An evolvable oestrogen receptor activity sensor: development of a modular system for integrating multiple genes into the yeast genome.
To study a gene interaction network, we developed a gene-targeting strategy that allows efficient and stable genomic integration of multiple genetic constructs at distinct target loci in the yeast genome. This gene-targeting strategy uses a modular plasmid with a recyclable selectable marker and a multiple cloning site into which the gene of interest is cloned, flanked by two long regions of homology to the target genomic locus that are generated using adaptamer primers. We used this strategy to integrate into a single yeast strain components of the oestrogen receptor (ER) signalling network, comprising the human ERalpha and three reporter genes driven by oestrogen response elements (EREs). The engineered strain contains multiple reporters of ligand-dependent receptor signalling, providing sensitive, reproducible, rapid, low-cost quantitative assays of ERalpha activity in order to screen potential receptor agonists. Further, because two of the ERE-driven reporter genes are required for growth in deficient media, the strain's growth rate-and therefore its fitness-depends on ligand-induced ERalpha activity. This evolvable oestrogen receptor activity sensor (EERAS) can therefore provide the foundation of a long-term experimental evolution strategy to elucidate ER structure-function relations and ligand-receptor evolution.
Fox
Jennifer E
JE
Center for Ecology and Evolutionary Biology, University of Oregon, Eugene, OR 97403, USA.
Bridgham
Jamie T
JT
Bovee
Toine F H
TF
Thornton
Joseph W
JW
D019020:Directed Molecular Evolution / Q000379:methods*; D004958:Estradiol / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000235:genetics* / Q000502:physiology; D053263:Gene Regulatory Networks; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors; D016681:Genome, Fungal; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology; D014170:Transformation, Genetic
D047628:Estrogen Receptor alpha; D049452:Green Fluorescent Proteins; D004958:Estradiol