PT  - JOURNAL ARTICLE
AU  - Zach Hensel
TI  - A plasmid-based <em>Escherichia coli</em> gene expression system with cell-to-cell variation below the extrinsic noise limit
AID  - 10.1101/192963
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 192963
4099  - http://biorxiv.org/content/early/2017/09/23/192963.short
4100  - http://biorxiv.org/content/early/2017/09/23/192963.full
AB  - 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.