BMC Research Notes (Dec 2018)
Attempted use of PACE for riboswitch discovery generates three new translational theophylline riboswitch side products
- Zachary M. Shaver,
- Stephanie S. Bent,
- Steven R. Bilby,
- Michael Brown,
- Anna Buser,
- Itzayana G. Cuellar,
- Athena J. Davis,
- Lindsay Doolan,
- Fatima C. Enriquez,
- Autumn Estrada,
- Shelby Herner,
- J. Cody Herron,
- Andrew M. Hunn,
- Madison Hunter,
- Hartlee Johnston,
- Owen Koucky,
- Christian C. Mackley,
- Dylan Maghini,
- Devin Mattoon,
- Haden T. McDonald,
- Hannah Sinks,
- Austin J. Sprague,
- David Sullivan,
- Altan Tutar,
- Avery Umphreys,
- Chris Watson,
- Daniel Zweerink,
- Laurie J. Heyer,
- Jeffrey L. Poet,
- Todd T. Eckdahl,
- A. Malcolm Campbell
Affiliations
- Zachary M. Shaver
- Department of Biology, Davidson College
- Stephanie S. Bent
- Department of Biology, Davidson College
- Steven R. Bilby
- Department of Biology, Missouri Western State University
- Michael Brown
- Department of Computer Science, Math, and Physics, Missouri Western State University
- Anna Buser
- Department of Biology, Davidson College
- Itzayana G. Cuellar
- Department of Biology, Davidson College
- Athena J. Davis
- Department of Biology, Missouri Western State University
- Lindsay Doolan
- Department of Biology, Missouri Western State University
- Fatima C. Enriquez
- Department of Biology, Davidson College
- Autumn Estrada
- Department of Computer Science, Math, and Physics, Missouri Western State University
- Shelby Herner
- Department of Biology, Missouri Western State University
- J. Cody Herron
- Department of Biology, Davidson College
- Andrew M. Hunn
- Department of Biology, Missouri Western State University
- Madison Hunter
- Department of Biology, Davidson College
- Hartlee Johnston
- Department of Biology, Davidson College
- Owen Koucky
- Department of Biology, Davidson College
- Christian C. Mackley
- Department of Biology, Missouri Western State University
- Dylan Maghini
- Department of Biology, Davidson College
- Devin Mattoon
- Department of Computer Science, Math, and Physics, Missouri Western State University
- Haden T. McDonald
- Department of Computer Science, Math, and Physics, Missouri Western State University
- Hannah Sinks
- Department of Biology, Davidson College
- Austin J. Sprague
- Department of Computer Science, Math, and Physics, Missouri Western State University
- David Sullivan
- Department of Computer Science, Math, and Physics, Missouri Western State University
- Altan Tutar
- Department of Math and Computer Science, Davidson College
- Avery Umphreys
- Department of Biology, Missouri Western State University
- Chris Watson
- Department of Biology, Missouri Western State University
- Daniel Zweerink
- Department of Computer Science, Math, and Physics, Missouri Western State University
- Laurie J. Heyer
- Department of Math and Computer Science, Davidson College
- Jeffrey L. Poet
- Department of Computer Science, Math, and Physics, Missouri Western State University
- Todd T. Eckdahl
- Department of Biology, Missouri Western State University
- A. Malcolm Campbell
- Department of Biology, Davidson College
- DOI
- https://doi.org/10.1186/s13104-018-3965-6
- Journal volume & issue
-
Vol. 11,
no. 1
pp. 1 – 5
Abstract
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
- Phage-assisted continuous evolution (PACE)
- Riboswitch
- Synthetic biology
- Metabolic engineering
- Theophylline
- Xanthine