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
Clement T. Y. Chan, Brent R. Bill, Ali Azghani
Masters of Science in Biology
Martini, Catherine A., "Testing the Combined Module Swapping and Repair by Modification Strategies: A Step Toward Universal Toolbox" (2020). Biology Theses. Paper 63.