Journal: bioRxiv

Article Title: Functional metagenomic discovery of novel tetracycline and acarbose resistance genes from low biomass samples using METa assembly

doi: 10.1101/2024.06.29.601325

Figure Lengend Snippet: A) General steps in the preparation and application of a functional metagenomic library: 1) Extraction of metagenomic DNA from a source microbiome. 2) Fragmentation of metagenomic DNA to preferred size range. 3) Packaging of inserts into expression vectors. 4) Transformation of host cells with vector library. 5) Screen or selection of functional metagenomic library for a phenotype of interest. 6) Collection of screened or selected metagenomic fragments. 7) Sequencing of selected inserts. 8) Open reading frame calling and annotation to identify potential genes underlying phenotypes of interest. B) Steps 2) and 3) above are modified in Mosaic ends tagmentation (METa) assembly. Fragmentation is achieved using Tn5 transposase tagmentation with mosaic end sequence oligos. Tagmented DNA is gap filled by polymerase and cloned into an expression vector with matching mosaic end sequences defining the cloning site using assembly cloning ( e.g., Gibson assembly). C) and D) The chemical structures of the antibiotic C) tetracycline and the diabetes pharmaceutical D) acarbose.

Article Snippet: An aliquot of the ZymoBIOMICS fecal reference standard (Zymo Research, D6323) was used as a model source for a fecal microbiome swab functional metagenomic library.

Techniques: Functional Assay, Extraction, Expressing, Transformation Assay, Plasmid Preparation, Selection, Sequencing, Modification, Clone Assay

Journal: bioRxiv

Article Title: Functional metagenomic discovery of novel tetracycline and acarbose resistance genes from low biomass samples using METa assembly

doi: 10.1101/2024.06.29.601325

Figure Lengend Snippet:

Article Snippet: An aliquot of the ZymoBIOMICS fecal reference standard (Zymo Research, D6323) was used as a model source for a fecal microbiome swab functional metagenomic library.

Techniques: Functional Assay

Journal: bioRxiv

Article Title: Functional metagenomic discovery of novel tetracycline and acarbose resistance genes from low biomass samples using METa assembly

doi: 10.1101/2024.06.29.601325

Figure Lengend Snippet: A) Gene schematics of the TET1, TET3, and TET13 metagenomic DNA fragments with predicted efflux pumps highlighted (TET1 red, TET3 blue, TET13 gray). Other predicted open reading frame are shown as empty arrows and plasmid backbone markers are highlighted as follows: Promoter (green), mosaic end sequence (orange), terminator (red). B) Maximum likelihood phylogenetic tree of CARD MFS efflux pumps with predicted TET1, TET3, and TET13 amino acid sequences highlighted. Microbroth dilution assays performed un quadruplicate for C) tetracycline or D) chloramphenicol resistance with calculated 50% inhibition concentration (IC 50 ) values graphed as insets. For statistical comparisons: * p <0.05, ** p <0.005, and *** p <0.0005.

Article Snippet: An aliquot of the ZymoBIOMICS fecal reference standard (Zymo Research, D6323) was used as a model source for a fecal microbiome swab functional metagenomic library.

Techniques: Plasmid Preparation, Sequencing, Inhibition, Concentration Assay

Journal: bioRxiv

Article Title: Functional metagenomic discovery of novel tetracycline and acarbose resistance genes from low biomass samples using METa assembly

doi: 10.1101/2024.06.29.601325

Figure Lengend Snippet: A) Gene schematics of the ACA1 and ACA7 metagenomic DNA fragments with the predicted effector genes highlighted (ACA1 red and ACA7 blue). Predicted genes captured on the same DNA fragment are shown as empty arrows. Plasmid backbone elements are: Promoter (green), mosaic end sequence (orange), terminator (red). B) Maximum likelihood phylogenetic tree of the predicted proteins for ACA1 (red) and ACA7 (blue) in the context of known acarbose degrading or modifying enzymes ( i.e., kinases). C) and D) Culture densities with or without addition of 256 μg/ml acarbose after 24 hours of growth in minimal media with C) 0.4% glucose or D) 0.4% maltose as carbon sources. Pairwise statistical comparisons for the effect of acarbose addition were made for each strain and culture condition: * p <0.05, ** p <0.005, and *** p <0.0005.

Article Snippet: An aliquot of the ZymoBIOMICS fecal reference standard (Zymo Research, D6323) was used as a model source for a fecal microbiome swab functional metagenomic library.

Techniques: Plasmid Preparation, Sequencing