Collins, Cynthia Hollie (2006) Directed evolution of the transcriptional activator LuxR. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-12272005-160649
LuxR-type proteins are responsible for density-dependent transcriptional regulation in quorum-sensing systems that employ acyl-homoserine lactones (acyl-HSLs) as signal molecules. The Vibrio fischeri lux operon, which includes LuxR, has been well studied and shown to function in E. coli. The components of the lux operon have been used to engineer de novo genetic circuits because they provide a versatile intercellular communications system. We have used directed evolution to engineer LuxR to generate new components for engineering genetic circuits with a wide range of communications functions and to explore the evolution of LuxR specificity and response.
We developed and validated a screening system to identify mutants of LuxR that activate gene expression with non-cognate acyl-HSLs. We screened libraries of luxR mutants for variants exhibiting increased gene activation in response to octanoyl-HSL (C8HSL). Eight LuxR variants were identified that showed a 100-fold increase in sensitivity to C8HSL. These variants displayed increased sensitivities to a broadened range of acyl-HSLs while maintaining a wild-type or greater response to LuxR’s cognate signal, 3-oxo-hexanoyl-homoserine lactone (3OC6HSL).
To generate a LuxR with a new signaling specificity, we used a dual selection system to identify LuxR variants that either activate gene expression (ON) or do not (OFF) under desired sets of conditions. The dual selection system was evaluated prior to its use, and a 490-fold enrichment in functional clones per round of ON/OFF selection was observed. We used the dual selection system to identify a LuxR variant that responds to straight-chain acyl-HSLs, but no longer responds to 3OC6HSL. A single mutation in the N terminal signal-binding domain reduces the response to acyl-HSLs having a carbonyl substituent at the third carbon of the acyl chain.
We used the dual selection system to identify LuxR variants that activate transcription upon binding to a promoter containing a mutated operator site. An amino acid position critical for modulating the DNA-binding specificity of LuxR for the new DNA target was identified. The dual selection system provides a rapid and reliable method for identifying LuxR variants that have the desired response, or lack thereof, to a given set of acyl-HSL signals or DNA targets.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||gene regulation; LuxR; quorum sensing; synthetic biology; transcription|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Major Option:||Biochemistry and Molecular Biophysics|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||19 December 2005|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||29 Dec 2005|
|Last Modified:||26 Dec 2012 03:15|
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