Synthetic biology is an emergent field incorporating aspects of computer science molecular biology-based methodologies in a systems biology context, taking naturally occurring cellular systems, pathways, and molecules, and selectively engineering them for the generation of novel or beneficial synthetic behaviour. This study described the construction of a novel synthetic gene circuit, which utilises the inducible downstream transcriptional activation properties of the pheromone-response pathway in the budding yeast Saccharomyces cerevisiae as the basis for initiation. The circuit was composed of three novel yeast expression plasmids; (1) a reporter plasmid in which the luciferase reporter gene was fused to the iron response element (IRE), and expressed under the control of the pheromone-inducible FUS1 promoter, (2) a repressor plasmid which constitutively expressed the mammalian iron response protein (IRP), which can bind to the IRE in the luciferase mRNA transcript, blocking translation, and (3) a de-repressor plasmid which also utilised the pheromone-inducible FUS1 promoter to express the bacterial LexA protein that represses transcription of the IRP gene, and thereby de-represses luciferase translation. Yeast cultures were propagated in media that selected for cells containing all three plasmid components of the gene circuit. In these cells, during vegetative growth conditions, reporter gene translation is constitutively repressed by IRP until addition of pheromone. Upon pheromone-induction, the pheromone response pathway up-regulated the expression of the LexA protein which represses transcription of IRP, enabling the translation of luciferase, which is itself up-regulated by the pheromone response pathway. The combination of the repressors functioned to increase the ratio of induction of the reporter gene between pheromone-induced and un-induced states. Proteins and mRNA species expressed by each plasmid were semi-quantified using SDS-PAGE, Western blot, and RT-qPCR. Luciferase expression was measured using an in vitro whole cell luminescence assay, and the data used to define the circuit 'output'. Metabolic control analysis was used prior to building the circuit in silico, and identified the transcription of IRP, as well as the IRP protein half-life as significant control points for increasing the expression of luciferase in vivo. Modelling resulted in the development of multiple variations of the circuit, incorporating strong and weak constitutive promoters for the IRP. For the degradation rate, the IRP was fused with a degradation tag from the PEST rich C-terminal residue of the Cln2 protein, forming IRPPEST , with approximately a 10-fold reduced half-life compared to wild type. By varying the promoter strength and half-life of the IRP, the circuit could be tuned in terms of the amplitude and period of luciferase expression during pheromone induction. Simulated annealing and Hooke-Jeeves algorithms were used to estimate model parameter values from the experimental luminescence data, refining the modelling such that it produced accurate time course simulation of the circuit output. While further characterisation of the individual components would be advantageous, the construction of the system represents a completed cycle of extensive modelling, experimentation, and further model refinement.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:553382 |
| Date | January 2012 |
| Creators | Checkley, Stephen |
| Contributors | Pedrosa Mendes, Pedro |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/engineering-tuneable-gene-circuits-in-yeast(71dda344-8802-4862-9b29-1a671f4c96ab).html |
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