Fourteen Ways to Reroute Cooperative Communication in the Lactose Repressor: Engineering Regulatory Proteins with Alternate Repressive Functions.
The lactose repressor (LacI) is a classic genetic switch that has been used as a fundamental component in a host of synthetic genetic networks. To expand the function of LacI for use in the development of novel networks and other biotechnological applications, we engineered alternate communication in the LacI scaffold via laboratory evolution. Here we produced 14 new regulatory elements based on the LacI topology that are responsive to isopropyl β-d-1-thiogalactopyranoside (IPTG) with variation in repression strengths and ligand sensitivities-on solid media. The new variants exhibit repressive as well as antilac (i.e., inverse-repression + IPTG) functions and variations in the control of gene output upon exposure to different concentrations of IPTG. In addition, examination of this collection of variants in solution results in the controlled output of a canonical florescent reporter, demonstrating the utility of this collection of new regulatory proteins under standard conditions.
Department of Molecular Biophysics & Biochemistry, Yale University , New Haven, Connecticut 06520, United States.
David H
DH
Richards
Department of Chemical & Environmental Engineering, Yale University , New Haven, Connecticut 06520, United States.
Sarai
S
Meyer
School of Chemical & Biomolecular Engineering, Georgia Institute of Technology , Atlanta, Georgia 30332, United States.
Corey J
CJ
Wilson
D000494:Allosteric Regulation / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D056968:Lac Repressors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D000072417:Protein Domains; D015202:Protein Engineering
D056968:Lac Repressors; D007544:Isopropyl Thiogalactoside
antilac; gene regulation; protein engineering