A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit

PLoS One. 2017 Oct 30;12(10):e0187259. doi: 10.1371/journal.pone.0187259. eCollection 2017.

Abstract

Experiments in synthetic biology and microbiology can benefit from protein expression systems with low cell-to-cell variability (noise) and expression levels precisely tunable across a useful dynamic range. Despite advances in understanding the molecular biology of microbial gene regulation, many experiments employ protein-expression systems exhibiting high noise and nearly all-or-none responses to induction. I present an expression system that incorporates elements known to reduce gene expression noise: negative autoregulation and bicistronic transcription. I show by stochastic simulation that while negative autoregulation can produce a more gradual response to induction, bicistronic expression of a repressor and gene of interest can be necessary to reduce noise below the extrinsic limit. I synthesized a plasmid-based system incorporating these principles and studied its properties in Escherichia coli cells, using flow cytometry and fluorescence microscopy to characterize induction dose-response, induction/repression kinetics and gene expression noise. By varying ribosome binding site strengths, expression levels from 55-10,740 molecules/cell were achieved with noise below the extrinsic limit. Individual strains are inducible across a dynamic range greater than 20-fold. Experimental comparison of different regulatory networks confirmed that bicistronic autoregulation reduces noise, and revealed unexpectedly high noise for a conventional expression system with a constitutively expressed transcriptional repressor. I suggest a hybrid, low-noise expression system to increase the dynamic range.

MeSH terms

  • Escherichia coli / genetics*
  • Flow Cytometry
  • Gene Expression*
  • Plasmids*
  • Stochastic Processes
  • Synthetic Biology

Grants and funding

This work was financially supported by: Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI), by national funds through FCT - Fundação para a Ciência e a Tecnologia, and by the Dean's Research Unit at the Okinawa Institute of Science and Technology (OIST). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.