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A multi-platform flow device for microbial (co-) cultivation and microscopic analysis.

Hesselman MC, Odoni DI, Ryback BM, de Groot S, van Heck RG, Keijsers J, Kolkman P, Nieuwenhuijse D, van Nuland YM, Sebus E, Spee R, de Vries H, Wapenaar MT, Ingham CJ, Schroën K, Martins dos Santos VA, Spaans SK, Hugenholtz F, van Passel MW - PLoS ONE (2012)

Bottom Line: The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups.Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish.The device was designed to be affordable, reusable, and above all, versatile.

View Article: PubMed Central - PubMed

Affiliation: Wageningen UR iGEM 2011 Team, Wageningen University, Wageningen, The Netherlands.

ABSTRACT
Novel microbial cultivation platforms are of increasing interest to researchers in academia and industry. The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups. Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish. It provides control over (co-)culturing conditions similar to a chemostat, while allowing organisms to be observed microscopically. The device was designed to be affordable, reusable, and above all, versatile. To test its functionality and general utility, we performed multiple experiments with Escherichia coli cells harboring synthetic gene circuits and were able to quantitatively study emerging expression dynamics in real-time via fluorescence microscopy. Furthermore, we demonstrated that the device provides a unique environment for the cultivation of nematodes, suggesting that the device could also prove useful in microscopy studies of multicellular microorganisms.

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Related in: MedlinePlus

Oscillating GFP expression observed in microdish wells.Fluorescent E. coli cells in the wells of the microdish showing variations in signal strength over time. The graph depicts variations plotted with the image analysis and processing tool ImageJ. The x-axis represents time, and the y-axis represents fluorescence (in arbitrary units and with a variance of maximally 0.01 for the normalised data of 5 wells). Below the graph are microscopic images of fluorescent bacteria in the cultivation chip wells at different intervals using identical illumination conditions and CCD camera exposure times. The time points at which the images were taken are indicated with an asterisk.
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pone-0036982-g003: Oscillating GFP expression observed in microdish wells.Fluorescent E. coli cells in the wells of the microdish showing variations in signal strength over time. The graph depicts variations plotted with the image analysis and processing tool ImageJ. The x-axis represents time, and the y-axis represents fluorescence (in arbitrary units and with a variance of maximally 0.01 for the normalised data of 5 wells). Below the graph are microscopic images of fluorescent bacteria in the cultivation chip wells at different intervals using identical illumination conditions and CCD camera exposure times. The time points at which the images were taken are indicated with an asterisk.

Mentions: Certain intercellular signalling networks consisting of regulatory feedback loops can result in synchronized oscillatory gene expression. However, the emergence of such phenomena is generally not very robust, and often contingent on specific environmental parameters. Temporal variations in GFP expression produced by E. coli microcolonies harboring such a signalling construct were detected using fluorescence microscopy. The averaged and normalized fluorescent emission intensities plotted against time clearly depict synchronized oscillatory behavior with a period of approximately 1 h (Figure 3). Since the aim of this experiment was to detect relative changes in fluorescence intensities over time in order to confirm the oscillatory properties of the construct rather than measurement of absolute GFP expression values, it was not necessary to normalize the measured intensities against the cell density.


A multi-platform flow device for microbial (co-) cultivation and microscopic analysis.

Hesselman MC, Odoni DI, Ryback BM, de Groot S, van Heck RG, Keijsers J, Kolkman P, Nieuwenhuijse D, van Nuland YM, Sebus E, Spee R, de Vries H, Wapenaar MT, Ingham CJ, Schroën K, Martins dos Santos VA, Spaans SK, Hugenholtz F, van Passel MW - PLoS ONE (2012)

Oscillating GFP expression observed in microdish wells.Fluorescent E. coli cells in the wells of the microdish showing variations in signal strength over time. The graph depicts variations plotted with the image analysis and processing tool ImageJ. The x-axis represents time, and the y-axis represents fluorescence (in arbitrary units and with a variance of maximally 0.01 for the normalised data of 5 wells). Below the graph are microscopic images of fluorescent bacteria in the cultivation chip wells at different intervals using identical illumination conditions and CCD camera exposure times. The time points at which the images were taken are indicated with an asterisk.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351485&req=5

pone-0036982-g003: Oscillating GFP expression observed in microdish wells.Fluorescent E. coli cells in the wells of the microdish showing variations in signal strength over time. The graph depicts variations plotted with the image analysis and processing tool ImageJ. The x-axis represents time, and the y-axis represents fluorescence (in arbitrary units and with a variance of maximally 0.01 for the normalised data of 5 wells). Below the graph are microscopic images of fluorescent bacteria in the cultivation chip wells at different intervals using identical illumination conditions and CCD camera exposure times. The time points at which the images were taken are indicated with an asterisk.
Mentions: Certain intercellular signalling networks consisting of regulatory feedback loops can result in synchronized oscillatory gene expression. However, the emergence of such phenomena is generally not very robust, and often contingent on specific environmental parameters. Temporal variations in GFP expression produced by E. coli microcolonies harboring such a signalling construct were detected using fluorescence microscopy. The averaged and normalized fluorescent emission intensities plotted against time clearly depict synchronized oscillatory behavior with a period of approximately 1 h (Figure 3). Since the aim of this experiment was to detect relative changes in fluorescence intensities over time in order to confirm the oscillatory properties of the construct rather than measurement of absolute GFP expression values, it was not necessary to normalize the measured intensities against the cell density.

Bottom Line: The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups.Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish.The device was designed to be affordable, reusable, and above all, versatile.

View Article: PubMed Central - PubMed

Affiliation: Wageningen UR iGEM 2011 Team, Wageningen University, Wageningen, The Netherlands.

ABSTRACT
Novel microbial cultivation platforms are of increasing interest to researchers in academia and industry. The development of materials with specialized chemical and geometric properties has opened up new possibilities in the study of previously unculturable microorganisms and has facilitated the design of elegant, high-throughput experimental set-ups. Within the context of the international Genetically Engineered Machine (iGEM) competition, we set out to design, manufacture, and implement a flow device that can accommodate multiple growth platforms, that is, a silicon nitride based microsieve and a porous aluminium oxide based microdish. It provides control over (co-)culturing conditions similar to a chemostat, while allowing organisms to be observed microscopically. The device was designed to be affordable, reusable, and above all, versatile. To test its functionality and general utility, we performed multiple experiments with Escherichia coli cells harboring synthetic gene circuits and were able to quantitatively study emerging expression dynamics in real-time via fluorescence microscopy. Furthermore, we demonstrated that the device provides a unique environment for the cultivation of nematodes, suggesting that the device could also prove useful in microscopy studies of multicellular microorganisms.

Show MeSH
Related in: MedlinePlus