Browsing by Author "Hirning, Andrew John"

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    Dynamic Studies of Synthetic Transcriptional Networks
    (2017-12-01) Hirning, Andrew John; Bennett, Matthew R
    Within synthetic biology, orthogonal genetic circuits are created using a small collection of transcription factors. Although the absolute number of transcription factors is small, the number of mutants explored for each transcription factor is greater by orders of magnitude. However, nearly all synthetic networks created utilize the wild-type transcription factor. The large number of published mutants for each transcription factor represent an equal number of variant networks with possibly varied behavior. Study of these networks can increase knowledge about how different aspects of protein structure/function impact the function of genetic networks. For this work, we chose two sets of mutants/variants for the transcriptional repressor LacI - a set of point mutants with varying operator affinity and a library of chimeric repressors that respond to unique inducers. We developed novel microfluidic devices for studying dynamic gene expression in single cells. First, we discuss attempts to alter the function of a two-gene oscillator by mutating LacI to modulate the operator affinity of the protein. We selected mutants with varying affinities for lacO1 and tested the mutant oscillators. We hypothesized the oscillatory period would increase as the affinity of LacI for O1 decreased. Instead, we observed the oscillatory period responded in a non-monotonic fashion to decreasing affinity. This behavior can be explained by a second operator site (lacOsym) in the synthetic promoter used in the oscillator. Our selected mutants do not vary in affinity in the same way for Osym as O1. Second, we discuss efforts to measure temporal responses of genetic logic gates. Utilizing chimeric repressors that bind LacI-responsive promoters, we generated three genetic logic gates (AND, IMPLY, and NOT). To study these gates, we created a bespoke microfluidic device with two independent, time-varying inducer inputs. Each gate was driven with a square wave of inducer across a period range of 20-240 minutes. We find that “ligand” gates (AND, IMPLY) constructed with chimeric repressors give reliable responses over a wide range of driving frequencies, whereas an inverted, “transcriptional” gate (NOT) responds over a narrower range, probably due to excess protein production coupled with degradation steps required for state transition in “transcriptional” gates.