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    Engineering Tuneable Gene Circuits in Yeast

    Checkley, Stephen

    [Thesis]. Manchester, UK: The University of Manchester; 2012.

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    Abstract

    Synthetic biology is an emergent field incorporating aspects of computer sciencemolecular biology-based methodologies in a systems biology context, taking naturallyoccurring cellular systems, pathways, and molecules, and selectively engineering themfor the generation of novel or beneficial synthetic behaviour. This study described theconstruction of a novel synthetic gene circuit, which utilises the inducible downstreamtranscriptional activation properties of the pheromone-response pathway in the buddingyeast Saccharomyces cerevisiae as the basis for initiation. The circuit was composedof three novel yeast expression plasmids; (1) a reporter plasmid in which the luciferasereporter gene was fused to the iron response element (IRE), and expressed under thecontrol of the pheromone-inducible FUS1 promoter, (2) a repressor plasmid whichconstitutively expressed the mammalian iron response protein (IRP), which can bind tothe IRE in the luciferase mRNA transcript, blocking translation, and (3) a de-repressorplasmid which also utilised the pheromone-inducible FUS1 promoter to express thebacterial 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 threeplasmid components of the gene circuit. In these cells, during vegetative growthconditions, reporter gene translation is constitutively repressed by IRP until additionof 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 pheromoneresponse pathway. The combination of the repressors functioned to increase the ratioof induction of the reporter gene between pheromone-induced and un-induced states.Proteins and mRNA species expressed by each plasmid were semi-quantified usingSDS-PAGE, Western blot, and RT-qPCR. Luciferase expression was measured using anin 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 identifiedthe transcription of IRP, as well as the IRP protein half-life as significant controlpoints for increasing the expression of luciferase in vivo. Modelling resulted inthe development of multiple variations of the circuit, incorporating strong and weakconstitutive promoters for the IRP. For the degradation rate, the IRP was fused with adegradation tag from the PEST rich C-terminal residue of the Cln2 protein, formingIRPPEST , with approximately a 10-fold reduced half-life compared to wild type. Byvarying the promoter strength and half-life of the IRP, the circuit could be tuned in termsof the amplitude and period of luciferase expression during pheromone induction.Simulated annealing and Hooke-Jeeves algorithms were used to estimate model pa-rameter values from the experimental luminescence data, refining the modelling suchthat it produced accurate time course simulation of the circuit output. While furthercharacterisation of the individual components would be advantageous, the constructionof the system represents a completed cycle of extensive modelling, experimentation,and further model refinement.

    Bibliographic metadata

    Type of resource:
    Content type:
    Form of thesis:
    Type of submission:
    Degree type:
    Doctor of Philosophy
    Degree programme:
    PhD Chemical Engineering and Analytical Science (48 months)
    Publication date:
    Location:
    Manchester, UK
    Total pages:
    268
    Abstract:
    Synthetic biology is an emergent field incorporating aspects of computer sciencemolecular biology-based methodologies in a systems biology context, taking naturallyoccurring cellular systems, pathways, and molecules, and selectively engineering themfor the generation of novel or beneficial synthetic behaviour. This study described theconstruction of a novel synthetic gene circuit, which utilises the inducible downstreamtranscriptional activation properties of the pheromone-response pathway in the buddingyeast Saccharomyces cerevisiae as the basis for initiation. The circuit was composedof three novel yeast expression plasmids; (1) a reporter plasmid in which the luciferasereporter gene was fused to the iron response element (IRE), and expressed under thecontrol of the pheromone-inducible FUS1 promoter, (2) a repressor plasmid whichconstitutively expressed the mammalian iron response protein (IRP), which can bind tothe IRE in the luciferase mRNA transcript, blocking translation, and (3) a de-repressorplasmid which also utilised the pheromone-inducible FUS1 promoter to express thebacterial 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 threeplasmid components of the gene circuit. In these cells, during vegetative growthconditions, reporter gene translation is constitutively repressed by IRP until additionof 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 pheromoneresponse pathway. The combination of the repressors functioned to increase the ratioof induction of the reporter gene between pheromone-induced and un-induced states.Proteins and mRNA species expressed by each plasmid were semi-quantified usingSDS-PAGE, Western blot, and RT-qPCR. Luciferase expression was measured using anin 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 identifiedthe transcription of IRP, as well as the IRP protein half-life as significant controlpoints for increasing the expression of luciferase in vivo. Modelling resulted inthe development of multiple variations of the circuit, incorporating strong and weakconstitutive promoters for the IRP. For the degradation rate, the IRP was fused with adegradation tag from the PEST rich C-terminal residue of the Cln2 protein, formingIRPPEST , with approximately a 10-fold reduced half-life compared to wild type. Byvarying the promoter strength and half-life of the IRP, the circuit could be tuned in termsof the amplitude and period of luciferase expression during pheromone induction.Simulated annealing and Hooke-Jeeves algorithms were used to estimate model pa-rameter values from the experimental luminescence data, refining the modelling suchthat it produced accurate time course simulation of the circuit output. While furthercharacterisation of the individual components would be advantageous, the constructionof the system represents a completed cycle of extensive modelling, experimentation,and further model refinement.
    Thesis main supervisor(s):
    Language:
    en

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    Record metadata

    Manchester eScholar ID:
    uk-ac-man-scw:155841
    Created by:
    Checkley, Stephen
    Created:
    14th February, 2012, 16:36:30
    Last modified by:
    Checkley, Stephen
    Last modified:
    16th March, 2012, 11:46:28