Efflux Pump Control Alters Synthetic Gene Circuit Function

• Published in: ACS synthetic biology 2016-07, Vol.5 (7), p.619-631
• Main Authors: Diao, Junchen, Charlebois, Daniel A, Nevozhay, Dmitry, Bódi, Zoltán, Pál, Csaba, Balázsi, Gábor
• Format: Article
• Language: English
• Subjects: ATP-Binding Cassette Transporters - genetics; ATP-Binding Cassette Transporters - metabolism; Dose-Response Relationship, Drug; Doxycycline - administration & dosage; Doxycycline - pharmacology; Gene Expression Regulation, Fungal - drug effects; Gene Regulatory Networks; Genes, Synthetic; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Models, Theoretical; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Saccharomyces cerevisiae - drug effects; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Tetracycline - pharmacology
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.5b00154

Synthetic biology aims to design new biological systems for predefined purposes, such as the controlled secretion of biofuels, pharmaceuticals, or other chemicals. Synthetic gene circuits regulating an efflux pump from the ATP-binding cassette (ABC) protein family could achieve this. However, ABC efflux pumps can also drive out intracellular inducer molecules that control the gene circuits. This will introduce an implicit feedback that could alter gene circuit function in ways that are poorly understood. Here, we used two synthetic gene circuits inducible by tetracycline family molecules to regulate the expression of a yeast ABC pump (Pdr5p) that pumps out the inducer. Pdr5p altered the dose–responses of the original gene circuits substantially in Saccharomyces cerevisiae. While one aspect of the change could be attributed to the efflux pumping function of Pdr5p, another aspect remained unexplained. Quantitative modeling indicated that reduced regulator gene expression in addition to efflux pump function could fully explain the altered dose–responses. These predictions were validated experimentally. Overall, we highlight how efflux pumps can alter gene circuit dynamics and demonstrate the utility of mathematical modeling in understanding synthetic gene circuit function in new circumstances.