Synthetic biology involves the creation of artificial gene and networks to program new cell behaviors. This nascent field is in need of well-characterized genetic components from which complex genetic circuits can be constructed. The quorum-sensing transcriptional-activator LuxR is activated by binding a small molecule, N- acyl homoserine lactone (AHL).
The aim of this proposal is to use directed evolution to create a collection of LuxR variants that respond to AHL analogs with high specificity. From this work, we anticipate identifying amino acids in LuxR which modulate AHL specificity. AHL analogs will be synthesized according to literature methods and tested for ability to activate LuxR-wt gene transcription. The mutational methods of error-prone PCR and DNA shuffling will be used to generate LuxR variants for multiple rounds of directed evolution. Screening and a dual selection strategy will be used to identify LuxR variants in which specificity is refocused for an AHL analog. AHL binding properties and dimerization of LuxR variants will be analyzed to gain insight in the mechanism of LuxR activation or inhibition. The novel LuxR variants will also be useful for constructing synthetic genetic circuits in which gene expression can be manipulated by external agents. ? ?
| Tracewell, Cara A; Arnold, Frances H (2009) Directed enzyme evolution: climbing fitness peaks one amino acid at a time. Curr Opin Chem Biol 13:3-9 |
