a-frame insert  (Med Associates Inc)

Journal: bioRxiv

Article Title: Discovery of novel antimicrobial resistance genes in food and fertiliser using a high-throughput gene capture and functional screening platform

doi: 10.64898/2026.03.15.711940

Figure Lengend Snippet: (A) Plasmid maps of the cassette capture system. The capture vector, pVR106 (pUS250 -ccdB-attI1-ccdA ) (Table S1), contains a ccdB gene, with an attI1 recombination site embedded within it, under the control of an L-arabinose inducible promoter, allowing counter-selection of successful, site-specific insertion events. Additionally, this plasmid contains the antitoxin gene ccdA under a cumate-inducible promoter to protects cells against the toxic CcdB. Plasmid pVR110 (pttQ- intI1 ) contains intI1 under the control of an IPTG-inducible promoter. (B) Workflow for the recovery and characterisation of the cassette metagenome from different environments. Gene cassettes were amplified from diverse environmental DNA sources (mixed salad leaves, prawn gut, sea water, and horse manure fertiliser), circularised and transformed into recipient cells expressing IntI1. Integration of a cassette into the attI1 site disrupts the ccdB reading frame, ensuring that cells containing a captured cassette survive counter-selection. The resulting library was subjected to two parallel analyses: targeted functional screening to identify AMR genes, and untargeted nanopore sequencing of the PCR-amplified plasmid DNA (using ccdB -in-fow/rev junction primers) to characterise the diversity of the captured cassette pool. Schematic created with BioRender.com and modified using assets from Adobe Stock.

Article Snippet: A series of in-frame attI1 insertion variants were synthesised (Twist Bioscience, USA) to identify integration sites that preserved marker lethality (Table S2).

Techniques: Plasmid Preparation, Control, Selection, Amplification, Transformation Assay, Expressing, Functional Assay, Nanopore Sequencing, Modification

Journal: bioRxiv

Article Title: Discovery of novel antimicrobial resistance genes in food and fertiliser using a high-throughput gene capture and functional screening platform

doi: 10.64898/2026.03.15.711940

Figure Lengend Snippet: (A) Captured gene cassette arrays and their corresponding environmental sources. Long arrows indicate open reading frames (ORFs) and their transcriptional orientation, maroon arrows represent attC sites and short green arrows represent the attI1 site. (B) Antimicrobial resistance (AMR) profile of bleomycin resistance genes, AlphaFold3 model of IblA and structural superimposition of IblA (blue) onto its structural homolog AF-Q53795-F1 (yellow). (C) AMR profile of ImzA, AlphaFold3 model and structural superimposition of ImzA (blue) onto its structural homolog AF-A0A0S8E1S1-F1 (yellow). (D) AMR profiles for the known AMR-conferring genes (in parentheses): rifampicin ( arr-3 ), aminoglycoside acetyltransferase ( aacA6 ), and trimethoprim ( dfrA1 ). Bar charts display the fold-increase in minimum inhibitory concentration (MIC) relative to the negative control (pVR106- fuGFP ) (Table S1). The antimicrobial concentrations (µg/mL) are indicated above each bar, and biological replicates (n = 3) are represented by asterisks.

Article Snippet: A series of in-frame attI1 insertion variants were synthesised (Twist Bioscience, USA) to identify integration sites that preserved marker lethality (Table S2).

Techniques: Concentration Assay, Negative Control