Team:Vanderbilt/Project
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<img src="https://static.igem.org/mediawiki/parts/8/82/VU_experiment_1_diagram.png" align=left alt="First few experiments" width="500" style="padding-bottom:0.5em; float:left" /> | <img src="https://static.igem.org/mediawiki/parts/8/82/VU_experiment_1_diagram.png" align=left alt="First few experiments" width="500" style="padding-bottom:0.5em; float:left" /> | ||
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Our project had several co-dependent sub-project that were all worked on in parallel. These can roughly be divided into two categories: the first involving work on our synthase genes and the second involving the construction of a new, specially designed plasmid vector. We tried two different team structures over the year to see which would give the best results. For the Spring, we had the original idea of dividing members into independent groups, each working on a specific terpene. Each group was headed by a single group manager who would teach 4-5 new members the protocol that was to be preformed and then supervise that the work was carried out correctly. On occasion either the group managers or the organization president or wetware director would also given lessons to teach members about the techniques and theory involved at each step. All group managers were in turn trained by either the president or wetware director, both of whom had come with the experiment, acquired all the necessary primers and reagents, wrote up the protocol, and had preformed it prior to any group-phase work for the purposes of troubleshooting and predicting where issues may come up. The president or wetware director also helped the group manager in being present during all experiments for answering questions, preparing materials, and other forms of assistance. | Our project had several co-dependent sub-project that were all worked on in parallel. These can roughly be divided into two categories: the first involving work on our synthase genes and the second involving the construction of a new, specially designed plasmid vector. We tried two different team structures over the year to see which would give the best results. For the Spring, we had the original idea of dividing members into independent groups, each working on a specific terpene. Each group was headed by a single group manager who would teach 4-5 new members the protocol that was to be preformed and then supervise that the work was carried out correctly. On occasion either the group managers or the organization president or wetware director would also given lessons to teach members about the techniques and theory involved at each step. All group managers were in turn trained by either the president or wetware director, both of whom had come with the experiment, acquired all the necessary primers and reagents, wrote up the protocol, and had preformed it prior to any group-phase work for the purposes of troubleshooting and predicting where issues may come up. The president or wetware director also helped the group manager in being present during all experiments for answering questions, preparing materials, and other forms of assistance. | ||
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Each group first planted seeds under the appropriate soil, humidity, and temperature conditions at the Vanderbilt Greenhouse. Once the majority of these grew into saplings with green leaves, Samples were flash frozen in liquid nitrogen in preperation for a genomic DNA extraction. After the extractions, nanodrop concentration readings and agarose gels confirmed the presence of high molecular weight genomic DNA. Groups then ran a PCR on their genomic DNA with primers specific to their synthase gene. More gels were run to check for PCR product. At this point, the semester was coming to an end, so groups were disbanded before most managed to isolate their synthase gene. Over the summer, the president and wetware director continued troubleshooting those genes which were not amplifying and eventually got each to the point where consistent PCR product bands were produced. | Each group first planted seeds under the appropriate soil, humidity, and temperature conditions at the Vanderbilt Greenhouse. Once the majority of these grew into saplings with green leaves, Samples were flash frozen in liquid nitrogen in preperation for a genomic DNA extraction. After the extractions, nanodrop concentration readings and agarose gels confirmed the presence of high molecular weight genomic DNA. Groups then ran a PCR on their genomic DNA with primers specific to their synthase gene. More gels were run to check for PCR product. At this point, the semester was coming to an end, so groups were disbanded before most managed to isolate their synthase gene. Over the summer, the president and wetware director continued troubleshooting those genes which were not amplifying and eventually got each to the point where consistent PCR product bands were produced. | ||
+ | <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. |
Revision as of 05:33, 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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