Mathematical modeling of a single‐cell enzyme assay

Kd, Wittrup; Je, Bailey

doi:10.1002/bit.260350511

Cited by 9 publications

(5 citation statements)

References 22 publications

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“…One class of artificial gene-based memory systems uses bistable transcriptional switches to record transient environmental signals transmitted either directly through one of the transcription factors in the switch or indirectly through a distinct trigger element (12)(13)(14)(15)(16). Such memory devices may be used as sophisticated reporters to record a biological event in an organism and interrogate the system at a later time.…”

mentioning

confidence: 99%

Programmable bacteria detect and record an environmental signal in the mammalian gut

Kotula

Kerns

Shaket

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

324

302

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Significance The human microbiota represents the trillions of bacteria that live on the skin, in the oral, nasal, and aural cavities, and throughout the gastrointestinal tract. The species that live in the gastrointestinal tract, the gut microbiota, closely interact with host cells and have a profound impact on health. To develop tools to effectively monitor the gut microbiota and ultimately help in disease diagnosis, we have engineered Escherichia coli to sense and record environmental stimuli, and demonstrated that E. coli with such memory systems can survive and function in the mammalian gut. This work demonstrates that E. coli can be engineered into living diagnostics capable of nondestructively probing the mammalian gut.

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mentioning

confidence: 99%

Programmable bacteria detect and record an environmental signal in the mammalian gut

Kotula

Kerns

Shaket

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

324

302

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“…This new model is different from a previous model developed for cytometry studies . First, the assumptions required for the previous model were no longer needed in our on-chip single-cell experiments for the following reasons: (a) The external substrate concentration is indeed time-invariant as the single cell is continually bathed in 12 μM FDA solution in the microchip.…”

Section: Resultsmentioning

confidence: 99%

“…This efflux is particularly strong in the FDA system as compared to other FDA derivatives . Dynamic studies of FDA metabolism in yeast have been performed by flow cytometry, , but these studies could not completely reveal the complexity of this complex influx−hydrolysis (by esterase)−efflux process.…”

mentioning

confidence: 99%

A Three-Dimensional Flow Control Concept for Single-Cell Experiments on a Microchip. 2. Fluorescein Diacetate Metabolism and Calcium Mobilization in a Single Yeast Cell As Stimulated by Glucose and pH Changes

Peng

2004

Anal. Chem.

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Using a three-dimensional flow control concept to manipulate and retain a single yeast cell in a microchip, we were able to study the kinetics of intracellular metabolism and calcium mobilization at the single-cell level, as stimulated by glucose and pH changes. As a model study, the fluorogenic substrate fluorescein diacetate (FDA) was chosen to study how the intracellular carboxylesterase metabolize it. A single yeast cell was first cultured in the microchip. Thereafter, under a constant concentration of FDA, influx of FDA into the yeast cell occurred and FDA was hydrolyzed or metabolized. It was found that changes in both pH and glucose stimulated the FDA metabolism in a yeast cell, and the stimuli can elicit multiple responses from the cell. Since it was carried out within the microchip, the whole experiment on one single yeast cell could last for as long as 10 h. The dormant cell, budding cell, and pretreated budding cell (in low-pH buffer) of yeast resulted in different responses. Experimental data provided details on the FDA metabolism at the single-cell level and revealed strong correlations between FDA metabolism and calcium mobilization. Furthermore, efflux of the FDA metabolite fluorescein could start spontaneously if there was glucose in the medium. The experiments on a single cell were of the "human cell conservation" style because the cell responded to the reagent changes implemented by the human researcher. A mathematical model was also developed to study the influx-hydrolysis-efflux processes of the FDA metabolism using single-cell fluorescent data. These long overdue single-cell experiments are now rendered possible using the three-dimensional flow control in the microchip.

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“…However, the accumulation of the fluorescent product in single yeast cells does not follow Michaelis-Menten kinetics. Diffusion limitation rather than enzyme activity may determine the level of single-cell fluorescence [206,207]. To quantitate /3galactosidase activity in Escherichia coli cells, Nit et al [135] trapped individual cells in agarose microbeads, let them form a microcolony, and measured the enzymatic activity of the whole colony by FCM after a vital permeabilization step and the use of the chromogenic substrate fluorescein di-/3-Dgalactopyranoside.…”

Section: Measurement Of Cell Physiological Activities: a Promising Nementioning

confidence: 99%

Recent advances of flow cytometry in fundamental and applied microbiology

Fouchet

Jayat

Héchard³

et al. 1993

Biology of the Cell

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This review focuses on the recent applications of flow cytometry (FCM) in microbiological research (1987-mid 1992). It tries to give a scope of the important breakthroughs which occurred in this field during this period. The technical difficulties of microorganism analysis by flow cytometry is briefly appraised. The significance and the limits of the different microbial cell parameters attainable by flow analyses are systematically evaluated: light scatter for cell size and structure, fluorescence measurements for quantification of cellular components, microbial antigen detection and cell physiological activity estimation. Emphasis is given on the new technological advances which appeared in the last two years. The second part of the review is devoted to the analysis of the usefulness of flow cytometric approach in the different fields of microbiology: fundamental studies in microbial physiology, differentiation, microbial ecology and aquatic sciences, medical microbiology, parasitology, microbial pharmacology and biotechnology.

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Mathematical modeling of a single‐cell enzyme assay

Cited by 9 publications

References 22 publications

Programmable bacteria detect and record an environmental signal in the mammalian gut

Programmable bacteria detect and record an environmental signal in the mammalian gut

A Three-Dimensional Flow Control Concept for Single-Cell Experiments on a Microchip. 2. Fluorescein Diacetate Metabolism and Calcium Mobilization in a Single Yeast Cell As Stimulated by Glucose and pH Changes

Recent advances of flow cytometry in fundamental and applied microbiology

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