a vector containing the murine adam12 and mcherry expression constructs flanked by sequences recognized by the sleeping beauty transposase  (VectorBuilder GmbH)

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: Life Science Alliance

Article Title: CTPS2 regulates CTP synthetase activity by interacting with CTPS1

doi: 10.26508/lsa.202403117

Figure Lengend Snippet: (A) CTPS1+2-KO HEK cells were transiently co-transfected with plasmids coding GFP-CTPS2 (in green) and pLVX vectors for CTPS1, CTPS2, or CTPS1 H355A (expressing mCherry as a reporter of transfection in red) in the absence of any treatment. Data from one representative experiment of two independent experiments. Bar graphs on the right panel correspond to the filament–cytoophidium/cell quantification from the images on the left with indicated numbers of cells and filaments–cytoophidia counted (n=) in three independent fields/images for each condition independently of the mCherry staining. Magnification 40x, scale bar: 25 μm. (B, C) CTPS1+2-KO cells stably expressing GFP-CTPS1, GFP-CTPS2, GFP-CTPS1 H355A , or GFP-CTPS2 H355A . Cells were then maintained in culture without cytidine. (B) Histograms of GFP expression of different cell lines. Data in the right and left panels are from the same experiment with the same “nontransfected” control histogram, which is representative of two independent experiments. (C) Confluency curves as percentages (%) showing the proliferation of different cell lines. Confluency was measured using an IncuCyte Zoom system. Cells were seeded for 24 h, then treated or not (untreated) with the indicated concentrations of 3-deazauridine (3-DU) or 200 μM cytidine. (A) Data as means ± SD. Mann–Whitney statistical tests, * P < 0.05 and ** P < 0.01. n = is indicated in the panel.

Article Snippet: The cDNAs were verified by sequencing and inserted into a bicistronic lentiviral expression vector encoding mCherry as a reporter (pLVX-EF1α-IRES-mCherry Vector; Clontech).

Techniques: Transfection, Expressing, Staining, Stable Transfection, Control, MANN-WHITNEY

Journal: Life Science Alliance

Article Title: CTPS2 regulates CTP synthetase activity by interacting with CTPS1

doi: 10.26508/lsa.202403117

Figure Lengend Snippet: (A) FACS dot-plots of GFP and mCherry expression. (A, B) Bar graphs from flow-cytometry data as presented in (A), showing the percentage (%) of GFP+ alone, mCherry+ alone, and double-positive GFP+mCherry+cells.

Article Snippet: The cDNAs were verified by sequencing and inserted into a bicistronic lentiviral expression vector encoding mCherry as a reporter (pLVX-EF1α-IRES-mCherry Vector; Clontech).

Techniques: Expressing, Flow Cytometry

Journal: Life Science Alliance

Article Title: CTPS2 regulates CTP synthetase activity by interacting with CTPS1

doi: 10.26508/lsa.202403117

Figure Lengend Snippet: Histograms of mCherry reporter expression profiles of CTPS1-KO Jurkat cells transduced with CTPS1 WT or CTPS1 H355A after or not cytidine deprivation (no cytidine) during 3, 6, and 24 d. The CTPS1 H355A profiles are similar to those of CTPS1 WT.

Article Snippet: The cDNAs were verified by sequencing and inserted into a bicistronic lentiviral expression vector encoding mCherry as a reporter (pLVX-EF1α-IRES-mCherry Vector; Clontech).

Techniques: Expressing, Transduction