Journal: mSystems

Article Title: Preparation of functional metagenomic libraries from low biomass samples using METa assembly and their application to capture antibiotic resistance genes

doi: 10.1128/msystems.01039-25

Figure Lengend Snippet: Functional metagenomics and METa assembly. (A) The general steps in the preparation and application of a functional metagenomic library consist of the following. (1) Extraction of metagenomic DNA from a source microbiome. (2) Fragmentation of metagenomic DNA to the preferred size range. (3) Packaging of inserts into expression vectors. (4) Transformation of host cells with vector library. (5) Screen or selection of a 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 directly cloned (without amplification) into an expression vector with matching mosaic end sequences defining the cloning site using assembly cloning.

Article Snippet: The ZymoBIOMICS fecal standard, the source of our stool functional metagenomic library input DNA, has been sequenced and annotated for known antibiotic resistance genes by the Zymo corporation, but the data associated with this product do not list any known streptothricin resistance genes.

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

Journal: mSystems

Article Title: Preparation of functional metagenomic libraries from low biomass samples using METa assembly and their application to capture antibiotic resistance genes

doi: 10.1128/msystems.01039-25

Figure Lengend Snippet: Tetracycline selected metagenomic DNA fragments from an aquarium microbiome. (A) Gene schematics of the tetracycline-selected TET1, TET3, and TET13 metagenomic DNA fragments with predicted efflux pumps highlighted (TET1 teal, TET3 red, and TET13 blue). Other predicted open reading frames are shown as empty arrows, and plasmid backbone markers are highlighted as follows: promoter (green), mosaic end sequences (orange), and terminator (red). (B) Maximum likelihood consensus phylogenetic tree of MFS efflux pumps with predicted efflux pump amino acid sequences from TET1 (teal), TET3 (blue), and TET13 (red) fragments highlighted.

Article Snippet: The ZymoBIOMICS fecal standard, the source of our stool functional metagenomic library input DNA, has been sequenced and annotated for known antibiotic resistance genes by the Zymo corporation, but the data associated with this product do not list any known streptothricin resistance genes.

Techniques: Plasmid Preparation

Journal: mSystems

Article Title: Preparation of functional metagenomic libraries from low biomass samples using METa assembly and their application to capture antibiotic resistance genes

doi: 10.1128/msystems.01039-25

Figure Lengend Snippet: Efflux pump containing metagenomic DNA fragments from an aquarium confers tetracycline resistance. Microbroth dilution assay curves and calculated 50% inhibitory concentration (IC50) values for E. coli clones carrying the indicated metagenomic DNA fragments (TET1, TET3, or TET13). (A and B) tetracycline, (C and D) chloramphenicol, or (E and F) azithromycin. **** P < 0.0001, ** P < 0.005, * P < 0.05, n = 4 for all.

Article Snippet: The ZymoBIOMICS fecal standard, the source of our stool functional metagenomic library input DNA, has been sequenced and annotated for known antibiotic resistance genes by the Zymo corporation, but the data associated with this product do not list any known streptothricin resistance genes.

Techniques: Dilution Assay, Concentration Assay, Clone Assay

Journal: mSystems

Article Title: Preparation of functional metagenomic libraries from low biomass samples using METa assembly and their application to capture antibiotic resistance genes

doi: 10.1128/msystems.01039-25

Figure Lengend Snippet: Predicted novel streptothricin acetyltransferase from the human gut microbiome. (A) Gene schematics of the NTC1 metagenomic DNA fragment with the predicted acetyltransferase gene highlighted (SatB, blue). Predicted genes captured on the same DNA fragment are shown as empty arrows. Plasmid backbone elements are: Promoter (green), mosaic end sequence (orange), and terminator (red). (B) Maximum likelihood consensus phylogenetic tree of the predicted SatB protein (blue) in the context of known streptothricin acetyltransferases (SATs, NAT, and STAT) and aminoglycoside, chloramphenicol, virginiamycin, apramycin, and capreomycin acetyltransferases.

Article Snippet: The ZymoBIOMICS fecal standard, the source of our stool functional metagenomic library input DNA, has been sequenced and annotated for known antibiotic resistance genes by the Zymo corporation, but the data associated with this product do not list any known streptothricin resistance genes.

Techniques: Plasmid Preparation, Sequencing

Journal: mSystems

Article Title: Preparation of functional metagenomic libraries from low biomass samples using METa assembly and their application to capture antibiotic resistance genes

doi: 10.1128/msystems.01039-25

Figure Lengend Snippet: Streptothricin resistance is conferred by the NTC1 metagenomic DNA fragment and satB . (A) The results of a microbroth dilution assay of E. coli clones grown in the presence of variable nourseothricin concentrations (NTC1, E. coli carrying the NTC1 metagenomic DNA fragment; stat , E. coli expressing the stat streptothricin acetyltransferase; satB , E. coli expressing the predicted satB open reading frame from the NTC1 fragment). (B) IC50 values calculated from dose-response curves. **** P < 0.0001, *** P < 0.0005, ** P < 0.005, n = 4 for all.

Article Snippet: The ZymoBIOMICS fecal standard, the source of our stool functional metagenomic library input DNA, has been sequenced and annotated for known antibiotic resistance genes by the Zymo corporation, but the data associated with this product do not list any known streptothricin resistance genes.

Techniques: Dilution Assay, Clone Assay, Expressing