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

作者：Hensel Zach^*

来源：PLos One, 2017, 12(10): e0187259.

DOI：10.1371/journal.pone.0187259

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.

• 出版日期2017-10-30