Biosensor libraries harness large classes of binding domains for construction of allosteric transcriptional regulators

Javier F Juárez,Christopher D Johnston,George M Church,Stuart L Brown,Begoña Lecube-Azpeitia

doi:10.1038/s41467-018-05525-6

Javier F Juárez, Christopher D Johnston + Show 3 more

Open Access

https://doi.org/10.1038/s41467-018-05525-6

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Journal: Nature Communications	Publication Date: Aug 6, 2018
Citations: 50	License type: open-access

Affiliation: Harvard University

Abstract

The ability of bacteria to sense specific molecules within their environment and trigger metabolic responses in accordance is an invaluable biotechnological resource. While many transcription factors (TFs) mediating such processes have been studied, only a handful have been leveraged for molecular biology applications. To expand the repertoire of biotechnologically relevant sensors we present a strategy for the construction and testing of chimeric TF libraries, based on the fusion of highly soluble periplasmic binding proteins (PBPs) with DNA-binding domains (DBDs). We validate this concept by constructing and functionally testing two unique sense-and-respond regulators for benzoate, an environmentally and industrially relevant metabolite. This work will enable the development of tailored biosensors for novel synthetic regulatory circuits.

Highlights

The ability of bacteria to sense specific molecules within their environment and trigger metabolic responses in accordance is an invaluable biotechnological resource
The chimeric transcription factors (TFs) Q1 is another example of the generation of a new TF by the fusion of a DNA-binding domains (DBDs) to a protein phylogenetically related to its LBD23
For systematic construction of fusion TFs we generated libraries under the following two degrees of freedom: (a) 15 DBDs sourced from bacterial transcriptional repressors with a common architecture and known operator sequences; and (b) 15 ligand-binding domains (LBDs) from periplasmic binding proteins (PBPs) associated with ATP-binding cassette transporters

Summary

Introduction

The ability of bacteria to sense specific molecules within their environment and trigger metabolic responses in accordance is an invaluable biotechnological resource. To connect Nt-DBD and Ct-LBD domains with a diverse set of LNK sequences, we developed a novel cloning strategy based on the ligase cycling/chain (LCR) reaction assembly method[39]. The fusion genes assembled by eLCR were PCR amplified with oligonucleotides that incorporated adapter sequences homologous to the expression vectors pCKTRBS or pCKTRBSOD (Supplementary Data 1).

Results

Conclusion