Direct measurements of IPTG enable analysis of the induction behavior of E. coli in high cell density cultures

Fernández-Castañé, Alfred; Caminal, Glòria; López-Santı́n, Josep

doi:10.1186/1475-2859-11-58

Cited by 24 publications

(32 citation statements)

References 34 publications

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“…Results (see Figure 2B-C) showed that IPTG is not degraded in this condition and the inducer disappearance in the medium of MG1655-Z1 was actually due to uptake by LacY. These results confirm the high stability of the IPTG molecule, previously found in literature in different conditions [16,19,20], and the role of lactose permease in IPTG uptake by LacY + strains. None of the tested environmental factors have been found to affect IPTG stability.…”

Section: Resultssupporting

confidence: 87%

Half-life measurements of chemical inducers for recombinant gene expression

et al. 2014

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BackgroundInducible promoters are widely spread genetic tools for triggering, tuning and optimizing the expression of recombinant genes in engineered biological systems. Most of them are controlled by the addition of a specific exogenous chemical inducer that indirectly regulates the promoter transcription rate in a concentration-dependent fashion. In order to have a robust and predictable degree of control on promoter activity, the degradation rate of such chemicals should be considered in many applications like recombinant protein production.ResultsIn this work, we use whole-cell biosensors to assess the half-life of three commonly used chemical inducers for recombinant Escherichia coli: Isopropyl β-D-1-thiogalactopyranoside (IPTG), anhydrotetracycline (ATc) and N-(3-oxohexanoyl)-L-homoserine lactone (HSL). A factorial study was conducted to investigate the conditions that significantly contribute to the decay rate of these inducers. Temperature has been found to be the major factor affecting ATc, while medium and pH have been found to highly affect HSL. Finally, no significant degradation was observed for IPTG among the tested conditions.ConclusionsWe have quantified the decay rate of IPTG, ATc and HSL in many conditions, some of which were not previously tested in the literature, and the main effects affecting their degradation were identified via a statistics-based framework. Whole-cell biosensors were successfully used to conduct this study, yielding reproducible measurements via simple multiwell-compatible assays. The knowledge of inducer degradation rate in several contexts has to be considered in the rational design of synthetic biological systems for improving the predictability of induction effects, especially for prolonged experiments.

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Section: Resultssupporting

confidence: 87%

Half-life measurements of chemical inducers for recombinant gene expression

et al. 2014

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“…Thus, each inducer takes on average 31.7 min (with 90% confidence, between 18 min and 104 min) to travel from the periplasm to the cytoplasm. Meanwhile, the leaky expression of the target gene (prior to induction) can be estimated to be B0.03 RNA per h per cell (using (12)), in agreement with the measured leakiness (o0.1 RNA per h per cell). 5,43 As a side note, the stochasticity of these events is also visible from the data.…”

Section: Transport Rate Of Iptg Through the Inner Membranesupporting

confidence: 65%

“…Due to the high rate of the forward reaction and inducer abundance, when the intracellular inducer concentration changes, we assume that reaction (12) reaches equilibrium before any binding event between R and Pr (1) can occur. Due to the high rate of the forward reaction and inducer abundance, when the intracellular inducer concentration changes, we assume that reaction (12) reaches equilibrium before any binding event between R and Pr (1) can occur.…”

Section: Model Of Inducer Repressor Interactionsmentioning

confidence: 99%

“…[7][8][9][10][11] Early studies of this system focused on the observation of the target gene's expression at the steady state, as a function of the inducer concentration in the media. Also, direct measurements of inducer levels in cells and media 12 are only accurate on high-density cultures able to deplete the media of inducers, which causes the cellular intake kinetics to vary over time. These studies were, in general, conducted in the regime of low IPTG concentration (usually below 0.5 mM), where the mean expression rate of the target gene exhibits a close-to-linear dependence on the intracellular inducer level.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2015

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From in vivo single-event measurements of the transient and steady-state transcription activity of a single-copy lac-ara-1 promoter in Escherichia coli, we characterize the intake kinetics of its inducer (IPTG) from the media. We show that the empirical data are well-fit by a model of intake assuming a bilayer membrane, with the passage through the second layer being rate-limiting, coupled to a stochastic, sub-Poissonian, multi-step transcription process. Using this model, we show that for a wide range of extracellular inducer levels (up to 1.25 mM) the intake process is diffusive-like, suggesting unsaturated membrane permeability. Inducer molecules travel from the periplasm to the cytoplasm in, on average, 31.7 minutes, strongly affecting cells' response time. The novel methodology followed here should aid the study of cellular intake mechanisms at the single-event level.

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“…11,13 Several solutions to reduce the metabolic burden by adapting the induction strategy were reported. In general, the inducer concentration can be reduced to decrease the product formation rate, [14][15][16][17] the optimal IPTG to biomass ratio can be identified, 19,20 and the inducer can be fed to the reaction mixture during expression phase to adapt the cells to recombinant protein expression. 13,[21][22][23] Nevertheless, the optimal induction strategy for many target proteins differ significantly.…”

Section: Introductionmentioning

confidence: 99%

Parallel steady state studies on a milliliter scale accelerate fed‐batch bioprocess design for recombinant protein production with Escherichia coli

Schmideder

Weuster‐Botz

2016

Biotechnology Progress

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In general, fed-batch processes are applied for recombinant protein production with Escherichia coli (E. coli). However, state of the art methods for identifying suitable reaction conditions suffer from severe drawbacks, i.e. direct transfer of process information from parallel batch studies is often defective and sequential fed-batch studies are time-consuming and cost-intensive. In this study, continuously operated stirred-tank reactors on a milliliter scale were applied to identify suitable reaction conditions for fed-batch processes. Isopropyl β-d-1-thiogalactopyranoside (IPTG) induction strategies were varied in parallel-operated stirred-tank bioreactors to study the effects on the continuous production of the recombinant protein photoactivatable mCherry (PAmCherry) with E. coli. Best-performing induction strategies were transferred from the continuous processes on a milliliter scale to liter scale fed-batch processes. Inducing recombinant protein expression by dynamically increasing the IPTG concentration to 100 µM led to an increase in the product concentration of 21% (8.4 g L ) compared to an implemented high-performance production process with the most frequently applied induction strategy by a single addition of 1000 µM IPGT. Thus, identifying feasible reaction conditions for fed-batch processes in parallel continuous studies on a milliliter scale was shown to be a powerful, novel method to accelerate bioprocess design in a cost-reducing manner. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1426-1435, 2016.

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Direct measurements of IPTG enable analysis of the induction behavior of E. coli in high cell density cultures

Cited by 24 publications

References 34 publications

Half-life measurements of chemical inducers for recombinant gene expression

Half-life measurements of chemical inducers for recombinant gene expression

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

Parallel steady state studies on a milliliter scale accelerate fed‐batch bioprocess design for recombinant protein production with Escherichia coli

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