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  • Review Article
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Using movies to analyse gene circuit dynamics in single cells

Nature Reviews Microbiology volume 7, pages 383–392 (2009)Cite this article

Key Points

  • Movies, acquired by time-lapse microscopy, provide a powerful way to analyse the dynamics of genetic circuits at the level of individual cells.

  • The combination of time-lapse microscopy, quantitative image analysis and fluorescent protein reporters has enabled direct observation of various cellular components over time in individual cells. In conjunction with mathematical modelling, these techniques are now providing powerful insights into genetic circuit behaviour in diverse microbial systems.

  • Movies have proved crucial for our understanding of how phenotypic variability is generated in clonal populations, through noise in gene expression, heritability of cellular states, ageing and other mechanisms.

  • Movies allow researchers to analyse the dynamics of biochemical events, such as the production of mRNA and protein, down to the level of individual molecules.

  • Using strains engineered to produce fluorescent protein reporters for key genetic circuit components, researchers are now able to analyse the genetic circuits that give rise to differentiation and stress responses at the single-cell level.

Abstract

Many bacterial systems rely on dynamic genetic circuits to control crucial biological processes. A major goal of systems biology is to understand these behaviours in terms of individual genes and their interactions. However, traditional techniques based on population averages 'wash out' crucial dynamics that are either unsynchronized between cells or are driven by fluctuations, or 'noise', in cellular components. Recently, the combination of time-lapse microscopy, quantitative image analysis and fluorescent protein reporters has enabled direct observation of multiple cellular components over time in individual cells. In conjunction with mathematical modelling, these techniques are now providing powerful insights into genetic circuit behaviour in diverse microbial systems.

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Figure 1: circuit-driven versus noisy cells.
Figure 2: Tracking and segmenting single cells.
Figure 3: Automated lineage analysis reveals epigenetic states.
Figure 4: In vivo biochemistry.
Figure 5: Circuit-level dynamics.

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Acknowledgements

We thank A. Eldar, J. Young, L. Cai, C. Dalal and all members of the Elowitz groups for their feedback and suggestions. J.L. was supported by a Human Frontiers Fellowship. This work was supported by US National Institutes of Health grants R01GM079771 and P50 GM068763, National Science Foundation CAREER Award 0644463 and the Packard Foundation.

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Authors and Affiliations

  1. Division of Biology and Department of Applied Physics, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, 91125, California, USA

    James C. W. Locke & Michael B. Elowitz

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Correspondence to Michael B. Elowitz.

Supplementary information

Supplementary information S1 (movie) | Analysis of gene regulation in Escherichia coli.

This movie shows the dilution of a fusion protein composed of lambda repressor and yellow fluorescence protein (YFP) domains (red) in cells that also express cyan fluorescent protein (CFP; green) from a lambda–regulated promoter. As the cell grows into a micro–colony, the repressor is diluted and CFP expression increases. (MOV 190 kb)

Supplementary information S2 (movie) | Analysis of the competence differentiation circuit in Bacillus subtilis.

This movie shows a micro–colony of B. subtilis cells growing vegetatively (green cells), sporulating (to produce white phase–bright spores) and occasionally switching to the competent state (red cell). Here, one fluorescent protein, labelled green, reports expression of comS, a gene necessary, but not sufficient, for competence. The other fluorescent protein, labelled red, is activated by ComK during competence. (MOV 3175 kb)

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DATABASES

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Glossary

Noise

Fluctuations in molecular components. Noise arises owing to the low copy numbers of molecular species and the burst-like nature of transcription, among other mechanisms.

Segmentation

Breaking up a complex image into individual objects, such as cells.

Edge detection

A computational algorithm that identifies sharp changes in intensity associated with boundaries between objects, such as cells.

Thresholding

One of the simplest segmentation techniques, in which groups of pixels for which the intensity exceeds a defined cut-off value are identified.

Hough transform

An algorithm for identifying particular shapes, such as circular disks, in complex images. The Hough transform is useful in many segmentation systems.

Linear microfluidic chamber

A microfluidic device in which cells are confined to grow in a narrow groove. These devices facilitate analysis of cell lineages, as more closely related cells are located closer together.

Galactose utilization system

A system of genes used by yeast to control the uptake and metabolism of galactose. This system is characterized by several feedback loops, both positive and negative.

Repressilator

A synthetic genetic circuit designed to produce clock-like oscillations in the levels of its components. The circuit consists of a 'rock–scissors–paper' feedback loop of three repressors, in which the first represses the expression of the second, the second represses the expression of the third and the third represses the expression of the first.

Spo0A

The master transcriptional regulator for sporulation in Bacillus subtilis. Spo0A is controlled by phosphorylation and transcriptional regulation.

Frequency modulation

A way to encode information about the frequency of events or oscillations. Frequency modulation is often contrasted with amplitude modulation, in which signals are encoded by varying the magnitude of a signal. In engineering applications, such as broadcasting, the frequency-modulated signal is typically periodic (oscillatory). In the example of Crz1 it is the frequency of discrete stochastically timed bursts that is varied.

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Locke, J., Elowitz, M. Using movies to analyse gene circuit dynamics in single cells. Nat Rev Microbiol 7, 383–392 (2009). https://doi.org/10.1038/nrmicro2056

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