title: A bistable genetic switch based on designable DNA-binding domains.
abstract: Bistable switches are fundamental regulatory elements of complex systems, ranging from electronics to living cells. Designed genetic toggle switches have been constructed from pairs of natural transcriptional repressors wired to inhibit one another. The complexity of the engineered regulatory circuits can be increased using orthogonal transcriptional regulators based on designed DNA-binding domains. However, a mutual repressor-based toggle switch comprising DNA-binding domains of transcription-activator-like effectors (TALEs) did not support bistability in mammalian cells. Here, the challenge of engineering a bistable switch based on monomeric DNA-binding domains is solved via the introduction of a positive feedback loop composed of activators based on the same TALE domains as their opposing repressors and competition for the same DNA operator site. This design introduces nonlinearity and results in epigenetic bistability. This principle could be used to employ other monomeric DNA-binding domains such as CRISPR for applications ranging from reprogramming cells to building digital biological memory.
authors.affiliation: 1] Department of Biotechnology, National Institute of Chemistry, Hajdrihova 19, Ljubljana 1000, Slovenia [2] EN-FIST Centre of Excellence, Ljubljana 1000, Slovenia.
authors.forename: Tina
authors.initials: T
authors.lastname: Lebar
authors.affiliation: Slovenian iGEM Team 2012, National Institute of Chemistry and University of Ljubljana, Ljubljana 1000, Slovenia.
authors.forename: Urban
authors.initials: U
authors.lastname: Bezeljak
authors.affiliation: 1] Department of Biotechnology, National Institute of Chemistry, Hajdrihova 19, Ljubljana 1000, Slovenia [2] Slovenian iGEM Team 2012, National Institute of Chemistry and University of Ljubljana, Ljubljana 1000, Slovenia.
authors.forename: Anja
authors.initials: A
authors.lastname: Golob
authors.affiliation: Slovenian iGEM Team 2012, National Institute of Chemistry and University of Ljubljana, Ljubljana 1000, Slovenia.
authors.forename: Miha
authors.initials: M
authors.lastname: Jerala
authors.affiliation: 1] Department of Biotechnology, National Institute of Chemistry, Hajdrihova 19, Ljubljana 1000, Slovenia [2] Slovenian iGEM Team 2012, National Institute of Chemistry and University of Ljubljana, Ljubljana 1000, Slovenia.
authors.forename: Lucija
authors.initials: L
authors.lastname: Kadunc
authors.affiliation: Slovenian iGEM Team 2012, National Institute of Chemistry and University of Ljubljana, Ljubljana 1000, Slovenia.
authors.forename: Boštjan
authors.initials: B
authors.lastname: Pirš
authors.affiliation: 1] Slovenian iGEM Team 2012, National Institute of Chemistry and University of Ljubljana, Ljubljana 1000, Slovenia [2] Faculty of Computer and Information Science, University of Ljubljana, Večna pot 113, Ljubljana 1000, Slovenia.
authors.forename: Martin
authors.initials: M
authors.lastname: Stražar
authors.affiliation: Slovenian iGEM Team 2012, National Institute of Chemistry and University of Ljubljana, Ljubljana 1000, Slovenia.
authors.forename: Dušan
authors.initials: D
authors.lastname: Vučko
authors.affiliation: Slovenian iGEM Team 2012, National Institute of Chemistry and University of Ljubljana, Ljubljana 1000, Slovenia.
authors.forename: Uroš
authors.initials: U
authors.lastname: Zupančič
authors.affiliation: 1] Department of Biotechnology, National Institute of Chemistry, Hajdrihova 19, Ljubljana 1000, Slovenia [2] EN-FIST Centre of Excellence, Ljubljana 1000, Slovenia.
authors.forename: Mojca
authors.initials: M
authors.lastname: Benčina
authors.affiliation: Department of Biotechnology, National Institute of Chemistry, Hajdrihova 19, Ljubljana 1000, Slovenia.
authors.forename: Vida
authors.initials: V
authors.lastname: Forstnerič
authors.affiliation: 1] Department of Biotechnology, National Institute of Chemistry, Hajdrihova 19, Ljubljana 1000, Slovenia [2] EN-FIST Centre of Excellence, Ljubljana 1000, Slovenia.
authors.forename: Rok
authors.initials: R
authors.lastname: Gaber
authors.affiliation: 1] Department of Biotechnology, National Institute of Chemistry, Hajdrihova 19, Ljubljana 1000, Slovenia [2] EN-FIST Centre of Excellence, Ljubljana 1000, Slovenia.
authors.forename: Jan
authors.initials: J
authors.lastname: Lonzarić
authors.affiliation: 1] Department of Biotechnology, National Institute of Chemistry, Hajdrihova 19, Ljubljana 1000, Slovenia [2] EN-FIST Centre of Excellence, Ljubljana 1000, Slovenia.
authors.forename: Andreja
authors.initials: A
authors.lastname: Majerle
authors.affiliation: 1] Department of Biotechnology, National Institute of Chemistry, Hajdrihova 19, Ljubljana 1000, Slovenia [2] EN-FIST Centre of Excellence, Ljubljana 1000, Slovenia.
authors.forename: Alja
authors.initials: A
authors.lastname: Oblak
authors.affiliation: Department of Biotechnology, National Institute of Chemistry, Hajdrihova 19, Ljubljana 1000, Slovenia.
authors.forename: Anže
authors.initials: A
authors.lastname: Smole
authors.affiliation: 1] Department of Biotechnology, National Institute of Chemistry, Hajdrihova 19, Ljubljana 1000, Slovenia [2] EN-FIST Centre of Excellence, Ljubljana 1000, Slovenia.
authors.forename: Roman
authors.initials: R
authors.lastname: Jerala
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000737:chemistry*; D044127:Epigenesis, Genetic; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D018613:Microscopy, Confocal; D008962:Models, Theoretical; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013269:Stochastic Processes
chemical_list: D004247:DNA; D008156:Luciferases