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: Elucidating the kinetics of CRISPR–SaCas9 action to obtain effective HIV DNA excision with two gRNAs

doi: 10.1093/nar/gkag205

Figure Lengend Snippet: The gRNA targets for SaCas9 attack on the HIV DNA genome. ( A ) Positions of the antiviral gRNAs on the HIV proviral DNA are indicated, targeting both the positive and negative strands. ( B ) Antiviral activity of single SaCas9/gRNAs. The cleavage activity of each gRNA was evaluated by transiently co-transfecting HEK293T cells with plasmids encoding HIV-1 LAI and SaCas9/gRNA. Two days post-transfection, supernatants were collected, and viral gene expression was quantified by CA-p24 ELISA. Values were normalized to the SaCas9 reference (set as 100%) and are presented as the mean ± standard deviation (SD) of three independent experiments.

Article Snippet: The SaCas9 expression plasmid p6XHis_NLS-SaCas9 (Addgene #101086) was transformed into Escherichia coli Rosetta (DE3) competent cells (Novagen).

Techniques: Activity Assay, Transfection, Gene Expression, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Elucidating the kinetics of CRISPR–SaCas9 action to obtain effective HIV DNA excision with two gRNAs

doi: 10.1093/nar/gkag205

Figure Lengend Snippet: Monitoring HIV replication in SupT1 cells transduced with single SaCas9/gRNA. SupT1 cells transduced with lentiviral vectors encoding individual SaCas9/gRNAs were infected with HIV at day 0 at different multiplicities of infection (MOIs): ( A ) 0.1, ( B ) 1, and ( C ) 10. Six parallel cultures were established for each gRNA. HIV replication was monitored twice per week by assessing syncytia formation. Transparent circles indicate cultures in which viral replication led to syncytia formation (breakthrough infection), while black circles represent cultures with no observable viral replication. WT: wild-type SupT1 cells (non-transduced); CTRL: cells transduced with a negative control gRNA.

Article Snippet: The SaCas9 expression plasmid p6XHis_NLS-SaCas9 (Addgene #101086) was transformed into Escherichia coli Rosetta (DE3) competent cells (Novagen).

Techniques: Transduction, Infection, Negative Control

Journal: Nucleic Acids Research

Article Title: Elucidating the kinetics of CRISPR–SaCas9 action to obtain effective HIV DNA excision with two gRNAs

doi: 10.1093/nar/gkag205

Figure Lengend Snippet: Characterization of CRISPR–SaCas9 target site mutations in HIV breakthrough cultures. Supernatants from cultures exhibiting syncytia formation were collected at the peak of infection and used to infect fresh cells transduced with SaCas9/gRNA. After robust viral replication, cells were harvested, and total cellular DNA was extracted. The target region was amplified by PCR and analyzed by Sanger sequencing. Sequences were aligned to the LAI reference, with the wild-type sequence shown at the top and culture numbers indicated on the left. The target sequence is underlined, and the PAM sequence is highlighted in blue. Nucleotide substitutions are shown in red, predicted SaCas9 cleavage sites are indicated with black triangles, and corresponding amino acids are displayed on the right.

Article Snippet: The SaCas9 expression plasmid p6XHis_NLS-SaCas9 (Addgene #101086) was transformed into Escherichia coli Rosetta (DE3) competent cells (Novagen).

