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: bioRxiv

Article Title: External Globus Pallidus Arkypallidal Circuit Dynamics Gate Risk-Taking Behavior

doi: 10.64898/2026.03.20.713182

Figure Lengend Snippet: (a) Experimental diagram. Npas1-Cre-TdTomato mice received bilateral GPe injections of Cre-dependent AAV8-hSyn-DIO-hM4D(Gi)-mCherry or AAV8-hSyn-DIO-hM3D(Gq)-mCherry, with Cre-negative littermates serving as controls. All animals received C21 prior to testing, ensuring equivalent drug exposure across groups. Mice were tested on the elevated plus maze (EPM) 5 weeks post-surgery. Behavioral sessions were video-recorded for subsequent analysis. (b) Time spent in closed arms, center, and open arms during the EPM shows the expected preference for closed arms across all groups, with no effect of GPe NPAS1 manipulation on arm occupancy (two-way mixed ANOVA, maze compartment F (2,52) = 122.8, p = 2.003e-20; group F (2,26) = 1.546, p = 0.2319). (c) Number of open-arm entries does not differ across groups, indicating no effect of GPe NPAS1 manipulation on exploration of these areas (ANOVA, group F (2,26) = 1.048, p = 0.365). (d) Percent time spent in the open arms in the EPM is comparable across control, hM4D(Gi), and hM3D(Gq) mice, consistent with preserved global EPM performance (ANOVA, group F (2,55) = 0.4928, p = 0.6136). (e) Empirical cumulative distribution functions (ECDFs) of frame-wise horizontal movement in the open arms during the EPM show highly overlapping movement distributions across groups, illustrating the absence of gross shifts in locomotor behavior within high-risk regions of the maze. Pairwise Kolmogorov–Smirnov tests detected statistically significant but very small distributional differences (KS D = 0.02–0.05; FDR-corrected p < 0.001), consistent with negligible effect sizes that do not reflect meaningful differences in open-arm movement dynamics. (f) Distance traveled (two-way mixed ANOVA, group x time F (238,3094) = 0.8741, p = 0.9131), (g) speed (two-way mixed ANOVA, group x time F (238,3094) = 0.8742, p = 0.9129), and (h) acceleration (two-way mixed ANOVA, group x time F (238,3094) = 1.037, p = 0.3419) over time are comparable across control, hM4D(Gi), and hM3D(Gq) mice, indicating preserved global locomotor output across the session. Frame-wise polar histograms of heading direction during EPM show (i) control mice exhibit a modest but significant preference for a closed-arm-oriented heading (Rayleigh test, r = 0.005399, p = 0.001871). (j) hM4D(Gi) mice show a statistically significant, strong preferred closed arm heading (Rayleigh test, r = 0.01707, p = 1.225e-16). (k) In contrast, GPe NPAS1 hM4D(Gq) mice do not exhibit a statistically significant preferred heading orientation (Rayleigh test, r = 0.001264, p = 0.7505). (l) Pose features extracted from video tracking show bound box area across time was decreased for hM3D(Gq) mice compared to control mice across all EPM areas (two-way mixed ANOVA, group x time F (238,3094) = 1.180, p = 0.03497; post hoc control v hM3D(Gq) p = 0.03348). (m) Similarly, box aspect ratio over time was decreased for hM3D(Gq) mice compared to control mice (two-way mixed ANOVA, group x time F (238,3094) = 1.181, p = 0.03486; post hoc control v hM3D(Gq) p = 0.04464). (n) There were no group differences in the change in aspect ratio across time (two-way mixed ANOVA, group x time F (238,3094) = 0.08639, p = 0.06231). Dots represent individual data points, error bars or shaded bands represent standard error of the mean (SEM). For polar plots, 32 bins were computed to generate 11.25 degree bars for histogram densities.

Article Snippet: The Gi/o-coupled DREADD vector AAV8-hSyn-DIO-hM4D(Gi)-mCherry (plasmid #44362) and the Gq-coupled DREADD vector AAV8-hSyn-DIO-hM3D(Gq)-mCherry (plasmid #44361) were obtained from Addgene.

