Precise control of gene expression with small-molecular or physical inducers has been a central aim of synthetic biology in recent decades, and has led, for example, to dramatic improvements in the production of protein therapeutics. However, the number of inputs for inducible promoters and gene circuits is still limited, and new orthogonal inducers are needed to support the construction of more complex programmable systems in the fields of bioengineering and biocomputing. Here, two new gene switches inducible by the antibiotic simocyclinone D8 (SD8) and the flavonoid luteolin in mammalian cells are presented. Streptomyces antibioticus Tu 6040 has evolved the DNA gyrase inhibitor SD8, which, through the bacterial TetR-like transcriptional repressor SimR, also regulates the multidrug efflux pump SimX. Taking advantage of SimR, as well as the luteolin-binding TetR-like transcriptional repressor EmrR from Sinorhizobium meliloti, we optimized the amounts of transfected genes and DNA operator sites to engineer highly effective, orthogonal transcriptional OFF- as well as ON-switches triggered by SD8 and/or luteolin. We confirmed that SD8 and luteolin are not cytotoxic at the concentrations required for switching, and demonstrated the functionality of these gene switches in a range of biotechnologically relevant cell lines. These switches were combined to generate OR and AND Boolean logic gates, and we confirmed their modularity by the addition of the vanillic acid-responsive transcriptional repressor VanR to generate a three-input AND gate. These additions to the panoply of inducers available for synthetic biology are expected to facilitate advances in the fields of biocomputing, biopharmaceutical manufacturing, and biomedicine. (c) 2018 The Authors. AIChE Journal published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers.