De Novo Evolution of Transcriptional Regulation in Escherichia coli

Yoshimura, Daniel

Undergraduate Honors Thesis

De Novo Evolution of Transcriptional Regulation in Escherichia coli Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/undergraduate_honors_theses/kw52j964b

Abstract

Gene expression is a critical process involved in regulating many cellular responses and is thought to play an important role in evolution. The molecular mechanisms of how complex transcriptional regulation evolves have yet to be comprehensively characterized in real-time in a living organism. Here I created a system to study the de novo evolution of complex transcriptional regulation in Escherichia coli by imposing positive or negative selection on various target genes (argC, nfsA36_37, and tetA) by changing the growth conditions. These target genes were inserted into the genome at a site where they are under the control of a constitutive promoter and are thus highly expressed. Based on the initial growth experiments to identify optimal selective conditions, I decided that tetA was the most practical target gene upon which I could reliably impose both strong positive and negative selection. I determined that minimal medium containing tetracycline (20 µg/ml) was sufficient to apply positive selective pressure for TetA expression and medium containing NiCl₂ (19 µg/ml) or a combination of fusaric acid (18 µg/ml) and kanamycin (2 μg/ml) was sufficient to provide negative selective pressure against TetA expression. I hypothesize that by alternating the ΔproBA::tetA strain between conditions that impose positive and negative selection of TetA expression, transcriptional regulation that can adjust tetA transcription depending on the environment will be adaptive. Studying how E. coli evolves in this experimental model system may provide insights into the mechanisms by which complex transcriptional regulation evolves.

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