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ACS Synth Biol. 2018 Oct 19;7(10):2457-2467. doi: 10.1021/acssynbio.8b00242. Epub 2018 Sep 25.

Engineered Biosensors from Dimeric Ligand-Binding Domains.

Author information

1
Howard Hughes Medical Institute , University of Washington , Seattle , Washington 98195 , United States.
2
Department of Genome Sciences , University of Washington , Seattle , Washington 98195 , United States.
3
Department of Biochemistry , University of Washington , Seattle , Washington 98195 , United States.
4
Department of Medicine , University of Washington , Seattle , Washington 98195 , United States.

Abstract

Biosensors are important components of many synthetic biology and metabolic engineering applications. Here, we report a second generation of Saccharomyces cerevisiae digoxigenin and progesterone biosensors based on destabilized dimeric ligand-binding domains that undergo ligand-induced stabilization. The biosensors, comprising one ligand-binding domain monomer fused to a DNA-binding domain and another fused to a transcriptional activation domain, activate reporter gene expression in response to steroid binding and receptor dimerization. The introduction of a destabilizing mutation to the dimer interface increased biosensor dynamic range by an order of magnitude. Computational redesign of the dimer interface and functional selections were used to create heterodimeric pairs with further improved dynamic range. A heterodimeric biosensor built from the digoxigenin and progesterone ligand-binding domains functioned as a synthetic "AND"-gate, with 20-fold stronger response to the two ligands in combination than to either one alone. We also identified mutations that increase the sensitivity or selectivity of the biosensors to chemically similar ligands. These dimerizing biosensors provide additional flexibility for the construction of logic gates and other applications.

KEYWORDS:

biosensor; directed evolution; ligand-binding domain; logic gate; next generation sequencing; protein engineering; transcription factor

PMID:
30204430
PMCID:
PMC6317713
[Available on 2019-10-19]
DOI:
10.1021/acssynbio.8b00242

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