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<bTitle:<> “Advanced Synthetic Circuits for Logical Genetic Manipulation”
Committee:
Wilson Wong, PhD – BME (Advisor, Chair)
John Ngo, PhD – BME
Doug Densmore, PhD – ECE
Jerry Chen, PhD – Biology
Abstract:
Synthetic biological circuits have demonstrated incredible promise for engineering complex, logical control over gene expression. Recently, the Boolean Logic and Arithmetic through DNA Excision (BLADE) single-layer circuit design has enabled the fabrication of the most complex circuits to date in mammalian cells using site-specific recombinases (SSRs). While logic circuits have proven invaluable for cell-based computation, their digital (on or off) behavior lacks the ability to finely tune levels of gene expression. Additionally, it remains difficult to engineer reliable behavior in cells due to a lack of understanding into how circuit components will perform when coupled together. To address these challenges, I seek to develop a suite of BLADE/CRISPR (BLASR) interfacing circuits, concatenating SSR-based logic computation with the dynamic expression modulation afforded by the dCas9-VPR transcriptional activator. I will demonstrate the capabilities of the BLASR system by constructing two of the most complicated circuits to date in mammalian cells: a digital-to-analog converter and genetic multiplier circuit. I will also present a workflow for relating promoter strength (input) to SSR activity (output) to allow greater predictability of logic circuits in new contexts. To demonstrate the utility of these SSR transfer functions, I will identify several complex cell phenotypes using traditionally non-specific promoters by titrating recombinase activity independent of promoter strength, thereby mitigating off-target activity and producing the intended response in the desired cell type. This body of work will provide gene circuit tools for the continued advancement of precise gene expression control.
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