Hensel, Z. | Search bioRxiv

bioRxiv2017

New Cell Fate Potentials and Switching Kinetics Uncovered in a Classic Bistable Genetic Switch

Bistable switches are common gene regulatory motifs directing two mutually exclusive gene expression states, and consequently distinct cell fates. Theoretical studies suggest that the simple circuitry of bistable switches is sufficient to encode more than two cell fates due to the non-equilibrium, heterogeneous cellular environment, allowing a high degree of adaptation and differentiation. However, new cell fates arising from a classic bistable switch without rewiring the circuitry have not been experimentally observed. By developing a new, dual single-molecule gene-expression reporting system in live E. coli cells, we investigated the expression dynamics of two mutually repressing transcription factors, CI and Cro, in the classic genetic switch of bacteriophage {lambda}. We found that in addition to the two expected high-Cro and high-CI production states, there existed two new ones, in which neither CI nor Cro was produced, or both CI and Cro were produced. We constructed the corresponding potential landscape and mapped the transition kinetics between the four production states, providing insight into possible state-switching rates and paths. These findings uncover new cell fate potentials beyond the classical picture of {lambda} switch, and open a new window to explore the genetic and environmental origins of the cell fate decision-making process in gene regulatory networks.

biophysics↗

bioRxiv2017

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

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.

synthetic biology↗