In vivo single-molecule kinetics of activation and subsequent activity of the arabinose promoter

Abstract

Using a single-RNA detection technique in live Escherichia coli cells, we measure, for each cell, the waiting time for the production of the first RNA under the control of PBAD promoter after induction by arabinose, and subsequent intervals between transcription events. We find that the kinetics of the arabinose intake system affect mean and diversity in RNA numbers, long after induction. We observed the same effect on Plac/ara-1 promoter, which is inducible by arabinose or by IPTG. Importantly, the distribution of waiting times of Plac/ara-1 is indistinguishable from that of PBAD, if and only if induced by arabinose alone. Finally, RNA production under the control of PBAD is found to be a sub-Poissonian process. We conclude that inducer-dependent waiting times affect mean and cell-to-cell diversity in RNA numbers long after induction, suggesting that intake mechanisms have non-negligible effects on the phenotypic diversity of cell populations in natural, fluctuating environments.

Kinetics of the cellular intake of a gene expression inducer at high concentrations

“…[1][2][3] The kinetics of the transcriptional response to the introduction of inducers into the media depends both on the genetic target system [4][5][6] as well as on the mechanisms of the intake of the inducer into cells' cytoplasm. [1][2][3] The kinetics of the transcriptional response to the introduction of inducers into the media depends both on the genetic target system [4][5][6] as well as on the mechanisms of the intake of the inducer into cells' cytoplasm.…”

“…This technique has recently been used to characterize the transcription kinetics of some promoters in E. coli, 6,14 revealing that, e.g., P lac-ara-1 transcription initiation is a multi-stepped process and IPTG mainly affects one of the two rate-limiting steps, likely the closed complex formation. 5 We then use methods of statistical inference to derive from the empirical data a deterministic model of inducer intake through a bilayer membrane, 18 coupled with a stochastic, multi-step model of transcription. 5 We then use methods of statistical inference to derive from the empirical data a deterministic model of inducer intake through a bilayer membrane, 18 coupled with a stochastic, multi-step model of transcription.…”

Screening of an Escherichia coli promoter library for a phenylalanine biosensor

“…Thus, the design of suitable biosensors would provide a convenient tool for strain analysis and screening, and might be especially useful for the identification of non-intuitive targets for strain improvement (Binder et al 2012;Mahr et al 2015;Mustafi et al 2012). However, our study revealed that several parameters require careful consideration to set up a screening procedure for effector-responsive promoters, including the adaption to growth medium, the uptake and catabolism of effector molecules (Luo et al 2014;Payne 1977), the activation of the gene expression machinery (Bintu et al 2005b;Carey et al 2013;Fritz et al 2014;Mäkelä et al 2013), the maturation of fluorescent proteins (Craggs 2009;Iizuka et al 2011), and last but not least, the composition of the particular growth medium. Here, we used M9 minimal medium due to its lack of other amino acids and carbon source and its very low auto-fluorescence (Miller 1972).…”

Determining the Limitations and Benefits of Noise in Gene Regulation and Signal Transduction through Single Cell, Microscopy-Based Analysis

“…Using this approach, Golding and colleagues demonstrated that transcription in E. coli is consistent with a gene activation/inactivation model in that it occurs in quantal bursts, has geometrically distributed burst sizes, and has exponentially distributed time intervals. In addition to transcriptional bursting, studies based on single cell fluorescence microscopy in E. coli have identified sources of cell-to-cell variability in gene activation related to the cellular response to external stimuli, such as the kinetics of cellular uptake mechanisms for inducer molecules [36, 37] Specifically, studies of the arabinose utilization system in individual, living E. coli cells demonstrated that cell-to-cell heterogeneity in gene activation was influenced by the kinetics of arabinose uptake, which may be attributed to varying levels of uptake proteins in cells prior to arabinose induction [36, 37]. Single-cell bioluminescence measurements enabled Suter and colleagues to observe transcriptional bursting in real time in mammalian cells [30].…”