Journal: PLoS ONE

Article Title: Precision long-read metagenomics sequencing for food safety by detection and assembly of Shiga toxin-producing Escherichia coli in irrigation water

doi: 10.1371/journal.pone.0245172

Figure Lengend Snippet: Enriched irrigation water (Water) was artificially contaminated with 10-fold dilutions of E . coli EDL933_2 (+Ecoli) with a starting concentration of 7 x 10 8 CFU/ml (Water+Ecoli1). Reads were analyzed by the EPI2ME WIMP workflow. Bacterial species contributing more than 1% of the classified reads are shown and the sum of the remaining species identified are included as “Other”.

Article Snippet: We identified the antimicrobial resistance genes present in our sequenced genomes using the EPI2ME Fastq Antimicrobial Resistance workflow (Oxford Nanopore Technologies).

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Precision long-read metagenomics sequencing for food safety by detection and assembly of Shiga toxin-producing Escherichia coli in irrigation water

doi: 10.1371/journal.pone.0245172

Figure Lengend Snippet: A) Direct comparison of quantitative qPCR detection with de novo assembly limits by nanopore sequencing informs detection and classification of STECs. B) Pipeline for detection and classification of STECs in enriched irrigation water using nanopore sequencing and EPI2ME cloud-based services to identify reads of a desired taxa for de novo assembly with Flye and in silico analysis.

Article Snippet: We identified the antimicrobial resistance genes present in our sequenced genomes using the EPI2ME Fastq Antimicrobial Resistance workflow (Oxford Nanopore Technologies).

Techniques: Comparison, Nanopore Sequencing, In Silico

Journal: Scientific Reports

Article Title: Genome targeting by hybrid Flp-TAL recombinases

doi: 10.1038/s41598-020-74474-2

Figure Lengend Snippet: Evolution of Flp-TAL recombinases. ( A ) The inversion assay. The reporter bears the inversion cassette flanked by the recombination targets in the head-to-head orientation: FL-61 , FL-63 , or FL-71 (marked as RT ) and FRT *, which has the spacer either from FL-61 , FL-63 , or FL-71 , respectively. Upon the expression of a recombination-competent Flp variant, the cassette is inverted so the gene that encodes this variant can be PCR amplified (the small black arrows above the Flp variant gene and the inversion cassette indicate the location and orientation of the PCR primers). ( B ) The deletion assay. The deletion reporter bears the lacZα cassette flanked by the respective recombination targets in the head-to-tail orientation. If a Flp variant is able to delete the cassette, bacterial cells, in which the deletion event occurs, form white/partially white colonies when plated on the X-gal containing plates. ( C ) Mutations in the FV61, FV63, and FV71 variants. ( D ) Activity of FV61, FV63, and FV71 on the FL-61 , FL-63 , and FL-71 substrates. The assays were performed using the respective deletion reporters. ( E ) Electrophoregram of the plasmid DNA isolated from the colonies that were pooled from the respective plates shown in ( D ); the plasmid DNA was digested with HindIII that uniquely cuts the expression and reporter plasmids. Ctr, control vectors and vector combinations: Exp, Flpe expression vector; Rep, the deletion reporter that bears the lacZα cassette flanked by the FRT sequences; Exp + Rep, Flpe expression vector and the respective reporter, in which the lacZα cassette was completely deleted; M, DNA ladder (NEB, 2-log). The arrows below the electrophoregram point to the location of the respective vectors.

Article Snippet: LJ, RJ, the PCR analysis of the left and right junctions of pTarget integrated into the respective genomic sequences; M, DNA ladder (NEB, 2-log). ( C ) PCR analysis of the pooled hygro R colonies generated in the integration experiments with three combinations of the FV71-TAL recombinases: FV71-TAL(L24) + FV71-TAL(R15), FV71-TAL(L18) + FV71-TAL(R15) and FV71-TAL(L15) + FV71-TAL(R15).

Techniques: Expressing, Variant Assay, Amplification, DNA Deletion Assay, Activity Assay, Plasmid Preparation, Isolation

