CHARACTERIZATION OF A LIBRARY OF DEGENERATE OPERATOR SITES FOR THE TRANSCRIPTION FACTOR BETI FOR USE IN A MODEL-GUIDED DYNAMIC TIME-DELAY GENETIC CIRCUIT

Open Access
Author:
Clauer, Phillip
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Howard M Salis, Thesis Supervisor
  • Wendy Hanna-Rose, Honors Advisor
Keywords:
  • Synthetic Biology
  • Metabolic Engineering
  • Biological Engineering
Abstract:
Microorganisms, such as S. cerevisiae and E. coli, can be metabolically engineered to convert simple and cheap carbon sources to complex and valuable chemicals with industrial or medicinal relevance through endogenous or heterologous pathways. However, engineering an organism’s metabolism to overproduce a desirable chemical product is often difficult either because the siphoning of energy and carbon away from the organism’s central metabolism is untenable or the desired chemical product or one of the pathway’s intermediates presents toxicity to the engineered cell. To avoid these hurdles and increase titers, we present a dynamic transcription factor-based time-delay genetic circuit capable of temporally regulating multiple genes over the course of hours. To develop this synthetic dynamic genetic circuit, we developed a statistical thermodynamic quantitative model to demonstrate proof of concept. We then screened for and characterized a library of degenerate operator sites for the TetR homolog, BetI, which were then used to combinatorically demonstrate temporal control of fluorescent protein expression in E. coli. Finally, the expression space of BetI in the dynamic genetic circuit was explored to examine its effects on circuit dynamics. Our transcription factor-based time-delay genetic circuit has applications in autonomously coordinating the expression of multiple operons in metabolically engineered organisms with complex heterologous pathways.