Attempted use of PACE for riboswitch discovery generates three new translational theophylline riboswitch side products

Abstract

Objective: The purpose of this project was to use an in vivo method to discover riboswitches that are activated by new ligands. We employed phage-assisted continuous evolution (PACE) to evolve new riboswitches in vivo. We started with one translational riboswitch and one transcriptional riboswitch, both of which were activated by theophylline. We used xanthine as the new target ligand during positive selection followed by negative selection using theophylline. The goal was to generate very large M13 phage populations that contained unknown mutations, some of which would result in new aptamer specificity. We discovered side products of three new theophylline translational riboswitches with different levels of protein production.

Results: We used next generation sequencing to identify M13 phage that carried riboswitch mutations. We cloned and characterized the most abundant riboswitch mutants and discovered three variants that produce different levels of translational output while retaining their theophylline specificity. Although we were unable to demonstrate evolution of new riboswitch ligand specificity using PACE, we recommend careful design of recombinant M13 phage to avoid evolution of "cheaters" that short circuit the intended selection pressure.

Keywords: M13; Metabolic engineering; Phage-assisted continuous evolution (PACE); Riboswitch; Synthetic biology; Theophylline; Xanthine.

Fig. 1

PACE experimental design for the discovery of new RSs. a During positive selection, anhydrotetracycline (aTc) concentration is reduced while the new target ligand xanthine concentration remained constant. Mutagenesis cassette is induced using arabinose. The chemostat replenishes growth media and uninfected E. coli. b Negative selection employed a dominant gIII negative allele (gIII neg) induced by a steady concentration of theophylline to select against theophylline-specific riboswitches while gradually reducing aTc to maintain phage with xanthine-specific riboswitches

Fig. 2

Directed evolution of three new riboswitches. a Each new riboswitch was cloned into rClone Red and grown in triplicate over 10 h with one of three treatments as indicated. Each population was measured for RFP and cell density three times to produce an average end point value ± s.e.m. b Theophylline-induction values from three independent populations from a were normalized by comparing each construct to the fold induction of RS-C in the absence of theophylline. Error bars represent s.e.m

Fig. 3

Predicted 2D structures of RS-C and three new riboswitches. a The original RS-C sequence (left) was submitted to mFold to produce a predicted 2D structure and quantification of stability (ΔG); the boxed region is the predicted RBS. The three new riboswitch sequences were also evaluated by mFold with RBS denoted by boxed nucleotides. Yellow highlighting shows inserted bases and the blue A within a box indicates a base substitution conserved in all three new riboswitches. b RNA base pairing (dashed lines) between ribosome (red) and the complementary anti-parallel  riboswitch sequence (black) for the original RS-C as well as the three new RSs. The number of base pairs between rRNA and RS sequence is shown on the far right