A suppressor tRNA-mediated feedforward loop eliminates leaky gene expression in bacteria

Abstract Ligand-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...

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Bibliographic Details
Published in:Nucleic acids research 2021-03, Vol.49 (5), p.e25-e25
Main Authors: Ho, Joanne M L, Miller, Corwin A, Parks, Sydney E, Mattia, Jacob R, Bennett, Matthew R
Format: Article
Language:English
Subjects:
Arabinose - metabolism
AraC Transcription Factor - metabolism
Codon, Terminator
DNA-Binding Proteins - metabolism
Escherichia coli - genetics
Escherichia coli - metabolism
Escherichia coli Proteins - metabolism
Gene Expression Regulation, Bacterial
Lactic Acid - metabolism
Methods Online
Mutagenesis
Plasmids - genetics
Protein Biosynthesis
RNA, Catalytic
RNA, Transfer - chemistry
RNA, Transfer - metabolism
Transcription Factors - metabolism
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Summary:Abstract Ligand-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.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkaa1179