The emerging field of synthetic biology aims to use artificially designed genetic circuits to program living cells, much as engineers program a computer or control electronic or mechanical systems. This thesis focuses on the design and implementation of synthetic gene circuits in the bacterium Escherichia coli to create new cellular functions, and on the quantitative characterization and modelling of these circuits.
Though important in any engineering discipline, quantitative system characterization has been poorly explored in synthetic biology. We have performed a quantitative system characterization by implementing simple gene circuits in Escherichia coli. The work showed that the level and variability of gene expression varied across different cell strains, and we investigated how these effects manifested through the coupled effects of cell division, cellular growth rate, and plasmid copy number regulation. The work suggests that gene circuit modules from a standard library cannot be used universally; the cellular context and the time dependent dynamics must be considered when implementing gene circuits.
In order to work precisely as engineering devices, synthetic gene circuits must be appropriately tuned. One standard method of tuning genetic circuits requires altering the DNA sequences by extensive molecular biology work. Part of this thesis focuses on developing easily tunable gene circuits. A set of circuits were developed in E. coli where the shape of the chemically induced signal response curves can be tuned from a band structure to a sigmoidal structure simply by altering the temperature in a single system. Another set of circuits was developed which demonstrate a range of chemically tunable signal response curves along with multiple functions in a single device.
One of the ultimate goals of synthetic biology is to program living cell in a human-controlled way. To this end, I developed a set of genetic devices that could work as ‘in cell disease prevention devices,’ preventing an otherwise fatal viral infection in E. coli. The device displays a number of ‘device’ properties: being dormant under normal conditions, detecting the onset of the disease state, turning on automatically to prevent a lethal outcome, and being subject to external deactivation when desired.
The combination of design, characterization, and mathematical understanding explored in this work represents a contribution in the direction of developing synthetic biology as a well-founded engineering discipline.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/26128 |
| Date | 14 February 2011 |
| Creators | Bagh, Sangram |
| Contributors | McMillen, David |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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