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Team:CSU Fort Collins/Biosensor/
From 2014.igem.org
A Biosensor for Breakdown Confirmation
The CSU iGEM team’s focus this year was to find an efficient, environmentally-friendly way to used frying oil. The goal of the biosensor group’s project was to create a biosensor that will produce an easily-recognizable signal that will indicate when the breakdown of cooking oil occurs. Because we wanted the signal to be understandable by people both with and without scientific training, we decided to use a visual signal that would not require any interpretation beyond its presence or lack thereof. In order to find a way to make this signal we researched β-oxidation, the process by which fatty acids are broken down in the cell.
The process of β-oxidation has multiple products, but we chose to focus on acyl-CoA because of a particular paper we found in our research of the topic. In design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids, Dr. Fuzhong Zhang “developed a dynamic sensor-regulator system (DSRS) to produce fatty acid–based products in Escherichia coli, and demonstrated its use for biodiesel production” (Zhang, 1). We incorporated the promoters that he designed for his system into our own, because it was designed to regulate the expression of the red fluorescent protein (RFP) gene dependent on the presence or absence of Acyl-CoA. This would allow us the visual signal that we were looking for.
We started by breaking the promoter sequences into four oligonucleotides each and assembling the promoters from the synthesized oligos, a method we chose because of time and cost efficiency. Then, after checking for correct construction via electrophoresis, we incorporated our promoters into the pSB1C3 plasmid backbone via a restriction enzyme digest and ligation. We chose this method over the Gibson assembly that our coworkers used because our lab found that Gibson assembly is more difficult for us to perform, and because we were inserting our promoters one at a time into the plasmid. After insertion into the plasmid, we attempted to transform the plasmid into E. Coli.
We have not yet successfully transformed our plasmid with the inserted promoters into our E. Coli cells. Most of our difficulties came from the construction of the promoters, which failed multiple times. In order to bypass this in future work we intend to order the entire promoters sequenced and insert that into the plasmid along with the RFP gene and measure the fluorescence of the E. Coli when inserted into an oil rich environment.
1. Zhang, Fuzhong, et al. "Design of a Dynamic Sensor-regulator System for Production of Chemicals and Fuels Derived from Fatty Acids." Nature Biotechnology 30.4 (2012): 354-59. Web.
The process of β-oxidation has multiple products, but we chose to focus on acyl-CoA because of a particular paper we found in our research of the topic. In design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids, Dr. Fuzhong Zhang “developed a dynamic sensor-regulator system (DSRS) to produce fatty acid–based products in Escherichia coli, and demonstrated its use for biodiesel production” (Zhang, 1). We incorporated the promoters that he designed for his system into our own, because it was designed to regulate the expression of the red fluorescent protein (RFP) gene dependent on the presence or absence of Acyl-CoA. This would allow us the visual signal that we were looking for.
We started by breaking the promoter sequences into four oligonucleotides each and assembling the promoters from the synthesized oligos, a method we chose because of time and cost efficiency. Then, after checking for correct construction via electrophoresis, we incorporated our promoters into the pSB1C3 plasmid backbone via a restriction enzyme digest and ligation. We chose this method over the Gibson assembly that our coworkers used because our lab found that Gibson assembly is more difficult for us to perform, and because we were inserting our promoters one at a time into the plasmid. After insertion into the plasmid, we attempted to transform the plasmid into E. Coli.
We have not yet successfully transformed our plasmid with the inserted promoters into our E. Coli cells. Most of our difficulties came from the construction of the promoters, which failed multiple times. In order to bypass this in future work we intend to order the entire promoters sequenced and insert that into the plasmid along with the RFP gene and measure the fluorescence of the E. Coli when inserted into an oil rich environment.
1. Zhang, Fuzhong, et al. "Design of a Dynamic Sensor-regulator System for Production of Chemicals and Fuels Derived from Fatty Acids." Nature Biotechnology 30.4 (2012): 354-59. Web.
