Ho, Joanne M.L.Miller, Corwin A.Parks, Sydney E.Mattia, Jacob R.Bennett, Matthew R.2021-04-212021-04-212021Ho, Joanne M.L., Miller, Corwin A., Parks, Sydney E., et al.. "A suppressor tRNA-mediated feedforward loop eliminates leaky gene expression in bacteria." <i>Nucleic Acids Research,</i> 49, no. 5 (2021) Oxford University Press: https://doi.org/10.1093/nar/gkaa1179.https://hdl.handle.net/1911/110302Ligand-inducible genetic systems are the mainstay of synthetic biology, allowing gene expression to be controlled by the presence of a small molecule. However, ‘leaky’ gene expression in the absence of inducer remains a persistent problem. We developed a leak dampener tool that drastically reduces the leak of inducible genetic systems while retaining signal in Escherichia coli. Our system relies on a coherent feedforward loop featuring a suppressor tRNA that enables conditional readthrough of silent non-sense mutations in a regulated gene, and this approach can be applied to any ligand-inducible transcription factor. We demonstrate proof-of-principle of our system with the lactate biosensor LldR and the arabinose biosensor AraC, which displayed a 70-fold and 630-fold change in output after induction of a fluorescence reporter, respectively, without any background subtraction. Application of the tool to an arabinose-inducible mutagenesis plasmid led to a 540-fold change in its output after induction, with leak decreasing to the level of background mutagenesis. This study provides a modular tool for reducing leak and improving the fold-induction within genetic circuits, demonstrated here using two types of biosensors relevant to cancer detection and genetic engineering.engThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.Journal articlegkaa1179https://doi.org/10.1093/nar/gkaa1179