Techniques: CRISPR, Infection, Transduction, Amplification, Sequencing

Journal: Nucleic Acids Research

Article Title: Elucidating the kinetics of CRISPR–SaCas9 action to obtain effective HIV DNA excision with two gRNAs

doi: 10.1093/nar/gkag205

Figure Lengend Snippet: Proviral HIV DNA analysis within cultures that have successfully cured HIV by a single SaCas9/gRNA. ( A ) DNA was isolated 90 days post-HIV infection (MOI = 10) from SaCas9/gRNA-transduced cells. The target region was PCR-amplified, TA-cloned, and Sanger-sequenced. Sequences were aligned to HIV LAI sequence that is shown at the top, with the gRNA target underlined and PAM in blue. Cleavage sites are marked with black triangles. Substitutions are indicated in red, insertions in green (+n), and deletions as dashes (–n). Variants are classified as mutation, deletion, delin, or insertion, with frequencies indicated on the left. ( B ) Distribution and relative proportions of mutations induced by a single-SaCas9/gRNA cleavage. Duplicate sequences, potentially originating from PCR or TA cloning, were excluded from frequency calculations. ( C ) Analysis of deletions (left) and insertions (right), with their frequencies and average sizes indicated. ( D ) Characterization of delins (sequences containing both deletions and insertions at the same site). Deletions are shown as transparent circles on the left, insertions as black circles on the right, with connecting lines linking paired events. The x -axis represents mutation size; “2×” indicates the variation occurred twice. ( E ) Frequency distribution of insertion sizes within the delin group.

Article Snippet: The SaCas9 expression plasmid p6XHis_NLS-SaCas9 (Addgene #101086) was transformed into Escherichia coli Rosetta (DE3) competent cells (Novagen).

Techniques: Isolation, Infection, Amplification, Clone Assay, Sequencing, Mutagenesis, TA Cloning

Journal: Nucleic Acids Research

Article Title: Elucidating the kinetics of CRISPR–SaCas9 action to obtain effective HIV DNA excision with two gRNAs

doi: 10.1093/nar/gkag205

Figure Lengend Snippet: Dual SaCas9/gRNA-mediated attack on the HIV genome. ( A ) Schematic of the lentiviral construct expressing SaCas9 and gRNAs. The first gRNA is co-expressed with SaCas9, while the second is expressed independently. ( B ) Five gRNA combinations are shown, with positions on the HIV genome indicated by triangles; distances between target sites are specified. ( C ) Comparison of single versus dual SaCas9/gRNA effects in long-term HIV infection. Six parallel cultures (MOI = 10) were analyzed. Transparent circles indicate virus replication; black circles indicate absence of replication. Controls: SupT1 wild-type (WT) non-transduced cells; CTRL, cells expressing a negative control gRNA.

Article Snippet: The SaCas9 expression plasmid p6XHis_NLS-SaCas9 (Addgene #101086) was transformed into Escherichia coli Rosetta (DE3) competent cells (Novagen).

Techniques: Construct, Expressing, Comparison, Infection, Virus, Negative Control

Journal: Nucleic Acids Research

Article Title: Elucidating the kinetics of CRISPR–SaCas9 action to obtain effective HIV DNA excision with two gRNAs

doi: 10.1093/nar/gkag205

Figure Lengend Snippet: ( A ) Schematic of primers used to detect genomic changes. Double editing at the two gRNA targets was assessed separately using primers a + b or c + d. Inversions were detected with primers a + c or b + d, and full-length or excision events with primers a + d. Cellular DNA was isolated 90 days post-HIV infection (MOI = 10) from dual SaCas9/gRNA-transduced cells. PCR amplification, TA cloning, and Sanger sequencing were performed for panels B, C, and F. Limiting dilution was conducted on Gag2 + Pol4 and Gag3 + Pol4, with single-copy full-length amplification followed by Sanger sequencing (D and E). Sequences were aligned to the HIV LAI reference (WT at the top). The gRNA target is underlined, PAM is shown in blue, and cleavage sites are indicated by black triangles. Substitutions are indicated in red, insertions in green (+n), and deletions as dashes (–n). Segment excisions between the two gRNAs are marked by red triangles on the right.

Article Snippet: The SaCas9 expression plasmid p6XHis_NLS-SaCas9 (Addgene #101086) was transformed into Escherichia coli Rosetta (DE3) competent cells (Novagen).

