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Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries.

ABSTRACT: The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene expression, while the other approach uses the lambda phage transcription anti-termination protein N to limit transcription termination. A metagenomic library was constructed and functionally screened to identify genes conferring carbenicillin resistance to E. coli. The use of these enhanced expression systems resulted in a 6-fold increase in the frequency of carbenicillin resistant clones. Subcloning and sequence analysis showed that, besides ?-lactamases, efflux pumps are not only able contribute to carbenicillin resistance but may in fact be sufficient by themselves to convey carbenicillin resistance.

SUBMITTER: Terron-Gonzalez L

PROVIDER: S-EPMC3551230 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries.

Terrón-González L L Medina C C Limón-Mortés M C MC Santero E E

Scientific reports 20130122

The extraordinary potential of metagenomic functional analyses to identify activities of interest present in uncultured microorganisms has been limited by reduced gene expression in surrogate hosts. We have developed vectors and specialized E. coli strains as improved metagenomic DNA heterologous expression systems, taking advantage of viral components that prevent transcription termination at metagenomic terminators. One of the systems uses the phage T7 RNA-polymerase to drive metagenomic gene ...[more]

PMID: 23346364

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Project description:The in vitro selection of nucleic acid libraries has driven the discovery of RNA and DNA receptors (aptamers) and catalysts with tailor-made functional properties. Functional nucleic acids emerging from selections have been observed to possess an unusually high degree of secondary structure. In this study, we experimentally examined the relationship between the degree of secondary structure in a nucleic acid library and its ability to yield aptamers that bind protein targets. We designed a patterned nucleic acid library (denoted R*Y*) to enhance the formation of stem-loop structures without imposing any specific sequence or secondary structural requirement. This patterned library was predicted computationally to contain a significantly higher average folding energy compared to a standard, unpatterned N(60) library of the same length. We performed three different iterated selections for protein binding using patterned and unpatterned libraries competing in the same solution. In all three cases, the patterned R*Y* library was enriched relative to the unpatterned library over the course of the 9- to 10-round selection. Characterization of individual aptamer clones emerging from the three selections revealed that the highest affinity aptamer assayed arose from the patterned library for two protein targets, while in the third case, the highest affinity aptamers from the patterned and random libraries exhibited comparable affinity. We identified the binding motif requirements for the most active aptamers generated against two of the targets. The two binding motifs are 3.4- and 27-fold more likely to occur in the R*Y* library than in the N(60) library. Collectively, our findings suggest that researchers performing selections for nucleic acid aptamers and catalysts should consider patterned libraries rather than commonly used N(m) libraries to increase both the likelihood of isolating functional molecules and the potential activities of the resulting molecules.

Dataset Information

Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries.

Publications

Heterologous viral expression systems in fosmid vectors increase the functional analysis potential of metagenomic libraries.

Similar Datasets

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