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Single gene-based distinction of individual microbial genomes from a mixed population of microbial cells.

Tamminen MV, Virta MP - Front Microbiol (2015)

Bottom Line: The amplified genomes are labeled based on the presence of a target gene and differentiated from those that do not contain the gene by flow cytometry.The method provides a novel tool for enumerating functional cell populations in complex microbial communities.We envision that the method could be optimized for fluorescence-activated cell sorting to enrich genetic material of interest from complex environmental samples.

View Article: PubMed Central - PubMed

Affiliation: Department of Food and Environmental Sciences, University of Helsinki Helsinki, Finland.

ABSTRACT
Recent progress in environmental microbiology has revealed vast populations of microbes in any given habitat that cannot be detected by conventional culturing strategies. The use of sensitive genetic detection methods such as CARD-FISH and in situ PCR have been limited by the cell wall permeabilization requirement that cannot be performed similarly on all cell types without lysing some and leaving some nonpermeabilized. Furthermore, the detection of low copy targets such as genes present in single copies in the microbial genomes, has remained problematic. We describe an emulsion-based procedure to trap individual microbial cells into picoliter-volume polyacrylamide droplets that provide a rigid support for genetic material and therefore allow complete degradation of cellular material to expose the individual genomes. The polyacrylamide droplets are subsequently converted into picoliter-scale reactors for genome amplification. The amplified genomes are labeled based on the presence of a target gene and differentiated from those that do not contain the gene by flow cytometry. Using the Escherichia coli strains XL1 and MC1061, which differ with respect to the presence (XL1), or absence (MC1061) of a single copy of a tetracycline resistance gene per genome, we demonstrate that XL1 genomes present at 0.1% of MC1061 genomes can be differentiated using this method. Using a spiked sediment microbial sample, we demonstrate that the method is applicable to highly complex environmental microbial communities as a target gene-based screen for individual microbes. The method provides a novel tool for enumerating functional cell populations in complex microbial communities. We envision that the method could be optimized for fluorescence-activated cell sorting to enrich genetic material of interest from complex environmental samples.

No MeSH data available.


Related in: MedlinePlus

A procedure to create agarose picoreactors for single-genome amplification. An agarose layer is added onto polyacrylamide droplets that contain individual genomes (Figures 1 and 2). Multiple displacement amplification (MDA) is performed in an emulsion to ensure individual amplification of each genome. Black lines and black-filled circles represent target-less unamplified and amplified genomes, respectively. Red lines and red-filled circles represent target-containing unamplified and amplified genomes, respectively. White-filled circles represent empty picoreactors.
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Figure 3: A procedure to create agarose picoreactors for single-genome amplification. An agarose layer is added onto polyacrylamide droplets that contain individual genomes (Figures 1 and 2). Multiple displacement amplification (MDA) is performed in an emulsion to ensure individual amplification of each genome. Black lines and black-filled circles represent target-less unamplified and amplified genomes, respectively. Red lines and red-filled circles represent target-containing unamplified and amplified genomes, respectively. White-filled circles represent empty picoreactors.

Mentions: In the second part of the technique, an agarose layer is added to the polyacrylamide droplets, which are subsequently dissolved prior to performing MDA reactions (Figure 3). The polyacrylamide must be dissolved because its polymer structure is too dense to allow an efficient MDA reaction. Dissolving the polyacrylamide droplet yields microbial genomes in picoliter liquid volumes within an agarose layer. These agarose picoreactors are mixed with reagents for the MDA reaction and then mixed into an emulsion. Agarose is permeable to enzymes and small molecules but not to genomic or amplified DNA (Figure 4). Because the only DNA in the agarose picoreactors comes from single-genomes trapped in the polyacrylamide droplets, the picoreactors provide a relatively sterile environment for the MDA reaction. The sterility of the reaction is also enhanced by performing the MDA reaction in an emulsion in which each picoreactor occupies an individual reaction compartment. Moreover, the contamination can be further decreased by UV treatment of the MDA reagents (Woyke et al., 2011).


