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    Decisive Noise: Noisy Intercellular Signalling Analysed and Enforced Through Synthetic Biology

    Jackson, Victoria Jane

    [Thesis]. Manchester, UK: The University of Manchester; 2013.

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    Abstract

    Individual cells in a genetically identical population, exposed to the same environment, can show great variation in their protein expression levels. This is due to noise, which is inherent in many biological processes, due in part to the low molecule numbers and probabilistic interactions which lead to stochasticity. Much of the work in the field of noise and its propagation in gene expression networks, whether it is experimental, modelling or theoretical, has been conducted on networks/systems that occur within a single cell. However, cells do not exist solely in isolation and understanding how cells are able to coordinate their behaviour despite this noise is an interesting area of expansion for the field. In this study, a synthetic intercellular communication system was designed that allows the investigation of how noise is propagated in intercellular communication. The communication system consists of separate sender and receiver cells incorporating components of the Lux quorum sensing system of Vibrio fischeri. The sender cell was designed so that the production of the signalling molecule, 3-oxohexanoyl homoserine lactone, is able to be controlled by addition of isopropyl-β-D-thio-galactoside (IPTG) and monitored via a reporter gene. The receiver cell was designed with a dual reporter system to enable the response of the cell to the signalling molecule to be monitored and the intrinsic and extrinsic noise contributions to the total noise to be calculated. Sender and the receiver cells were engineered in Escherichia coli. The functionality of the receiver cells was tested in the presence of known concentrations of the signalling molecule. The population response and the noise characteristics of the receiver cells in the homogeneous environment were determined from single cell measurements. The functionality of the sender cells was tested in the presence of a range of IPTG concentrations and the induction of expression from the LacI-repressible promoter was monitored. Mathematical models of the system were developed. Stochastic simulations of the models were used to investigate any unexplained behaviour seen in the characterisation of the cells. The full functionality of the intercellular communication system was then tested by growing the receiver in the collected media of the induced sender cells. The response of the receiver cells to the signalling molecule in the media was again characterised using single cell measurements of the reporter expression levels. The analysis of mixed populations of the sender and receiver cells was hampered by the technical limitations of the instruments used for the single cell measurements. Difficulties were encountered in simultaneous and specific measurement of the three reporter genes. Two methods for overcoming this issue were proposed using microscopy, and one of these methods was shown to have potential in overcoming the issue.

    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:
    165
    Abstract:
    Individual cells in a genetically identical population, exposed to the same environment, can show great variation in their protein expression levels. This is due to noise, which is inherent in many biological processes, due in part to the low molecule numbers and probabilistic interactions which lead to stochasticity. Much of the work in the field of noise and its propagation in gene expression networks, whether it is experimental, modelling or theoretical, has been conducted on networks/systems that occur within a single cell. However, cells do not exist solely in isolation and understanding how cells are able to coordinate their behaviour despite this noise is an interesting area of expansion for the field. In this study, a synthetic intercellular communication system was designed that allows the investigation of how noise is propagated in intercellular communication. The communication system consists of separate sender and receiver cells incorporating components of the Lux quorum sensing system of Vibrio fischeri. The sender cell was designed so that the production of the signalling molecule, 3-oxohexanoyl homoserine lactone, is able to be controlled by addition of isopropyl-β-D-thio-galactoside (IPTG) and monitored via a reporter gene. The receiver cell was designed with a dual reporter system to enable the response of the cell to the signalling molecule to be monitored and the intrinsic and extrinsic noise contributions to the total noise to be calculated. Sender and the receiver cells were engineered in Escherichia coli. The functionality of the receiver cells was tested in the presence of known concentrations of the signalling molecule. The population response and the noise characteristics of the receiver cells in the homogeneous environment were determined from single cell measurements. The functionality of the sender cells was tested in the presence of a range of IPTG concentrations and the induction of expression from the LacI-repressible promoter was monitored. Mathematical models of the system were developed. Stochastic simulations of the models were used to investigate any unexplained behaviour seen in the characterisation of the cells. The full functionality of the intercellular communication system was then tested by growing the receiver in the collected media of the induced sender cells. The response of the receiver cells to the signalling molecule in the media was again characterised using single cell measurements of the reporter expression levels. The analysis of mixed populations of the sender and receiver cells was hampered by the technical limitations of the instruments used for the single cell measurements. Difficulties were encountered in simultaneous and specific measurement of the three reporter genes. Two methods for overcoming this issue were proposed using microscopy, and one of these methods was shown to have potential in overcoming the issue.
    Thesis main supervisor(s):
    Language:
    en

    Institutional metadata

    University researcher(s):
    Academic department(s):

    Record metadata

    Manchester eScholar ID:
    uk-ac-man-scw:187670
    Created by:
    Jackson, Victoria
    Created:
    15th February, 2013, 15:29:28
    Last modified by:
    Jackson, Victoria
    Last modified:
    2nd May, 2014, 19:26:38

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