The study of synthetic gene regulatory networks allows the isolation and investigation of components and motifs in natural regulatory networks. Many synthetic gene networks are regulated at the transcriptional level. In this work, two methods of regulating gene expression at the transcriptional level were studied with the objective of gaining finer control over network behaviour.
The first approach focuses on activation and repression of promoters by transcription factors. A synthetic repressor-activator network was engineered using the cI and cro genes and the PRM promoter from bacteriophage λ. The cI and cro genes activated and repressed PRM, respectively, and the monomeric red fluorescent protein (mrfp) gene reported PRM activity. Experimental testing showed an increase in mrfp expression in response to CI, a decrease in mrfp expression in response to Cro, and a differential output that reflected the relative concentrations of CI and Cro when both inputs were applied together. A positive feedback network was then created by placing a cI gene downstream of PRM. The network showed increased expression in response to CI and decreased expression in response to Cro. A negative feedback network was created by placing a cro gene downstream of PRM. Experimental testing showed decreased mrfp expression in response to both inputs.
The second approach employed two methods for tuning expression levels without modifying the genes or promoters. First, using a series of networks with tandem mrfp genes under the control of the PLtet0-1 promoter, it was demonstrated that magnitude and range of expression levels could be tuned by adjusting the number of genes in the operon. A network was tuned using this principle by placing luxR genes in tandem to increase the activity of the luxPR promoter. It was then demonstrated that the level of gene expression could be varied through the placement of the gene within an operon. Operons that were three, five and seven genes and contained one green fluorescent protein gene in the first, middle, or end position were created. By comparing green fluorescence levels in induced and uninduced networks, it was found that the gene closest to the promoter was the most inducible.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/24838 |
| Date | 01 September 2010 |
| Creators | Nagaraj, Seema |
| Contributors | Truong, Kevin |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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