Quantifying the accessibility of the metagenome by random expression cloning techniques
Esther M Gabor, Wynand B L Alkema, Dick B Janssen
Data Science for Life Science and health
The exploitation of the metagenome for novel biocatalysts by functional screening is determined by the ability to express the respective genes in a surrogate host. The probability of recovering a certain gene thereby depends on its abundance in the environmental DNA used for library construction, the chosen insert size, the length of the target gene, and the presence of expression signals that are functional in the host organism. In this paper, we present a set of formulas that describe the chance of isolating a gene by random expression cloning, taking into account the three different modes of heterologous gene expression: independent expression, expression as a transcriptional fusion and expression as a translational fusion. Genes of the last category are shown to be virtually inaccessible by shotgun cloning because of the low frequency of functional constructs. To evaluate which part of the metagenome might in this way evade exploitation, 32 complete genome sequences of prokaryotic organisms were analysed for the presence of expression signals functional in E. coli hosts, using bioinformatics tools. Our study reveals significant differences in the predicted expression modes between distinct taxonomic groups of organisms and suggests that about 40% of the enzymatic activities may be readily recovered by random cloning in E. coli.
Published - 11 Aug 2004
bacteria/enzymology
cloning, molecular/methods
computational biology/methods
escherichia coli/metabolism
gene library
genes/genetics
genomics/methods
species specificity
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Metagenome Medicine & Life Sciences
cloning Earth & Environmental Sciences
accessibility Earth & Environmental Sciences
Organism Cloning Medicine & Life Sciences
gene Earth & Environmental Sciences
molecular cloning Agriculture & Biology
Escherichia coli Medicine & Life Sciences
Gabor, E. M., Alkema, W. B. L., & Janssen, D. B. (2004). Quantifying the accessibility of the metagenome by random expression cloning techniques. Applied and environmental microbiology, 6(9), 879-986. https://doi.org/10.1111/j.1462-2920.2004.00640.x
Gabor, Esther M ; Alkema, Wynand B L ; Janssen, Dick B. / Quantifying the accessibility of the metagenome by random expression cloning techniques. In: Applied and environmental microbiology. 2004 ; Vol. 6, No. 9. pp. 879-986.
@article{d470b89efa6b4ccda792c678e4f20e25,
title = "Quantifying the accessibility of the metagenome by random expression cloning techniques",
abstract = "The exploitation of the metagenome for novel biocatalysts by functional screening is determined by the ability to express the respective genes in a surrogate host. The probability of recovering a certain gene thereby depends on its abundance in the environmental DNA used for library construction, the chosen insert size, the length of the target gene, and the presence of expression signals that are functional in the host organism. In this paper, we present a set of formulas that describe the chance of isolating a gene by random expression cloning, taking into account the three different modes of heterologous gene expression: independent expression, expression as a transcriptional fusion and expression as a translational fusion. Genes of the last category are shown to be virtually inaccessible by shotgun cloning because of the low frequency of functional constructs. To evaluate which part of the metagenome might in this way evade exploitation, 32 complete genome sequences of prokaryotic organisms were analysed for the presence of expression signals functional in E. coli hosts, using bioinformatics tools. Our study reveals significant differences in the predicted expression modes between distinct taxonomic groups of organisms and suggests that about 40% of the enzymatic activities may be readily recovered by random cloning in E. coli.",
keywords = "bacteria/enzymology, cloning, molecular/methods, computational biology/methods, escherichia coli/metabolism, gene expression, gene library, genes/genetics, genomics/methods, species specificity, microbiologie",
author = "Gabor, {Esther M} and Alkema, {Wynand B L} and Janssen, {Dick B}",
month = aug
journal = "Applied and environmental microbiology",
publisher = "American Society for Microbiology",
Gabor, EM, Alkema, WBL & Janssen, DB 2004, 'Quantifying the accessibility of the metagenome by random expression cloning techniques', Applied and environmental microbiology, vol. 6, no. 9, pp. 879-986. https://doi.org/10.1111/j.1462-2920.2004.00640.x
Quantifying the accessibility of the metagenome by random expression cloning techniques. / Gabor, Esther M; Alkema, Wynand B L; Janssen, Dick B.
In: Applied and environmental microbiology, Vol. 6, No. 9, 11.08.2004, p. 879-986.
T1 - Quantifying the accessibility of the metagenome by random expression cloning techniques
AU - Gabor, Esther M
AU - Alkema, Wynand B L
AU - Janssen, Dick B
N2 - The exploitation of the metagenome for novel biocatalysts by functional screening is determined by the ability to express the respective genes in a surrogate host. The probability of recovering a certain gene thereby depends on its abundance in the environmental DNA used for library construction, the chosen insert size, the length of the target gene, and the presence of expression signals that are functional in the host organism. In this paper, we present a set of formulas that describe the chance of isolating a gene by random expression cloning, taking into account the three different modes of heterologous gene expression: independent expression, expression as a transcriptional fusion and expression as a translational fusion. Genes of the last category are shown to be virtually inaccessible by shotgun cloning because of the low frequency of functional constructs. To evaluate which part of the metagenome might in this way evade exploitation, 32 complete genome sequences of prokaryotic organisms were analysed for the presence of expression signals functional in E. coli hosts, using bioinformatics tools. Our study reveals significant differences in the predicted expression modes between distinct taxonomic groups of organisms and suggests that about 40% of the enzymatic activities may be readily recovered by random cloning in E. coli.
AB - The exploitation of the metagenome for novel biocatalysts by functional screening is determined by the ability to express the respective genes in a surrogate host. The probability of recovering a certain gene thereby depends on its abundance in the environmental DNA used for library construction, the chosen insert size, the length of the target gene, and the presence of expression signals that are functional in the host organism. In this paper, we present a set of formulas that describe the chance of isolating a gene by random expression cloning, taking into account the three different modes of heterologous gene expression: independent expression, expression as a transcriptional fusion and expression as a translational fusion. Genes of the last category are shown to be virtually inaccessible by shotgun cloning because of the low frequency of functional constructs. To evaluate which part of the metagenome might in this way evade exploitation, 32 complete genome sequences of prokaryotic organisms were analysed for the presence of expression signals functional in E. coli hosts, using bioinformatics tools. Our study reveals significant differences in the predicted expression modes between distinct taxonomic groups of organisms and suggests that about 40% of the enzymatic activities may be readily recovered by random cloning in E. coli.
KW - bacteria/enzymology
KW - cloning, molecular/methods
KW - computational biology/methods
KW - escherichia coli/metabolism
KW - gene library
KW - genes/genetics
KW - genomics/methods
KW - species specificity
KW - microbiologie
JO - Applied and environmental microbiology
JF - Applied and environmental microbiology
Gabor EM, Alkema WBL, Janssen DB. Quantifying the accessibility of the metagenome by random expression cloning techniques. Applied and environmental microbiology. 2004 Aug 11;6(9):879-986. https://doi.org/10.1111/j.1462-2920.2004.00640.x