Techniques: Control

Journal: bioRxiv

Article Title: External Globus Pallidus Arkypallidal Circuit Dynamics Gate Risk-Taking Behavior

doi: 10.64898/2026.03.20.713182

Figure Lengend Snippet: (a) Experimental diagram. Npas1-Cre-TdTomato mice received bilateral GPe injections of Cre-dependent AAV8-hSyn-DIO-hM4D(Gi)-mCherry or AAV8-hSyn-DIO-hM3D(Gq)-mCherry, with Cre-negative littermates serving as controls. All animals received C21 prior to testing, ensuring equivalent drug exposure across groups. Mice were tested on the elevated plus maze (EPM) 5 weeks post-surgery. Behavioral sessions were video-recorded for subsequent analysis. (b) Time spent in closed arms, center, and open arms during the EPM shows the expected preference for closed arms across all groups, with no effect of GPe NPAS1 manipulation on arm occupancy (two-way mixed ANOVA, maze compartment F (2,52) = 122.8, p = 2.003e-20; group F (2,26) = 1.546, p = 0.2319). (c) Number of open-arm entries does not differ across groups, indicating no effect of GPe NPAS1 manipulation on exploration of these areas (ANOVA, group F (2,26) = 1.048, p = 0.365). (d) Percent time spent in the open arms in the EPM is comparable across control, hM4D(Gi), and hM3D(Gq) mice, consistent with preserved global EPM performance (ANOVA, group F (2,55) = 0.4928, p = 0.6136). (e) Empirical cumulative distribution functions (ECDFs) of frame-wise horizontal movement in the open arms during the EPM show highly overlapping movement distributions across groups, illustrating the absence of gross shifts in locomotor behavior within high-risk regions of the maze. Pairwise Kolmogorov–Smirnov tests detected statistically significant but very small distributional differences (KS D = 0.02–0.05; FDR-corrected p < 0.001), consistent with negligible effect sizes that do not reflect meaningful differences in open-arm movement dynamics. (f) Distance traveled (two-way mixed ANOVA, group x time F (238,3094) = 0.8741, p = 0.9131), (g) speed (two-way mixed ANOVA, group x time F (238,3094) = 0.8742, p = 0.9129), and (h) acceleration (two-way mixed ANOVA, group x time F (238,3094) = 1.037, p = 0.3419) over time are comparable across control, hM4D(Gi), and hM3D(Gq) mice, indicating preserved global locomotor output across the session. Frame-wise polar histograms of heading direction during EPM show (i) control mice exhibit a modest but significant preference for a closed-arm-oriented heading (Rayleigh test, r = 0.005399, p = 0.001871). (j) hM4D(Gi) mice show a statistically significant, strong preferred closed arm heading (Rayleigh test, r = 0.01707, p = 1.225e-16). (k) In contrast, GPe NPAS1 hM4D(Gq) mice do not exhibit a statistically significant preferred heading orientation (Rayleigh test, r = 0.001264, p = 0.7505). (l) Pose features extracted from video tracking show bound box area across time was decreased for hM3D(Gq) mice compared to control mice across all EPM areas (two-way mixed ANOVA, group x time F (238,3094) = 1.180, p = 0.03497; post hoc control v hM3D(Gq) p = 0.03348). (m) Similarly, box aspect ratio over time was decreased for hM3D(Gq) mice compared to control mice (two-way mixed ANOVA, group x time F (238,3094) = 1.181, p = 0.03486; post hoc control v hM3D(Gq) p = 0.04464). (n) There were no group differences in the change in aspect ratio across time (two-way mixed ANOVA, group x time F (238,3094) = 0.08639, p = 0.06231). Dots represent individual data points, error bars or shaded bands represent standard error of the mean (SEM). For polar plots, 32 bins were computed to generate 11.25 degree bars for histogram densities.

Article Snippet: The Gi/o-coupled DREADD vector AAV8-hSyn-DIO-hM4D(Gi)-mCherry (plasmid #44362) and the Gq-coupled DREADD vector AAV8-hSyn-DIO-hM3D(Gq)-mCherry (plasmid #44361) were obtained from Addgene.

Techniques: Control