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Team:Valencia UPV/Project/modules/switch
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<p class="subpart">Our Strategy</p><br/> | <p class="subpart">Our Strategy</p><br/> | ||
| - | <p>We designed a <span class="black-bold">genetic switch</span> to control the production of sex pheromones . Our inspiration came from previous projects in Synthetic Biology, which had already created genetic elements which resemble elements in electrical circuits, such as oscillators, memory elements or switches [1]. Our switch is only in ON mode when the plant is sprayed with CuSO4, commonly used as a fungicide and fertilizer in agriculture. This switch gives us total control over pheromone production, which will only be activated when mating season is coming.</p><br/><br/> | + | <p>We designed a <span class="black-bold">genetic switch</span> to control the <a class="normal-link-page" href="https://2014.igem.org/Team:Valencia_UPV/Project/modules/biosynthesis" target="_blank">production of sex pheromones</a> . Our inspiration came from previous projects in Synthetic Biology, which had already created genetic elements which resemble elements in electrical circuits, such as oscillators, memory elements or switches [1]. Our switch is only in ON mode when the plant is sprayed with CuSO4, commonly used as a fungicide and fertilizer in agriculture. This switch gives us total control over pheromone production, which will only be activated when mating season is coming.</p><br/><br/> |
<p class="subpart">The Design</p><br/> | <p class="subpart">The Design</p><br/> | ||
| - | <p>Unfortunately, we can’t just tell the plant to produce or not to produce the pheromones as we would tell a person to start walking. Molecular mechanisms are required to switch ON of OFF the production. We designed a genetic switch taking the Saccharomyces cerevisiae CUP1 regulatory system [2] as reference. Our copper-based genetic switch is made of two parts (figure 1):</p><br/><br/> | + | <p>Unfortunately, we can’t just tell the plant to produce or not to produce the pheromones as we would tell a person to start walking. Molecular mechanisms are required to switch ON of OFF the production. We designed a genetic switch taking the <i>Saccharomyces cerevisiae</i> CUP1 regulatory system [2] as reference. Our copper-based genetic switch is made of two parts (figure 1):</p><br/><br/> |
<ul style="list-style: square;"> | <ul style="list-style: square;"> | ||
| - | <li>A first transcriptional unit (TU1) which constitutively expresses a CUP2 - Gal4 Activation Domain fusion protein. CUP2 is a metalloresponsive transcription factor which changes conformation in the presence of copper ions and binds Upstream Activating Sequences (UAS) in its presence [3-5]. Gal4 Activation Domain (Gal4AD) stabilizes TFIID and enhances transcription [6].</li><br/><br/> | + | <li>A first transcriptional unit (TU1) which constitutively expresses a CUP2 - Gal4 Activation Domain fusion protein. <b>CUP2</b> is a metalloresponsive transcription factor which changes conformation in the presence of copper ions and binds Upstream Activating Sequences (<b>UAS</b>) in its presence [3-5]. Gal4 Activation Domain (<b>Gal4AD</b>) stabilizes TFIID and enhances transcription [6].</li><br/><br/> |
<li>A second transcriptional unit (TU2) expressing the gene of interest (CDS) under the regulation of a designed inducible promoter. The inducible promoter is formed by an UAS followed by the minimal cauliflower mosaic virus promoter P35s promoter (mini35S). A 68bp long untranslated region (UTR) is found between the promote and the CDS to improve mRNA stability and improve translation [7].</li><br/><br/> | <li>A second transcriptional unit (TU2) expressing the gene of interest (CDS) under the regulation of a designed inducible promoter. The inducible promoter is formed by an UAS followed by the minimal cauliflower mosaic virus promoter P35s promoter (mini35S). A 68bp long untranslated region (UTR) is found between the promote and the CDS to improve mRNA stability and improve translation [7].</li><br/><br/> | ||
</ul> | </ul> | ||
Revision as of 14:59, 14 October 2014
Project > Modules > Switch
The Idea
We think it’s illogical to have the Sexy Plant continuously producing sexual pheromones. They are not required during periods with no moth presence or sexual activity, so sending resources during these moments is suboptimal for the plant’s housekeeping metabolism. For that reason, we designed a strategy to have control on the moment when the pheromone is being produced.
Our Strategy
We designed a genetic switch to control the production of sex pheromones . Our inspiration came from previous projects in Synthetic Biology, which had already created genetic elements which resemble elements in electrical circuits, such as oscillators, memory elements or switches [1]. Our switch is only in ON mode when the plant is sprayed with CuSO4, commonly used as a fungicide and fertilizer in agriculture. This switch gives us total control over pheromone production, which will only be activated when mating season is coming.
The Design
Unfortunately, we can’t just tell the plant to produce or not to produce the pheromones as we would tell a person to start walking. Molecular mechanisms are required to switch ON of OFF the production. We designed a genetic switch taking the Saccharomyces cerevisiae CUP1 regulatory system [2] as reference. Our copper-based genetic switch is made of two parts (figure 1):
- A first transcriptional unit (TU1) which constitutively expresses a CUP2 - Gal4 Activation Domain fusion protein. CUP2 is a metalloresponsive transcription factor which changes conformation in the presence of copper ions and binds Upstream Activating Sequences (UAS) in its presence [3-5]. Gal4 Activation Domain (Gal4AD) stabilizes TFIID and enhances transcription [6].
- A second transcriptional unit (TU2) expressing the gene of interest (CDS) under the regulation of a designed inducible promoter. The inducible promoter is formed by an UAS followed by the minimal cauliflower mosaic virus promoter P35s promoter (mini35S). A 68bp long untranslated region (UTR) is found between the promote and the CDS to improve mRNA stability and improve translation [7].
How Does It Work?
CUP2-Gal4AD, the product from TU1, binds to the UAS in TU2 with its CUP2 region in the presence of copper ions due to the conformational change it suffers. Gal4AD is therefore brought close to mini35S in TU2 and enhances transcription. Transcription and therefore pheromone production will only take place at sufficient levels under the presence of copper ions (Operation in figure 1).
Figure 1. Genetic switch structure and operation. TU1 constitutively expresses the fusion protein CUP2-Gal4AD. This protein changes conformation in the presence of copper ions (CU2+) and binds to the Upstream Activation Sequence in TU2 in the CUP2 region. Gal4AD region of the fusion protein enhances transcription in TU2 and leads to the expression of the Gene of Interest.
Genetic Switch in Sexy Plant
This easy-to-control genetic switch is the perfect regulation for pheromone production in the Sexy Plant. It enables the user to activate production only when it’s required. Its implementation in the Sexy Plant provides a more optimal use of resources.
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References
- Wagner GJ (1991) Secreting glandular trichomes: more than just hairs. Plant Physiol 96: 675-679.
- CEnnajdaoui H, Vachon G, Giacalone C, Besse I, Sallaud C, et al. (2010) Trichome specific expression of the tobacco (Nicotiana sylvestris) cembratrien-ol synthase genes is controlled by both activating and repressing cis-regions. Plant Mol Biol 73: 673-685.
- 3. Sallaud C, Giacalone C, Topfer R, Goepfert S, Bakaher N, et al. (2012) Characterization of two genes for the biosynthesis of the labdane diterpene Z-abienol in tobacco (Nicotiana tabacum) glandular trichomes. Plant J 72: 1-17.
