Random Walks to Synthetic Riboswitches—A High‐Throughput Selection Based on Cell Motility

A major goal of chemical and synthetic biologists is to create ligand-dependent genetic-control systems to report on cellular metabolism, to construct synthetic gene circuits, or to reprogram cellular behavior. Because designing genetic switches de novo is challenging, many groups have employed directedevolution strategies to achieve these goals. 2] Our laboratory recently reported a high-throughput robotic screen that was used to identify synthetic riboswitches that displayed low background levels of gene expression in the absence of a ligand, and strongly activated gene expression in the presence of the ligand. We subsequently demonstrated that these riboswitches could control bacterial motility in a ligand-dependent fashion. Because the differences in cell motility at different ligand concentrations were easy to distinguish by using only a ruler, we asked whether motility differences could be the basis of a high-throughput selection to discover new synthetic riboswitches from large libraries. We envisioned that this method could equal, if not exceed, the throughput of our previously reported robotic screen, and could be performed at a fraction of the cost. Here we report an inexpensive and operationally simple selection method based on cell motility that not only approaches the throughput of a genetic selection, but also provides the quantitative nature of a genetic screen. We further show that this selection quickly identifies synthetic riboswitches that display low background levels of gene expression in the absence of a ligand, and robust increases in the presence of a ligand. We anticipate that motility-based selections will be generally useful in the discovery of rare events from large genetic libraries. There is good precedent for using motility to select for rare events. For decades, microbiologists have identified rare mutants by spotting cells at the center of a Petri dish containing semisolid media, and manually selecting cells that migrate abnormally. Capitalizing on these successes, Goulian and coworkers recently developed a motility-based selection to discover mutant chemoreceptors that could recognize a new ligand. Because a riboswitch displays two different phenotypes, depending on whether or not the ligand is present, a selection process must be able to quantitatively assay both the “on” and “off” states. Such counter-selections, which select for one phenotype and against another, are powerful, but can be challenging to implement. We envisioned that a motility-based selection could be used to discover “on” switches by selecting for mutants that do not move in the absence of the ligand, and then further selecting for mutants that move in the presence of the ligand. Of course, there are many ways that one can imagine setting up the experiment (for example, looking for “off” switches), but the key is to be able to rapidly and ACHTUNGTRENNUNGinexpensively determine the phenotype under two sets of conditions. To compare our motility selection to a more traditional screening technique, we assayed combinatorial libraries that were similar to those previously reported by our laboratory, which were comprised of four to eight randomized base pairs flanked by the mTCT8-4 theophylline aptamer and a fixed ribosome-binding site upstream of the b-galactosidase reporter gene. E. coli expressing these libraries were assayed by using a multistep process that used a robotic colony picker and an ACHTUNGTRENNUNGautomated liquid-handling system. While our previous assay identified several outstanding synthetic riboswitches, it relied on expensive robotics, in addition to large quantities of consumables. We anticipated that a motility-based selection could eliminate the need for specialized capital equipment, and identify synthetic riboswitches with only standard consumables. To select for riboswitches by using motility as the readout, we used cheZ as a reporter gene. CheZ plays a critical role in E. coli chemotaxis by dephosphorylating the CheY-P protein, which binds to the flagellar motor and causes cells to tumble. Optimal levels of CheZ are necessary for E. coli cells to migrate on semisolid media. If too little CheZ is present, the level of CheY-P will increase, and the cells will tumble incessantly and not migrate. If cells have excess CheZ, they will swim very smoothly and rarely tumble. Because cells that swim extremely smoothly can become embedded in the semisolid media, they cannot migrate. Thus it is critical to ensure that CheZ is not over-expressed in these assays. Since the strength of the promoter will ultimately dictate the maximum expression level of the cheZ gene, we began with two different promoters: a “weak” IS10 promoter and the tac promoter, which is 60to 100-fold stronger (S.T. unpublished results). We anticipated that the motility selections would readily reveal which promoter provides the appropriate CheZ expression level. Using cassette-based PCR mutagenesis, we constructed a library in which the mTCT8-4 theophylline aptamer was followed by ten consecutive randomized base pairs (N10) in the 5’ untranslated region (5’ UTR) of cheZ (Figure 1). We chose to assay an N10 library that lacked a preset ribosome-binding site for these experiments rather than our previously described “N8” library, for two reasons. First, we anticipated that this selection could more effectively sample the additional sequence space; and second, having the additional [a] S. Topp, Prof. J. P. Gallivan Department of Chemistry and Center for Fundamental and Applied Molecular Evolution Emory University 1515 Dickey Drive, Atlanta, GA 30322 (USA) Fax: (+1)404-727-6586 E-mail : justin.gallivan@emory.edu Supporting information for this article is available on the WWW under http://www.chembiochem. org or from the author.