mbp mcherry expression plasmid  (Addgene inc)

Journal: iScience

Article Title: Horizontal and vertical gene transfer shape the plasmid host range in surface-associated microbial systems

doi: 10.1016/j.isci.2026.115299

Figure Lengend Snippet: Hypothesis and experiment system (A) We hypothesize that vertical and horizontal gene transfer (VGT and HGT) are influenced by the characteristics of the potential recipient cell types and determine the proliferation and diversity of transconjugant cells. Because the potential recipient community comprises multiple cell types with varying growth traits and conjugation probabilities, we expect the resulting composition of transconjugant cells to be shaped by these cell type-specific traits. (B) Our experimental system consists of E . coli MG1655 lacI q -pLpp-mCherry as the plasmid donor strain and pB10 as the focal plasmid. pB10 donor cells express RFP from the chromosome and transconjugants express GFP from pB10.

Article Snippet: MBP- mCherry expression plasmid (Amp R ) , Addgene , Plasmid# 29747.

Techniques: Conjugation Assay, Plasmid Preparation

Journal: iScience

Article Title: Horizontal and vertical gene transfer shape the plasmid host range in surface-associated microbial systems

doi: 10.1016/j.isci.2026.115299

Figure Lengend Snippet: Transconjugant proportions and diversities after surface-associated conjugation assays for different environmental conditions (A) Proportion of transconjugant cells relative to total cells after surface-associated conjugation assays using the WWTP community as the potential recipient cell population. We conducted conjugation assays on 1×SWW, 10×SWW, or LB agar plates using E . coli MG1655 lacI q -pLpp-mCherry as the pB10 donor strain. (B) Relative abundances of bacterial class in the total potential recipient cell population (T) and the transconjugant cell population (TC) as identified by 16S rRNA gene sequencing. We separated and identified TC cells using FC-FACS-sorting of GFP-positive cells. (C) Normalized Shannon index of the transconjugant populations after surface-associated conjugation assays on 1×SWW, 10×SWW, or LB agar plates. We normalized the Shannon index of the TC populations to their corresponding T populations. (D) Principal coordinate analysis (PCoA) based on weighted UniFrac distances of T and TC populations after surface-associated conjugation assays on 1×SWW, 10×SWW, or LB agar plates. (E) Phylogenetic tree of transconjugant ASVs detected after surface-associated conjugation assays on 1×SWW, 10×SWW, or LB agar plates. The outer colored box denotes the bacterial phylum of each ASV, corresponding to the phylum-level groupings shown in panel (B). The inner heatmap box aligned with each tip shows the log 10 fold-changes in ASV abundance (TC relative to T) across the three conditions. For (A and C), each point is an independent biological replicate ( n = 3), horizontal bars are the means, error bars are ±1 standard deviation, and asterisks indicate statistically significant differences between the means based on two-way ANOVA with Holm correction (∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, ns = not significant). For (D), each point is an independent biological replicate ( n = 3).

Article Snippet: MBP- mCherry expression plasmid (Amp R ) , Addgene , Plasmid# 29747.

Techniques: Conjugation Assay, Sequencing, Standard Deviation

Journal: iScience

Article Title: Horizontal and vertical gene transfer shape the plasmid host range in surface-associated microbial systems

doi: 10.1016/j.isci.2026.115299

Figure Lengend Snippet: Transconjugant growth during surface-associated conjugation assays for different environmental conditions (A) Representative fluorescence microscopy images of transconjugant cells during surface-associated conjugation assays on LB agar plates. E . coli MG1655 lacI q -pLpp-mCherry is the pB10 donor strain and show red fluorescence. Transconjugant cells are green. The time indicated in the images refers to the point at which transconjugant cells first became detectable. (B) Normalized microcolony area ( A / a 0 ) plotted as a function of time during the surface-associated conjugation assays on LB agar plates. A is the total microcolony area and a 0 is the initial transconjugant area. Connected data points are for individual colonies ( n = 12). (C) Microcolony area at the endpoint of the mating assay (t = 24 h) for different environmental conditions. The half-violin and scatterplots present the sample distribution and individual microcolony measurements for surface-associated conjugation assays on different medium (n 1xSWW = 880, n 10xSWW = 664, n LB = 1,070, for microcolony number). We performed each experiment at least three independent experiments. Horizontal bars are the mean microcolony areas, error bars are the 99% confidence intervals, and asterisks indicate statistically significant differences between the means based on two-way ANOVA with Holm correction (∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, ns = not significant).

Article Snippet: MBP- mCherry expression plasmid (Amp R ) , Addgene , Plasmid# 29747.

Techniques: Conjugation Assay, Fluorescence, Microscopy

Journal: Nucleic Acids Research

Article Title: EEPD1 evolved a unique DNA clamping dimer protecting reversed replication forks

doi: 10.1093/nar/gkag188

Figure Lengend Snippet: EEPD1 binding to dynamic dsDNA intermediates requires dimeric full-length protein. ( A ) EMSA shows binding to 5′-Cy5-labeled 45-nt ssDNA or 45-bp dsDNA fragment (20 nM) at 10 mM KCl, with increasing EEPD1 titer. ( B ) MST measurements of EEPD1 binding affinity for various dsDNA structures at 50-mM KCl. The labeled strand is the same 5′-Cy5-labeled 45-nt oligo as in (A). ( C ) MST measurements of DNA bubble binding by truncated EEPD1 constructs. Domains are colored according to schematics in (A): (HhH) 2 in blue and green, EEP in orange. Losing one or two (HhH) 2 increasingly impairs binding. The N-terminal fragment (aa 31–201, purple trace) alone doesn’t bind DNA. ( D ) MST measurements of EEPD1 mutants’ binding to DNA bubble. H404A is pseudo active site mutation, W522A is dimer-disrupting mutation. (HhH) 2 -truncated construct Δ1–129 (from C, blue curve) is copied here for comparison. Indicated titers of StrepII-EEPD1 (also applying to truncations and mutants) assume a dimeric state. All MST dissociation constants K d are mean values with standard deviation from three independent experiments. Experiments of binding-defective groups were done at least twice. ND: not determined. ( E ) Model for how dsDNA (cartoon) could be encircled by EEPD1 dimer (surface and cartoon), based on overlays with APE1–dsDNA complex (PDB: 1DEW) onto either subunit of closed EEPD1 dimer (PDB: 9YXY). A “di-Trp-Pro” pocket, at the bottom of the DNA-binding channel, would lie next to the major groove of the DNA (zoom). A potential extra-helical base flipping is indicated by arrow. ( F ) The “di-Trp-Pro” pocket is 12-Å wide, formed by symmetrically related tryptophans and prolines in the dimer interface, could theoretically accommodate two bases. Residues that line this pocket have significant ET scores (red dots). ( G ) Experimental simulated annealing omit map shows a density (*) sandwiched between the tryptophans, which could be a docking site for aromatic DNA bases.

Article Snippet: MGC human EEPD1 complementary DNA (cDNA) was purchased from Dharmacon (GenBank: BC065518 ). pET StrepII TEV LIC cloning vector (1R) was a gift from Scott Gradia (Addgene #29 664).

Techniques: Binding Assay, Labeling, Construct, Mutagenesis, Comparison, Standard Deviation