Abstract

The crux of synthetic biology is the engineering of biological components to modulate the activity of specific DNA-based promoter(s) to drive gene expression; thus, providing a means to modulate pathways between signal detection and cellular response. However, the longstanding frustration of synthetic biologists has been the inability to transfer those engineered components between cellular systems — this lack of “modular universality” or “universal toolbox” impedes research by forcing a metaphorical reinvention of the wheel in new systems. The modular swapping strategy expanded the available “toolbox” with customizable hybrid repressors formed from “swapping” the DNA-recognition modules (DRMs) and environmental-sensing modules (ESMs) of LacI/GalR homologs — further refined by a computational model to more accurately predict compatible DRMs and ESMs. Nevertheless, the model was imperfect; modules predicted to be compatible resulted in poorly functioning hybrid repressors, implying disturbed amino acid-specific interactions at the module-module border. Thus, to improve functionality, the repair by modification strategy — in which one to three amino acids were mutated as determined by an offshoot of the computational model — was tested in combination with the module swapping strategy. To that end, mutant sets of five hybrid repressors (LacI-RbsR, LacI-ScrR, CelR-RbsR, PurR-GalR, and MalR-LacI) and native GalR were engineered and their functionality characterized. Induction experiments revealed successful repair in some mutant strains of LacI-RbsR, CelR-RbsR, and PurR-GalR but a worsening of functionality in the LacI-ScrR, GalR, and MalR-LacI mutant strains.

Date of publication

Fall 8-13-2020

Document Type

Thesis

Language

english

Persistent identifier

http://hdl.handle.net/10950/2654

Committee members

Clement T. Y. Chan, Brent R. Bill, Ali Azghani

Degree

Masters of Science in Biology

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