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

Autor: Shaver ZM; Department of Biology, Davidson College, Davidson, USA., Bent SS; Department of Biology, Davidson College, Davidson, USA., Bilby SR; Department of Biology, Missouri Western State University, Saint Joseph, USA., Brown M; Department of Computer Science, Math, and Physics, Missouri Western State University, Saint Joseph, USA., Buser A; Department of Biology, Davidson College, Davidson, USA., Cuellar IG; Department of Biology, Davidson College, Davidson, USA., Davis AJ; Department of Biology, Missouri Western State University, Saint Joseph, USA., Doolan L; Department of Biology, Missouri Western State University, Saint Joseph, USA., Enriquez FC; Department of Biology, Davidson College, Davidson, USA., Estrada A; Department of Computer Science, Math, and Physics, Missouri Western State University, Saint Joseph, USA., Herner S; Department of Biology, Missouri Western State University, Saint Joseph, USA., Herron JC; Department of Biology, Davidson College, Davidson, USA., Hunn AM; Department of Biology, Missouri Western State University, Saint Joseph, USA., Hunter M; Department of Biology, Davidson College, Davidson, USA., Johnston H; Department of Biology, Davidson College, Davidson, USA., Koucky O; Department of Biology, Davidson College, Davidson, USA., Mackley CC; Department of Biology, Missouri Western State University, Saint Joseph, USA., Maghini D; Department of Biology, Davidson College, Davidson, USA.; Department of Math and Computer Science, Davidson College, Davidson, USA., Mattoon D; Department of Computer Science, Math, and Physics, Missouri Western State University, Saint Joseph, USA., McDonald HT; Department of Computer Science, Math, and Physics, Missouri Western State University, Saint Joseph, USA., Sinks H; Department of Biology, Davidson College, Davidson, USA.; Department of Math and Computer Science, Davidson College, Davidson, USA., Sprague AJ; Department of Computer Science, Math, and Physics, Missouri Western State University, Saint Joseph, USA., Sullivan D; Department of Computer Science, Math, and Physics, Missouri Western State University, Saint Joseph, USA., Tutar A; Department of Math and Computer Science, Davidson College, Davidson, USA., Umphreys A; Department of Biology, Missouri Western State University, Saint Joseph, USA., Watson C; Department of Biology, Missouri Western State University, Saint Joseph, USA., Zweerink D; Department of Computer Science, Math, and Physics, Missouri Western State University, Saint Joseph, USA., Heyer LJ; Department of Math and Computer Science, Davidson College, Davidson, USA., Poet JL; Department of Computer Science, Math, and Physics, Missouri Western State University, Saint Joseph, USA., Eckdahl TT; Department of Biology, Missouri Western State University, Saint Joseph, USA., Campbell AM; Department of Biology, Davidson College, Davidson, USA. macampbell@davidson.edu.
Jazyk: angličtina
Zdroj: BMC research notes [BMC Res Notes] 2018 Dec 05; Vol. 11 (1), pp. 861. Date of Electronic Publication: 2018 Dec 05.
DOI: 10.1186/s13104-018-3965-6
Abstrakt: 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.
Databáze: MEDLINE
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