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Date of Award

2-2010

Document Type

Campus Access

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemical Engineering

First Advisor

Lianhong Sun

Second Advisor

Michael Henson

Third Advisor

Jeffrey Blanchard

Subject Categories

Biomedical Engineering and Bioengineering | Chemical Engineering

Abstract

Quorum-sensing systems enable autonomous activation of gene expression and have been widely used in the construction of artificial genetic circuits, but exhibit poor tunability. We anticipate that tunable quorum sensing would facilitate its application in synthetic biology and industrial biotechnology. We engineered LuxI, which synthesizes signal molecule, 3-oxo homoserine lactone (OHHL), using a protein engineering method referred to as directed evolution. An antibiotic-based genetic selection method was developed and two rounds of mutant library screening resulted in two mutants with approximately 80-fold increase in OHHL yields. To demonstrate the application of the genetic selection in engineering LuxI homologs, a similar method was applied to alter the substrate specificity of RhlI, a key enzyme of the RhlI-RhlR quorum-sensing system involved in the pathogenesis of Pseudomonas aeruginosa. In particular, production of its cognate signal molecule, BHL (butanoyl homoserine lactone), has been enhanced more than two-fold, and synthesis of additional signal molecule, HHL (hexanoyl homoserine lactone), has been improved from an undetectable level to a level comparable to BHL, indicating a significant change in substrate specificity.

We demonstrated the application of the engineered LuxI-LuxR quorum-sensing systems for regulation of gene expression. Although inducible gene expression systems are commonly used to enhance production of toxic proteins in lab cultures, the high cost of inducers prevents their application at an industrial scale, and therefore an auto-inducible and tunable gene expression system is an attractive alternative. Engineered LuxI-LuxR autoinduction system is highly tunable to a desired cell density for the optimal production of proteins of various toxicities. In particular, expression of Nun toxic protein when expressed under engineered LuxI-LuxR system with delayed autoinduction in the mid-late log phase was detectable on western blotting whereas constitute expression resulted in complete inhibition of cell growth. By coupling with the Cre- loxP site-specific recombination system, cell density-dependent deactivation of gene expression was also accomplished. Taking together, we have created a highly tunable and auto-inducible system for activating or deactivating gene expression. Using LuxI-LuxR and its homologous systems, we designed a synthetic symbiotic two species ecosystem for application to mixed cultures. Dynamic simulation and bifurcation analysis showed that the designed system admits a stable coexistence and the steady-state fraction of each species could be altered by varying signaling molecule synthesis rates. The design also revealed stable periodic solutions over certain domains of parameter space.

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