Single gene-based distinction of individual microbial genomes from a mixed population of microbial cells.

Tamminen MV, Virta MP - Front Microbiol (2015)

A procedure to create agarose picoreactors for single-genome amplification. An agarose layer is added onto polyacrylamide droplets that contain individual genomes (Figures 1 and 2). Multiple displacement amplification (MDA) is performed in an emulsion to ensure individual amplification of each genome. Black lines and black-filled circles represent target-less unamplified and amplified genomes, respectively. Red lines and red-filled circles represent target-containing unamplified and amplified genomes, respectively. White-filled circles represent empty picoreactors.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356102&req=5

Figure 3: A procedure to create agarose picoreactors for single-genome amplification. An agarose layer is added onto polyacrylamide droplets that contain individual genomes (Figures 1 and 2). Multiple displacement amplification (MDA) is performed in an emulsion to ensure individual amplification of each genome. Black lines and black-filled circles represent target-less unamplified and amplified genomes, respectively. Red lines and red-filled circles represent target-containing unamplified and amplified genomes, respectively. White-filled circles represent empty picoreactors.
Mentions: In the second part of the technique, an agarose layer is added to the polyacrylamide droplets, which are subsequently dissolved prior to performing MDA reactions (Figure 3). The polyacrylamide must be dissolved because its polymer structure is too dense to allow an efficient MDA reaction. Dissolving the polyacrylamide droplet yields microbial genomes in picoliter liquid volumes within an agarose layer. These agarose picoreactors are mixed with reagents for the MDA reaction and then mixed into an emulsion. Agarose is permeable to enzymes and small molecules but not to genomic or amplified DNA (Figure 4). Because the only DNA in the agarose picoreactors comes from single-genomes trapped in the polyacrylamide droplets, the picoreactors provide a relatively sterile environment for the MDA reaction. The sterility of the reaction is also enhanced by performing the MDA reaction in an emulsion in which each picoreactor occupies an individual reaction compartment. Moreover, the contamination can be further decreased by UV treatment of the MDA reagents (Woyke et al., 2011).

Bottom Line: The amplified genomes are labeled based on the presence of a target gene and differentiated from those that do not contain the gene by flow cytometry.The method provides a novel tool for enumerating functional cell populations in complex microbial communities.We envision that the method could be optimized for fluorescence-activated cell sorting to enrich genetic material of interest from complex environmental samples.

View Article: PubMed Central - PubMed

Affiliation: Department of Food and Environmental Sciences, University of Helsinki Helsinki, Finland.

ABSTRACT
Recent progress in environmental microbiology has revealed vast populations of microbes in any given habitat that cannot be detected by conventional culturing strategies. The use of sensitive genetic detection methods such as CARD-FISH and in situ PCR have been limited by the cell wall permeabilization requirement that cannot be performed similarly on all cell types without lysing some and leaving some nonpermeabilized. Furthermore, the detection of low copy targets such as genes present in single copies in the microbial genomes, has remained problematic. We describe an emulsion-based procedure to trap individual microbial cells into picoliter-volume polyacrylamide droplets that provide a rigid support for genetic material and therefore allow complete degradation of cellular material to expose the individual genomes. The polyacrylamide droplets are subsequently converted into picoliter-scale reactors for genome amplification. The amplified genomes are labeled based on the presence of a target gene and differentiated from those that do not contain the gene by flow cytometry. Using the Escherichia coli strains XL1 and MC1061, which differ with respect to the presence (XL1), or absence (MC1061) of a single copy of a tetracycline resistance gene per genome, we demonstrate that XL1 genomes present at 0.1% of MC1061 genomes can be differentiated using this method. Using a spiked sediment microbial sample, we demonstrate that the method is applicable to highly complex environmental microbial communities as a target gene-based screen for individual microbes. The method provides a novel tool for enumerating functional cell populations in complex microbial communities. We envision that the method could be optimized for fluorescence-activated cell sorting to enrich genetic material of interest from complex environmental samples.

No MeSH data available.


Related in: MedlinePlus