Techniques: Isolation, Infection, Amplification, TA Cloning, Sequencing

Journal: Nucleic Acids Research

Article Title: Elucidating the kinetics of CRISPR–SaCas9 action to obtain effective HIV DNA excision with two gRNAs

doi: 10.1093/nar/gkag205

Figure Lengend Snippet: A ) Pie charts show the outcomes of dual SaCas9-mediated targeting across different gRNA combinations, including excision and double editing (indels and delins) at both target sites. Single-editing events and WT) sequences were not detected. ( B ) The graph compares excision efficiency among the tested dual SaCas9/gRNA combinations, highlighting differences in excision outcomes. ( C ) Deletions and insertions are illustrated with their frequencies; deletions are shown on the left, insertions on the right, and average sizes are indicated. ( D ) Analysis of the delin group (sequences containing both deletions and insertions). Deletions are shown as transparent circles on the left, insertions as black circles on the right, connected by lines. The x -axis shows mutation size; “2×” indicates the same variation occurred twice. ( E ) Frequency distribution of insertion sizes within the delin group.

Article Snippet: The SaCas9 expression plasmid p6XHis_NLS-SaCas9 (Addgene #101086) was transformed into Escherichia coli Rosetta (DE3) competent cells (Novagen).

Techniques: Mutagenesis

Journal: Nucleic Acids Research

Article Title: Elucidating the kinetics of CRISPR–SaCas9 action to obtain effective HIV DNA excision with two gRNAs

doi: 10.1093/nar/gkag205

Figure Lengend Snippet: A ) Competitive proliferation assay of SaCas9/gRNA-transduced cells. As the construct encodes GFP, transduced cells were monitored by flow cytometry. Transduced cells were co-cultured with wild-type cells, and relative proliferation was assessed every 7 days for up to 42 days. ( B ) Detection of off-target effects by T7EI cleavage assay. Cleaved bands are indicated by triangles. The M lane indicates the DNA ladder. CTRL indicates control gRNA-transduced samples, while Gag2 and Pol5 correspond to HIV-infected SaCas9/gRNA-transduced cells.

Article Snippet: The SaCas9 expression plasmid p6XHis_NLS-SaCas9 (Addgene #101086) was transformed into Escherichia coli Rosetta (DE3) competent cells (Novagen).

Techniques: Proliferation Assay, Construct, Flow Cytometry, Cell Culture, Cleavage Assay, Control, Infection

Journal: Nucleic Acids Research

Article Title: Elucidating the kinetics of CRISPR–SaCas9 action to obtain effective HIV DNA excision with two gRNAs

doi: 10.1093/nar/gkag205

Figure Lengend Snippet: Kinetics of the SaCas9 RNP complex. ( A ) Left: Schematics of DNA constructs used in SaCas9 RNP beacon 1 and beacon 2 assays. Target sequences and PAMs are shown in red and green, respectively. Right: Diagrams of SaCas9/gRNA RNP interaction with the beacons. Fluorophore (F, green) and quencher (Q, black) are indicated. ( B ) Binding kinetics of SaCas9/gRNA RNP complexes with beacon 1. The x -axis shows time (s), with fluorescence measured every 2 s. The y -axis represents normalized fluorescence intensity (%), scaled from 0% (minimum) to 100% (maximum) within each subgroup. ( C ) Cleavage kinetics of SaCas9/gRNA RNP complexes with beacon 2, displayed as in panel (B). ( D ) Correlation between excision efficiency and SaCas9/gRNA RNP kinetics. The plot shows the relationship between in vitro kinetic parameters ( y -axis: dissociation constant, Kd) and excision efficiency in cured cultures, with excision efficiencies labeled as percentages.

Article Snippet: The SaCas9 expression plasmid p6XHis_NLS-SaCas9 (Addgene #101086) was transformed into Escherichia coli Rosetta (DE3) competent cells (Novagen).

Techniques: Construct, Binding Assay, Fluorescence, In Vitro, Labeling