A Biosensor Strategy for E. coli Based on Ligand-Dependent Stabilization

ACS Synth Biol. 2018 Sep 21;7(9):1990-1999. doi: 10.1021/acssynbio.8b00052. Epub 2018 Aug 14.


The engineering of microorganisms to monitor environmental chemicals or to produce desirable bioproducts is often reliant on the availability of a suitable biosensor. However, the conversion of a ligand-binding protein into a biosensor has been difficult. Here, we report a general strategy for generating biosensors in Escherichia coli that act by ligand-dependent stabilization of a transcriptional activator and mediate ligand concentration-dependent expression of a reporter gene. We constructed such a biosensor by using the lac repressor, LacI, as the ligand-binding domain and fusing it to the Zif268 DNA-binding domain and RNA polymerase omega subunit transcription-activating domain. Using error-prone PCR mutagenesis of lacI and selection, we identified a biosensor with multiple mutations, only one of which was essential for biosensor behavior. By tuning parameters of the assay, we obtained a response dependent on the ligand isopropyl β-d-1-thiogalactopyranoside (IPTG) of up to a 7-fold increase in the growth rate of E. coli. The single destabilizing mutation combined with a lacI mutation that expands ligand specificity to d-fucose generated a biosensor with improved response both to d-fucose and to IPTG. However, a mutation equivalent to the one that destabilized LacI in either of two structurally similar periplasmic binding proteins did not confer ligand-dependent stabilization. Finally, we demonstrated the generality of this method by using mutagenesis and selection to engineer another ligand-binding domain, MphR, to function as a biosensor. This strategy may allow many natural proteins that recognize and bind to ligands to be converted into biosensors.

Keywords: biosensors; directed evolution; ligand-dependent stabilization.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Biosensing Techniques / methods*
  • Escherichia coli / growth & development
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Fucose / pharmacology
  • Isopropyl Thiogalactoside / pharmacology
  • Lac Repressors / genetics
  • Lac Repressors / metabolism
  • Ligands
  • Mutagenesis
  • Transcriptional Activation / drug effects


  • Escherichia coli Proteins
  • Lac Repressors
  • LacI protein, E coli
  • Ligands
  • Fucose
  • Isopropyl Thiogalactoside