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Interplay of metagenomics and in vitro compartmentalization.

Ferrer M, Beloqui A, Vieites JM, Guazzaroni ME, Berger I, Aharoni A - Microb Biotechnol (2008)

Bottom Line: In recent years, the application of approaches for harvesting DNA from the environment, the so-called, 'metagenomic approaches' has proven to be highly successful for the identification, isolation and generation of novel enzymes.Functional screening for the desired catalytic activity is one of the key steps in mining metagenomic libraries, as it does not rely on sequence homology.In particular, we focus on the use of in vitro compartmentalization (IVC) approaches to address potential advantages and problems the merger of culture-independent and IVC techniques might bring on the mining of enzyme activities in microbial communities.

View Article: PubMed Central - PubMed

Affiliation: CSIC, Institute of Catalysis, Department of Applied Biocatalysis, Madrid, Spain. mferrer@icp.csic.es

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Selections by FACS sorting of double emulsion droplets. A gene library is transformed into bacteria, and the encoded proteins are expressed in the cytoplasm, the periplasm, or on the surface of the cells (Step 1). The bacteria are dispersed to form a water‐in‐oil (w/o) emulsion, with typically one cell per aqueous microdroplet. Alternatively, an in vitro transcription/translation reaction mixture containing a library of genes is dispersed to form a w/o emulsion with typically one gene per aqueous microdroplet. The genes are transcribed and translated within the microdroplets (Step 2). Proteins with enzymatic activity convert the non‐fluorescent substrate into a fluorescent product and the w/o emulsion is converted into a water‐in‐oil‐in‐water (w/o/w) emulsion (Step 3). Fluorescent microdroplets are separated from non‐fluorescent microdroplets using a fluorescence activated cell sorter (FACS) (Step 4). Bacteria or genes from fluorescent microdroplets which encode active enzymes are recovered and the bacteria are propagated or the DNA is amplified using the polymerase chain reaction. These bacteria or genes can then be re‐compartmentalized for further rounds of selection.
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f3: Selections by FACS sorting of double emulsion droplets. A gene library is transformed into bacteria, and the encoded proteins are expressed in the cytoplasm, the periplasm, or on the surface of the cells (Step 1). The bacteria are dispersed to form a water‐in‐oil (w/o) emulsion, with typically one cell per aqueous microdroplet. Alternatively, an in vitro transcription/translation reaction mixture containing a library of genes is dispersed to form a w/o emulsion with typically one gene per aqueous microdroplet. The genes are transcribed and translated within the microdroplets (Step 2). Proteins with enzymatic activity convert the non‐fluorescent substrate into a fluorescent product and the w/o emulsion is converted into a water‐in‐oil‐in‐water (w/o/w) emulsion (Step 3). Fluorescent microdroplets are separated from non‐fluorescent microdroplets using a fluorescence activated cell sorter (FACS) (Step 4). Bacteria or genes from fluorescent microdroplets which encode active enzymes are recovered and the bacteria are propagated or the DNA is amplified using the polymerase chain reaction. These bacteria or genes can then be re‐compartmentalized for further rounds of selection.

Mentions: In vitro compartmentalization is based on water‐in‐oil emulsions, where the water phase is dispersed in the oil phase to form microscopic aqueous compartments. Each droplet contains, on average, a single gene, and serves as an artificial cell allowing for transcription, translation and the activity of the resulting proteins, to take place within the compartment. The oil phase remains largely inert and restricts the diffusion of genes and proteins between compartments (Fig. 3). The droplet volume (∼5 femtoliter) enables a single DNA molecule to be transcribed and translated (Griffiths and Tawfik, 2006), as well as the detection of single enzyme molecules (Griffiths and Tawfik, 2003). The high capacity of the system (> 1010 in 1 ml of emulsion), the ease of preparing emulsions and their high stability over a broad range of temperatures, render IVC an attractive system for HTS of enzymes, as well as for many other HT genetic and genomic manipulations (for a recent review, see Griffiths and Tawfik, 2006).


