitat Rovira I Virgili (URV), Apr. 2007. Web. 10 Dec. 2011. <http://genomes.urv.es/OPTIMIZER/>.

The Missouri Farmstead Assessment System. "WQ654 Assessing the Risk of Groundwater Contamination From Petroleum Product Storage | University of Missouri Extension."University of Missouri Extension Home. University of Missouri, Oct. 1995. Web. 28 Oct. 2011. <http://extension.missouri.edu/p/WQ654>.

The Shell Company of Australia Limited. "Material Safety Data Sheet for Shell Unleaded Petrol." Shell.com. Shell, 4 Aug. 2010. Web. 27 Oct. 2011. <http://www-static.shell.com/static/aus/downloads/fuels/msds/msds_shellunleadedpetrol.pdf>.

Van Beilen, J.B., S. Panke, S. Lucchini, A.G. Franchini, M. Rothlisberger, and B. Witholt. "Analysis of Pseudomonas Putida Alkane Degradation Gene Clusters and ﬂanking Insertion Sequences : Evolution and Regulation of the Alk Genes." Microbiology 147 (2001): 1621-630.

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