Genetic selection provides the most powerful method to assay large libraries of biomolecules for function, and harnessing the power of genetic selection for the detection of specific, non-endogenous small molecule targets would greatly facilitate the cloning of biosynthesis genes, the directed evolution of enzymes, and the engineering of metabolic pathways. While significant progress has been made in developing in vivo genetic selections for small molecule targets, there remain limitations to both the sizes of libraries and the types of molecules and chemical transformations that may be selected for. This proposal details a comprehensive effort to develop a method capable of performing genetic screens and selections for non-endogenous small molecule targets in bacteria. The proposed method addresses many of the limitations of current methods and rests on two well-established principles: The first is that it is possible to use in vitro selection to generate RNA sequences (aptamers) that tightly and specifically recognize small molecules, while discriminating against related structures. The second is that small molecule metabolites can interact with messenger RNAs to modulate gene expression in vivo through a mechanism known as riboswitch control. Results from our lab and from others show that it is possible to combine these ideas to generate synthetic riboswitches that recognize non-endogenous small molecules and control gene expression in E. coli. Herein we propose a method for producing synthetic riboswitches capable of mediating genetic selections for small molecules. This approach will be validated by using synthetic riboswitch-mediated'genetic selections to clone and direct the evolution of enzymes within two important alkaloid biosynthesis pathways. Concurrent mechanistic studies will provide evidence of how the new synthetic riboswitches function and will likely illuminate principles that govern the function of natural riboswitches in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM074070-05
Application #
7765577
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Fabian, Miles
Project Start
2006-02-01
Project End
2012-01-31
Budget Start
2010-02-01
Budget End
2012-01-31
Support Year
5
Fiscal Year
2010
Total Cost
$226,499
Indirect Cost
Name
Emory University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Reynoso, Colleen M K; Miller, Mark A; Bina, James E et al. (2012) Riboswitches for intracellular study of genes involved in Francisella pathogenesis. MBio 3:
Seeliger, Jessica C; Topp, Shana; Sogi, Kimberly M et al. (2012) A riboswitch-based inducible gene expression system for mycobacteria. PLoS One 7:e29266
Sinha, Joy; Reyes, Samuel J; Gallivan, Justin P (2010) Reprogramming bacteria to seek and destroy an herbicide. Nat Chem Biol 6:464-70
Mishler, Dennis M; Topp, Shana; Reynoso, Colleen M K et al. (2010) Engineering bacteria to recognize and follow small molecules. Curr Opin Biotechnol 21:653-6
Topp, Shana; Reynoso, Colleen M K; Seeliger, Jessica C et al. (2010) Synthetic riboswitches that induce gene expression in diverse bacterial species. Appl Environ Microbiol 76:7881-4
Topp, Shana; Gallivan, Justin P (2010) Emerging applications of riboswitches in chemical biology. ACS Chem Biol 5:139-48
Lynch, Sean A; Topp, Shana; Gallivan, Justin P (2009) High-throughput screens to discover synthetic riboswitches. Methods Mol Biol 540:321-33
Lynch, Sean A; Gallivan, Justin P (2009) A flow cytometry-based screen for synthetic riboswitches. Nucleic Acids Res 37:184-92
Gallivan, Justin P (2009) A versatile actor finds a new role. Chem Biol 16:1128-9
Topp, Shana; Gallivan, Justin P (2007) Guiding bacteria with small molecules and RNA. J Am Chem Soc 129:6807-11

Showing the most recent 10 out of 12 publications