Interplay of metagenomics and in vitro compartmentalization.

Ferrer M, Beloqui A, Vieites JM, Guazzaroni ME, Berger I, Aharoni A - Microb Biotechnol (2008)

Selections by FACS sorting of double emulsion droplets. A gene library is transformed into bacteria, and the encoded proteins are expressed in the cytoplasm, the periplasm, or on the surface of the cells (Step 1). The bacteria are dispersed to form a water‐in‐oil (w/o) emulsion, with typically one cell per aqueous microdroplet. Alternatively, an in vitro transcription/translation reaction mixture containing a library of genes is dispersed to form a w/o emulsion with typically one gene per aqueous microdroplet. The genes are transcribed and translated within the microdroplets (Step 2). Proteins with enzymatic activity convert the non‐fluorescent substrate into a fluorescent product and the w/o emulsion is converted into a water‐in‐oil‐in‐water (w/o/w) emulsion (Step 3). Fluorescent microdroplets are separated from non‐fluorescent microdroplets using a fluorescence activated cell sorter (FACS) (Step 4). Bacteria or genes from fluorescent microdroplets which encode active enzymes are recovered and the bacteria are propagated or the DNA is amplified using the polymerase chain reaction. These bacteria or genes can then be re‐compartmentalized for further rounds of selection.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815420&req=5

f3: Selections by FACS sorting of double emulsion droplets. A gene library is transformed into bacteria, and the encoded proteins are expressed in the cytoplasm, the periplasm, or on the surface of the cells (Step 1). The bacteria are dispersed to form a water‐in‐oil (w/o) emulsion, with typically one cell per aqueous microdroplet. Alternatively, an in vitro transcription/translation reaction mixture containing a library of genes is dispersed to form a w/o emulsion with typically one gene per aqueous microdroplet. The genes are transcribed and translated within the microdroplets (Step 2). Proteins with enzymatic activity convert the non‐fluorescent substrate into a fluorescent product and the w/o emulsion is converted into a water‐in‐oil‐in‐water (w/o/w) emulsion (Step 3). Fluorescent microdroplets are separated from non‐fluorescent microdroplets using a fluorescence activated cell sorter (FACS) (Step 4). Bacteria or genes from fluorescent microdroplets which encode active enzymes are recovered and the bacteria are propagated or the DNA is amplified using the polymerase chain reaction. These bacteria or genes can then be re‐compartmentalized for further rounds of selection.
Mentions: In vitro compartmentalization is based on water‐in‐oil emulsions, where the water phase is dispersed in the oil phase to form microscopic aqueous compartments. Each droplet contains, on average, a single gene, and serves as an artificial cell allowing for transcription, translation and the activity of the resulting proteins, to take place within the compartment. The oil phase remains largely inert and restricts the diffusion of genes and proteins between compartments (Fig. 3). The droplet volume (∼5 femtoliter) enables a single DNA molecule to be transcribed and translated (Griffiths and Tawfik, 2006), as well as the detection of single enzyme molecules (Griffiths and Tawfik, 2003). The high capacity of the system (> 1010 in 1 ml of emulsion), the ease of preparing emulsions and their high stability over a broad range of temperatures, render IVC an attractive system for HTS of enzymes, as well as for many other HT genetic and genomic manipulations (for a recent review, see Griffiths and Tawfik, 2006).

Bottom Line: In recent years, the application of approaches for harvesting DNA from the environment, the so-called, 'metagenomic approaches' has proven to be highly successful for the identification, isolation and generation of novel enzymes.Functional screening for the desired catalytic activity is one of the key steps in mining metagenomic libraries, as it does not rely on sequence homology.In particular, we focus on the use of in vitro compartmentalization (IVC) approaches to address potential advantages and problems the merger of culture-independent and IVC techniques might bring on the mining of enzyme activities in microbial communities.

View Article: PubMed Central - PubMed

Affiliation: CSIC, Institute of Catalysis, Department of Applied Biocatalysis, Madrid, Spain. mferrer@icp.csic.es

Show MeSH
Related in: MedlinePlus