Team:Valencia UPV/Project/modules/switch

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Project > Modules > Switch



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).



switch

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.








References


  1. Khalil AS, Collins JJ (2010) Synthetic biology: applications come of age. Nat Rev Genet 11: 367-379.
  2. Butt TR, Sternberg EJ, Gorman JA, Clark P, Hamer D, et al. (1984) Copper metallothionein of yeast, structure of the gene, and regulation of expression. Proc Natl Acad Sci U S A 81: 3332-3336.
  3. Mett VL, Lochhead LP, Reynolds PH (1993) Copper-controllable gene expression system for whole plants. Proc Natl Acad Sci U S A 90: 4567-4571.
  4. Thiele DJ (1988) ACE1 regulates expression of the Saccharomyces cerevisiae metallothionein gene. Mol Cell Biol 8: 2745-2752.
  5. Szczypka MS, Thiele DJ (1989) A cysteine-rich nuclear protein activates yeast metallothionein gene transcription. Mol Cell Biol 9: 421-429.
  6. Chen JL, Attardi LD, Verrijzer CP, Yokomori K, Tjian R (1994) Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activators. Cell 79: 93-105.
  7. Kovtun AA, Shirokikh NE, Gudkov AT, Spirin AS (2007) The leader sequence of tobacco mosaic virus RNA devoid of Watson-Crick secondary structure possesses a cooperatively melted, compact conformation. Biochem Biophys Res Commun 358: 368-372.




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