Limits...
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.

Show MeSH

Related in: MedlinePlus

Co-cultivation of cells in the flow device.Expression of GFP increases over time in cells growing in microdish wells after adding inducer (Acyl homoserine lactone (AHL) producing) and RFP expressing cells to the bottom compartment of the flow device. The intensity of the light emitted from five wells was quantified using imageJ and normalized against the background. No RFP was detected in the top chamber, indicating that the inducer-cells added to the bottom chamber did not come in contact with the top, and GFP expression was induced by diffusion of the inducer (AHL) through the microdish.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3351485&req=5

pone-0036982-g005: Co-cultivation of cells in the flow device.Expression of GFP increases over time in cells growing in microdish wells after adding inducer (Acyl homoserine lactone (AHL) producing) and RFP expressing cells to the bottom compartment of the flow device. The intensity of the light emitted from five wells was quantified using imageJ and normalized against the background. No RFP was detected in the top chamber, indicating that the inducer-cells added to the bottom chamber did not come in contact with the top, and GFP expression was induced by diffusion of the inducer (AHL) through the microdish.

Mentions: Initial measurements of the inducible cells in the top compartment showed hardly detectable amounts of basally expressed GFP. After addition of the inducer culture in the lower compartment, a rapid increase of GFP expression was observed, both in the microsieve (Figure 4) and the microdish (Figure 5). The observed increase was too rapid to be the result of cell growth alone. This can be concluded from the observation that cells constitutively expressing GFP do not display increases in fluorescence of a comparable rate and magnitude (data not shown). In order to validate the barrier function of the two platforms, the suspension of inducer cells was spiked with E. coli cells expressing RFP. Additionally to the GFP measurements, the top compartment was monitored for presence of RFP. Leakage was not observed, indicating that the edges of the respective growing platforms were sealed properly.


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)

Co-cultivation of cells in the flow device.Expression of GFP increases over time in cells growing in microdish wells after adding inducer (Acyl homoserine lactone (AHL) producing) and RFP expressing cells to the bottom compartment of the flow device. The intensity of the light emitted from five wells was quantified using imageJ and normalized against the background. No RFP was detected in the top chamber, indicating that the inducer-cells added to the bottom chamber did not come in contact with the top, and GFP expression was induced by diffusion of the inducer (AHL) through the microdish.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0036982-g005: Co-cultivation of cells in the flow device.Expression of GFP increases over time in cells growing in microdish wells after adding inducer (Acyl homoserine lactone (AHL) producing) and RFP expressing cells to the bottom compartment of the flow device. The intensity of the light emitted from five wells was quantified using imageJ and normalized against the background. No RFP was detected in the top chamber, indicating that the inducer-cells added to the bottom chamber did not come in contact with the top, and GFP expression was induced by diffusion of the inducer (AHL) through the microdish.
Mentions: Initial measurements of the inducible cells in the top compartment showed hardly detectable amounts of basally expressed GFP. After addition of the inducer culture in the lower compartment, a rapid increase of GFP expression was observed, both in the microsieve (Figure 4) and the microdish (Figure 5). The observed increase was too rapid to be the result of cell growth alone. This can be concluded from the observation that cells constitutively expressing GFP do not display increases in fluorescence of a comparable rate and magnitude (data not shown). In order to validate the barrier function of the two platforms, the suspension of inducer cells was spiked with E. coli cells expressing RFP. Additionally to the GFP measurements, the top compartment was monitored for presence of RFP. Leakage was not observed, indicating that the edges of the respective growing platforms were sealed properly.

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