pcr amplicon sequencing  (Microsynth ag)

Journal: bioRxiv

Article Title: Bridge recombinase enables versatile rewriting of bacterial genomes

doi: 10.64898/2026.04.29.721476

Figure Lengend Snippet: (A) Schematic of IS 621 -mediated recombination and possible recombination classes with single bRNAs. Nucleotide-resolution of the donor DNA (dDNA) and target DNA (tDNA) substrates during an “insertion”-style recombination. (B) Schematic of the split kanamycin assay, wherein a split kanamycin gene is used to assess the recombinase-mediated integration of different-sized plasmids into a programmed locus. (C) Fraction of colonies surviving kanamycin selection over total transformants is shown. We observed that insertion efficiency did not statistically vary with insertion size (one-way ANOVA, p-value = 0.35). (D) An alignment of whole genome sequencing reads from a representative 141.8 kb recombinant colony is mapped across the in silico predicted map. (E) bRNAs are programmed to invert or excise the galK locus. Following transformation of pEdit, recombinants (fraction resistant to 2-DOG) are quantified. (F) bRNAs are designed to excise increasing portions of the colibactin biosynthetic gene cluster. hsvTK is inserted into the left flank of the pathway to mediate quantification. Upon transformation of pEdit, excision efficiencies are quantified as fraction resistant to dP. The fraction of post-selection clones with correct excision are quantified via colony PCR. (G) To assess genome-wide rearrangements, the counterselectable hsvTK gene was randomly inserted across the E. coli MG1655 genome via Himar transposition; bRNAs are programmed to recombine between the native galK locus and hsvTK insertion library, the outcome being an excision or inversion based on the orientation of hsvTK insertion. Upon recombinase induction, recombined cells are selected on 2-DOG- and dP-containing media. (H) 67 colonies were randomly selected for recombination mapping via whole genome sequencing. Within the genome plot, inner ribbons represent observed inversions (red) and excisions (blue); the inner ring shows the distribution of initial hsvTK insertions; the outer ring highlights essential genes; external spots note rearranged clones validated though whole genome sequencing. (I) rRNA operons were collapsed into a single contig to enable mapping of inversions at these identical loci. (J) Rearrangements proximal to galK are enlarged. (K) Representative Mauve alignment of a mutant colony encoding a 2.3 Mb inversion (as verified by whole-genome sequencing) against the reference genome E. coli MG1655, verifying on-target inversion and the absence of other structural rearrangements. All measurements of fraction resistant over total transformants or resistant CFUs are the mean ± sd of three biological replicates. Limit of detection (LOD) of each assay is represented as a dashed line.

Article Snippet: Individual colonies were analyzed by cPCR and PCR amplicon sequencing (Plasmidsaurus) to confirm the reconstituted kanR junction.

Techniques: Selection, Sequencing, Recombinant, In Silico, Transformation Assay, Clone Assay, Genome Wide, Mutagenesis

Journal: bioRxiv

Article Title: Bridge recombinase enables versatile rewriting of bacterial genomes

doi: 10.64898/2026.04.29.721476

Figure Lengend Snippet: (A) Plasmid design and transformation method for phyla-specific pEdit vectors. The bRNA targets a universally-present 14mer in the 16S rRNA gene. Insertions are quantified via antibiotic selection. (B) Unrooted order-level tree of all GTDB prokaryotic taxa, with edited strains and phyla highlighted. (C) Insertions across diverse species are validated through sequencing of the 16S rRNA locus. (D) Top axis: for strains transformed with replicating vectors, the percent of transformant CFUs with PCR-verified integration at 16S, is plotted (black). Bottom axis: for strains transformed with non-replicating vectors, the combined frequency of transient transfer and insertion of the cargo plasmid is quantified (white), as antibiotic-resistant CFUs divided by total recipients. In parallel, a replicative vector is transformed to quantify transformation efficiency, allowing calculation of transformation rate-normalized editing efficiency (black) (mean ± sd of three biological replicates). ** indicates a suitable transformation efficiency control vector was not available for normalization. (E) Sequence similarity and off-target profile of bridge recombinase-mediated integration across diverse bacteria. Genomic DNA from transformed populations (>200 CFUs) was analyzed by AP-PCR to map cargo insertion sites. Insertions were classified as target-like or donor-like and plotted by their Levenshtein distance from the programmed target sequence (x-axis) versus percent of reads (y-axis). Insertions at 16S rRNA are highlighted in pink; donor-like insertions are shown in red; other target-like insertions are shown as open circles. Sequence logos depict aligned target-like insertion sites for each species with bases matching the programmed target sequence are highlighted in pink.

Article Snippet: Individual colonies were analyzed by cPCR and PCR amplicon sequencing (Plasmidsaurus) to confirm the reconstituted kanR junction.

Techniques: Plasmid Preparation, Transformation Assay, Selection, Sequencing, Control, Bacteria

Journal: bioRxiv

Article Title: Bridge recombinase enables versatile rewriting of bacterial genomes

doi: 10.64898/2026.04.29.721476

Figure Lengend Snippet: (A) Expression of two bRNAs and formation of two distinct synaptic complexes permit programmable DNA TRADE editing. (B) Dual bRNAs are designed to disrupt sfGFP and mRFP with a catP replacement; incomplete single recombination reactions at sfGFP, mRFP , or off-target sites are expected to integrate the full donor plasmid. (C) Following recombination, chloramphenicol-selected CFUs are phenotyped by fluorescence to determine recombination type. Double recombinants are the minority product across multiple bRNA pairs. (D) Orthogonal dinucleotide core sequences are predicted to suppress cross-reactivity between bRNAs with native CT:CT cores. (E) Following transformation and IS 621 induction, AP-PCR and deep sequencing are used to map the insertion sites of both Donor A and Donor B . Observed insertion sites are classified by sequence similarity to the true programmed Target A and Target B , or to off-target sites resembling Target A , Target B , Donor A , or Donor B . Cross-targets are defined as the insertion of Donor A into any B-like sequences and vice-versa. The log 2 fraction of reads from each category is shown as a heat map. (F) Cross-target reaction rates are highlighted as a bar graph for each tested bRNA pair. (G) Aggregated cross-target reaction rates from 60 independent reactions across 10 bRNA pairs are plotted. Marker colors correspond to Donor A and Donor B . (H) Incomplete single recombination reactions, predicted to integrate the donor plasmid backbone, can be counterselected by placing the conditional toxin hsvTK on the backbone. (I) Following recombination, dP-selected clones are phenotyped by fluorescence to determine recombination types. (J-K) TRADE editing of the sfGFP - mRFP - kanR and colibactin BGC using hsvTK counterselection and orthogonal cores. The fractions of post-selection CFUs with the correct double recombination are quantified. For all plots, mean ± s.d. of three biological replicates are shown.

Article Snippet: Individual colonies were analyzed by cPCR and PCR amplicon sequencing (Plasmidsaurus) to confirm the reconstituted kanR junction.

Techniques: Expressing, Plasmid Preparation, Fluorescence, Transformation Assay, Sequencing, Marker, Clone Assay, Selection