Team:Vanderbilt/Project
From 2014.igem.org
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<img src="https://static.igem.org/mediawiki/parts/f/fd/VU_experiment_2_diagram.png" align=right alt="First few experiments" width="500" style="padding-bottom:0.5em; float:right" /> | <img src="https://static.igem.org/mediawiki/parts/f/fd/VU_experiment_2_diagram.png" align=right alt="First few experiments" width="500" style="padding-bottom:0.5em; float:right" /> | ||
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- | Once we had clear banding patterns, it became clear that the number of introns in each of our genes (a variable which was unknown since most of the plants we worked with have not had their genomes sequenced) was too great for cloning and expression to be practical. Therefore, as soon as the fall semester began, we shifted strategy to isolating RNA from our plants. This RNA could be converted to cDNA by reverse transcription, which would eliminate the issue with introns we were having. Several of our greenhouse plants were no longer available, so we reduced the number of target terpenes we were focusing on. After extracting RNA and running an RT-PCR, several samples produced bands that corresponded roughly to where the synthase gene should be. These were gel extracted. Because almost every synthase gene had restriction sites in them that prevented them from being RFC10 compatible, we ligated the genes in pUC19 for site directed mutagenesis. After that, a second processing step would have been necessary to add the correct restriction sites to each gene to allow them to be integrated into pSB1C3 as a biobrick. | + | Once we had clear banding patterns, it became clear that the number of introns in each of our genes (a variable which was unknown since most of the plants we worked with have not had their genomes sequenced) was too great for cloning and expression to be practical. Therefore, as soon as the fall semester began, we shifted strategy to isolating RNA from our plants. This RNA could be converted to cDNA by reverse transcription, which would eliminate the issue with introns we were having. Several of our greenhouse plants were no longer available, so we reduced the number of target terpenes we were focusing on. After extracting RNA and running an RT-PCR, several samples produced bands that corresponded roughly to where the synthase gene should be. These were gel extracted. Because almost every synthase gene had restriction sites in them that prevented them from being RFC10 compatible, we ligated the genes in pUC19 for site directed mutagenesis. After that, a second processing step would have been necessary to add the correct restriction sites to each gene to allow them to be integrated into pSB1C3 as a biobrick. In the interest of time, we synthesized a codon-optimized santalene synthase gene in order to skip these RFC10 processing steps, even though we had already successfully reverse transcribed cDNA of the santalene synthase gene. |
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In the spring concurrent to work by the terpene groups, work began on plasmid construction. This was preformed by the president, wetware director, and a handful of others rather than in group format. Each gene cassette for our final plasmid was first identified in an existing, readily available plasmid. All of these cassettes were extracted by PCR using those plasmids as templates. Overlap extension PCR was then done on the gel-purified product to add restriction sites and homology regions for the purposes of eventually combining all of the cassettes together into a single plasmid. By the end of the summer, only one final fragment remained to be inserted to complete the intermediate plasmid pVU14004. Upon the successful creation of pVU14004, several restriction enzyme sites had to be removed by site directed mutagensis in order to make the plasmid RFC10 compatible. | In the spring concurrent to work by the terpene groups, work began on plasmid construction. This was preformed by the president, wetware director, and a handful of others rather than in group format. Each gene cassette for our final plasmid was first identified in an existing, readily available plasmid. All of these cassettes were extracted by PCR using those plasmids as templates. Overlap extension PCR was then done on the gel-purified product to add restriction sites and homology regions for the purposes of eventually combining all of the cassettes together into a single plasmid. By the end of the summer, only one final fragment remained to be inserted to complete the intermediate plasmid pVU14004. Upon the successful creation of pVU14004, several restriction enzyme sites had to be removed by site directed mutagensis in order to make the plasmid RFC10 compatible. |
Revision as of 19:37, 16 October 2014
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Introduction
The production of plant essential oils and their derivatives represents an over 9 billion dollar industry when considering just their applications in the food and fragrance industries 1. A staggering 23 million kilograms of citrus oil alone are produced worldwide each year. Up until only a couple decades ago, the production of these essential oils was done exclusively by chemical extraction from plant material. However, the sudden emergence of synthetic biology a versatile and efficient tool has the potential to transform this immense industry, the products of which nearly everyone will come in contact with on a daily basis.
Terpene biosynthesis in plants is part of larger pathways that metabolize isoprenoid intermediates. Genes encoding for enzymes known as synthases catalyze the terminal step in these pathways, from a precursor (commonly farnesyl pyrophosphate (FPP) or garnyl pyrophosphate (GPP)) to the final terpene product. As it happens, two well established and genetically manipulable model organisms- the bacterium Escherichia coli and baker's yeast Saccharomyces cerevisiae- produce moderate amounts of GPP and FPP as part of their endogenous non-mevalonate pathway (MEP) and mevalonate pathway (MEV) respectively3. All that is required for either of these organisms to begin producing terepenes is to introduce that single synthase gene.
Several factors contributed to the difficulty we experienced during the final phase of the project. First, member engagement suffered a significant decline between the spring and fall semesters, to the point where only a small handful of people were left to preform all experiments. Second, the late realization that we had to change our cloning strategy to modified cDNA inserts effectively meant we had to start anew in late August despite having what was a good head start when we began in early March. Third, the RFC10 requirements added a substantial dimension of difficulty to the project since all of our starting material (both the extracted gene cassettes for plasmid construction and the synthase genes) contained multiple sites that made them incompatible with the biobrick standard. Nevertheless, our team accomplished an enormous amount during our first year in competition.
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