Biology subjects

Noireaux, V.

Publications and source records attributed to Noireaux, V..

21 records · Page 2Linked to original sources

bioRxiv2018

Anomalous Scaling of Gene Expression in Confined Cell-Free Reactions

Cellular surface breaks the symmetry of molecular diffusion across membrane. Here, we study how steric interactions between the surface and the bulk of cell-sized emulsion droplets alters gene expression emulated by a cell-free transcription/translation (TXTL) system. The concentration of synthesized reporter proteins in droplets of radius R shows an anomalous geometric scaling of R4 different from the expected normal size-dependence of R3. Given that TXTL becomes less efficient at thin surface layer, a mathematical model explains anomalous size-dependence found in experiment. The surface of cell-sized compartment thus plays a regulatory role for cell-free gene expression.

synthetic biology↗

bioRxiv2017

Rapid and scalable characterization of CRISPR technologies using an E. coli cell-free transcription-translation system

CRISPR-Cas systems have offered versatile technologies for genome engineering, yet their implementation has been outpaced by the ongoing discovery of new Cas nucleases and anti-CRISPR proteins. Here, we present the use of E. coli cell-free transcription-translation systems (TXTL) to vastly improve the speed and scalability of CRISPR characterization and validation. Unlike prior approaches that require protein purification or live cells, TXTL can express active CRISPR machinery from added plasmids and linear DNA, and TXTL can output quantitative dynamics of DNA cleavage and gene repression. To demonstrate the applicability of TXTL, we rapidly measure guide RNA-dependent DNA cleavage and gene repression for single- and multi-effector CRISPR-Cas systems, accurately predict the strength of gene repression in E. coli, quantify the inhibitory activity of anti-CRISPR proteins, and develop a fast and scalable high-throughput screen for protospacer-adjacent motifs. These examples underscore the potential of TXTL to facilitate the characterization and application of CRISPR technologies across their many uses.

synthetic biology↗

bioRxiv2017

Tuning of recombinant protein expression in Escherichia coli by manipulating transcription, translation initiation rates and incorporation of non-canonical amino acids

Protein synthesis in cells has been thoroughly investigated and characterized over the past 60 years. However, some fundamental issues remain unresolved, including the reasons for genetic code redundancy and codon bias. In this study, we changed the kinetics of the E. coli transcription and translation processes by mutating the promoter and ribosome binding domains and by using genetic code expansion. The results expose a counterintuitive phenomenon, whereby an increase in the initiation rates of transcription and translation lead to a decrease in protein expression. This effect can be rescued by introducing slow translating codons into the beginning of the gene, by shortening gene length or by reducing initiation rates. Based on the results, we developed a biophysical model, which suggests that the density of co-transcriptional translation plays a role in bacterial protein synthesis. These findings indicate how cells use codon bias to tune translation speed and protein synthesis.

synthetic biology↗