Directed evolution of AraC for improved compatibility of arabinose- and lactose-inducible promoters.
Synthetic biological systems often require multiple, independently inducible promoters in order to control the expression levels of several genes; however, cross talk between the promoters limits this ability. Here, we demonstrate the directed evolution of AraC to construct an arabinose-inducible (P(BAD)) system that is more compatible with IPTG (isopropyl-beta-D-1-thiogalactopyranoside) induction of a lactose-inducible (P(lac)) system. The constructed system is 10 times more sensitive to arabinose and tolerates IPTG significantly better than the wild type. Detailed studies indicate that the AraC dimerization domain and C terminus are important for the increased sensitivity of AraC to arabinose.
Lee
Sung Kuk
SK
Department of Chemical Engineering, University of California, Berkeley, California 94720, USA.
Chou
Howard H
HH
Pfleger
Brian F
BF
Newman
Jack D
JD
Yoshikuni
Yasuo
Y
Keasling
Jay D
JD
D051777:AraC Transcription Factor / Q000187:drug effects* / Q000235:genetics; D001089:Arabinose / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000187:drug effects* / Q000235:genetics; D019143:Evolution, Molecular; D007544:Isopropyl Thiogalactoside / Q000378:metabolism / Q000494:pharmacology*; D007785:Lactose / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D009876:Operon; D011401:Promoter Regions, Genetic / Q000187:drug effects* / Q000502:physiology
D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D029968:Escherichia coli Proteins; D007544:Isopropyl Thiogalactoside; D001089:Arabinose; D007785:Lactose