Dual Genetic Selection of Synthetic Riboswitches in Escherichia coli
This chapter describes a genetic selection strategy to engineer synthetic riboswitches that can chemically regulate gene expression in Escherichia coli. Riboswitch libraries are constructed by randomizing the nucleotides that potentially comprise an expression platform and fused to the hybrid selection/screening marker tetA–gfpuv. Iterative ON and OFF selections are performed under appropriate conditions that favor the survival or the growth of the cells harboring the desired riboswitches. After the selection, rapid screening of individual riboswitch clones is performed by measuring GFPuv fluorescence without subcloning. This optimized dual genetic selection strategy can be used to rapidly develop synthetic riboswitches without detailed computational design or structural knowledge.
Key wordsRiboswitch Aptamer Gene regulation RNA engineering Translational regulation
We thank the previous members of our group, in particular, Dr. Norihito Muranaka and Dr. Vandana Sharma who contributed to the development of the dual genetic selection methods. The work was supported by National Science Foundation.