Journal: Scientific Reports

Article Title: Genome targeting by hybrid Flp-TAL recombinases

doi: 10.1038/s41598-020-74474-2

Figure Lengend Snippet: Flp-TAL variants target FRT -like sequences FL-61 , FL-63 , and FL-71 in their native chromosomal environment. ( A ) Schematics of the integration assays. The reporter pTarget can be integrated either into FL-61 , FL-63 , or FL-71 depending on the specificity of the Flp-TAL recombinase and the version of pTarget: FL-61 / FL-71 or FL-63 / FL-71 . Upon integration of pTarget into FL-61 or FL-63 the resultant cells become hygromycin resistant and red, while upon integration into FL-71 , the cells become hygromycin resistant and green (images of the individual expanded hygro R /red and hygro R /green colonies are shown as examples). The analysis of the individual colonies was performed in two biological replicates. LJ and RJ mark the locations of the left and right junctions of the integrated reporter and genomic DNA, respectively; diagnostic PCR at these locations was used to determine the authenticity of the reporter integration. ( B ) PCR analysis of the pooled hygro R colonies generated in the experiments with the Flp-TAL recombinase, the ‘plain’ recombinase variant, and the ‘empty’ expression vector. LJ, RJ, the PCR analysis of the left and right junctions of pTarget integrated into the respective genomic sequences; M, DNA ladder (NEB, 2-log). ( C ) PCR analysis of the pooled hygro R colonies generated in the integration experiments with three combinations of the FV71-TAL recombinases: FV71-TAL(L24) + FV71-TAL(R15), FV71-TAL(L18) + FV71-TAL(R15) and FV71-TAL(L15) + FV71-TAL(R15). The diagnostic PCR analysis was performed at the right junction.

Article Snippet: LJ, RJ, the PCR analysis of the left and right junctions of pTarget integrated into the respective genomic sequences; M, DNA ladder (NEB, 2-log). ( C ) PCR analysis of the pooled hygro R colonies generated in the integration experiments with three combinations of the FV71-TAL recombinases: FV71-TAL(L24) + FV71-TAL(R15), FV71-TAL(L18) + FV71-TAL(R15) and FV71-TAL(L15) + FV71-TAL(R15).

Techniques: Diagnostic Assay, Generated, Variant Assay, Expressing, Plasmid Preparation, Genomic Sequencing

Journal: Scientific Reports

Article Title: Genome targeting by hybrid Flp-TAL recombinases

doi: 10.1038/s41598-020-74474-2

Figure Lengend Snippet: Flp-TAL recombinase can delete large genome DNA fragments. ( A ) Schematic of the deletion assay. Upon expression of FV71-TAL in cells, in which pTarget was pre-integrated into FL-61 (Fig. A), the DNA fragment located between two FL-71 sequences can be deleted (this DNA fragment contains ~ 4.7 kb of reporter DNA and ~ 10 kb of genome DNA). ( B ) PCR analysis of the FV71-TAL treated cells. The deletion activity was analyzed in three biological replicates (see Supplemental Fig. ). FL-61 (LJ) and FL-71 (RJ), the PCR analysis of the respective left and right vector-genomic junctions of the integrated pTarget before and after the treatment with FV71-TAL. M, DNA ladder (NEB 2-log). ( C ) Sequencing of the deletion-specific PCR product FL-71 (RJ) confirmed its identity.

Article Snippet: LJ, RJ, the PCR analysis of the left and right junctions of pTarget integrated into the respective genomic sequences; M, DNA ladder (NEB, 2-log). ( C ) PCR analysis of the pooled hygro R colonies generated in the integration experiments with three combinations of the FV71-TAL recombinases: FV71-TAL(L24) + FV71-TAL(R15), FV71-TAL(L18) + FV71-TAL(R15) and FV71-TAL(L15) + FV71-TAL(R15).

Techniques: DNA Deletion Assay, Expressing, Activity Assay, Plasmid Preparation, Sequencing

Journal: Scientific Reports

Article Title: Genome targeting by hybrid Flp-TAL recombinases

doi: 10.1038/s41598-020-74474-2

Figure Lengend Snippet: Target specificity of the Flp variants within Flp-TAL is determined by the TAL module. ( A ) pTarget co-transfected with either FV61-TAL, FV63-TAL, or FV71-TAL into HEK-293 cells efficiently targets only the intended FRT -like sequence. ( B ) The TAL modules in the Flp-TAL recombinases FV71-TAL61 and FV71-TAL63 variants deliver the FV71 variant to the FRT -like sequences FL-61 and FL-63 but not to FL-71 . The diagnostic PCR analyses were performed at the respective right junctions between the integrated pTarget and genomic DNA. M, DNA ladder (NEB, 2-log).

Article Snippet: LJ, RJ, the PCR analysis of the left and right junctions of pTarget integrated into the respective genomic sequences; M, DNA ladder (NEB, 2-log). ( C ) PCR analysis of the pooled hygro R colonies generated in the integration experiments with three combinations of the FV71-TAL recombinases: FV71-TAL(L24) + FV71-TAL(R15), FV71-TAL(L18) + FV71-TAL(R15) and FV71-TAL(L15) + FV71-TAL(R15).

Techniques: Transfection, Sequencing, Variant Assay, Diagnostic Assay