Hensel, Zach2020-02-062020-02-062017-10-011932-6203PURE: 4073691PURE UUID: ae89e1d5-2f64-4734-937a-900057ae5988Scopus: 85032740883PubMed: 29084263ORCID: /0000-0002-4348-6229/work/69844408http://hdl.handle.net/10362/92359Experiments 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.3450835engGeneral Biochemistry,Genetics and Molecular BiologyGeneral Agricultural and Biological Sciencesjournal article10.1371/journal.pone.0187259https://www.scopus.com/pages/publications/85032740883https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0187259&type=printable