Publicação
A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit
| dc.contributor.author | Hensel, Zach | |
| dc.contributor.institution | Molecular, Structural and Cellular Microbiology (MOSTMICRO) | |
| dc.contributor.institution | Instituto de Tecnologia Química e Biológica António Xavier (ITQB) | |
| dc.contributor.pbl | PLOS - Public Library of Science | |
| dc.date.accessioned | 2020-02-06T23:30:43Z | |
| dc.date.available | 2020-02-06T23:30:43Z | |
| dc.date.issued | 2017-10-01 | |
| dc.description.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. | en |
| dc.description.version | published | |
| dc.format.extent | 3450835 | |
| dc.identifier.doi | 10.1371/journal.pone.0187259 | |
| dc.identifier.issn | 1932-6203 | |
| dc.identifier.other | PURE: 4073691 | |
| dc.identifier.other | PURE UUID: ae89e1d5-2f64-4734-937a-900057ae5988 | |
| dc.identifier.other | Scopus: 85032740883 | |
| dc.identifier.other | PubMed: 29084263 | |
| dc.identifier.other | ORCID: /0000-0002-4348-6229/work/69844408 | |
| dc.identifier.uri | http://hdl.handle.net/10362/92359 | |
| dc.identifier.url | https://www.scopus.com/pages/publications/85032740883 | |
| dc.identifier.url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0187259&type=printable | |
| dc.language.iso | eng | |
| dc.peerreviewed | yes | |
| dc.subject | General Biochemistry,Genetics and Molecular Biology | |
| dc.subject | General Agricultural and Biological Sciences | |
| dc.title | A plasmid-based Escherichia coli gene expression system with cell-to-cell variation below the extrinsic noise limit | en |
| dc.type | journal article | |
| degois.publication.issue | 10 | |
| degois.publication.title | PLoS ONE | |
| degois.publication.volume | 12 | |
| dspace.entity.type | Publication | |
| rcaap.rights | openAccess |
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