crispri backbone pljr962 125  (Addgene inc)

Journal: BMC Microbiology

Article Title: Effects of CwlM, a peptidoglycan synthesis regulator, on beta-lactam tolerance and host-pathogen interactions

doi: 10.1186/s12866-025-04548-6

Figure Lengend Snippet: CRISPRi-mediated silencing of the cwlM gene in M. smegmatis and resulting phenotypical and mRNA expression differences. a sgRNA-mediated repression of the cwlM gene in M. smegmatis , detailing target site (+ 467 bps) and the employed PAM strength (PAM14). b The local context of the cwlM gene in M. smegmatis . The cwlM gene is non-operonic, being preceded by the trx gene and succeeded by MSMEG_6936 , which is transcribed in the opposite direction. c Relative gene expression of cwlM normalized to sigA , with (stripped bars) and without (smooth bars) 100 ng/mL of ATc treatment for 6 h, evaluated through qRT-PCR assays ( n = 2). PLJR962 is a non-targeting empty vector control. The mRNA expression levels of the calibrator sample ( M. smegmatis WT) are shown as dashed lines. The error bars represent the standard error of the mean (SEM). Comparisons between multiple groups were made using one-way ANOVA, with significance levels: * P < 0.05; ** P < 0.01. d Spotting dilution assays of the negative controls ( M. smegmatis WT, PLJR962) and of the cwlM knockdown mutant ( n = 3). The first spots were normalized to an OD 600 of 0.001, and the subsequent spots correspond to two-fold serial dilutions

Article Snippet: Escherichia coli ( E. coli ) strains were grown in Luria-Bertani (LB) media (Merck), overnight (ON) at 37 °C, to amplify the CRISPRi backbone PLJR962 (Addgene #115162) and for cloning.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Plasmid Preparation, Control, Knockdown, Mutagenesis

Journal: BMC Microbiology

Article Title: Effects of CwlM, a peptidoglycan synthesis regulator, on beta-lactam tolerance and host-pathogen interactions

doi: 10.1186/s12866-025-04548-6

Figure Lengend Snippet: Antibiotic susceptibility assays of the negative controls and the cwlM knockdown mutant. Heatmap of the fold differences in the minimum inhibitory concentrations (MICs; in µg/mL) between the cwlM knockdown mutant, the non-targeting control PLJR962 and M. smegmatis WT, with and without 100 ng/mL of ATc. The red shades depict an increase in the MIC while the blue shades depict a decrease in the MIC, when compared to the WT strain. AMX, amoxicillin; CLA, clavulanate; CTX, cefotaxime; EMB, ethambutol; INH, isoniazid; MEM, meropenem; VAN, vancomycin

Article Snippet: Escherichia coli ( E. coli ) strains were grown in Luria-Bertani (LB) media (Merck), overnight (ON) at 37 °C, to amplify the CRISPRi backbone PLJR962 (Addgene #115162) and for cloning.

Techniques: Knockdown, Mutagenesis, Control

Journal: bioRxiv

Article Title: Phase separation of mycobacterial Rho factor is associated with acid stress

doi: 10.1101/2025.08.28.672908

Figure Lengend Snippet: (A) Wild-type strain harbouring the CRISPRi system with different sgRNAs for the promoter region of rho and three positive controls ( DNAβ, MSMEG_0317 , and rho ). (B) Wild-type strain containing the CRISPRi plasmid and a second copy of rho . Strains with pJR962 derivatives expressing sgRNAs were grown to the stationary phase in TSBT in the absence of aTc. The cells were serially diluted 10-fold, and 5 µL of each dilution was spotted onto TSA without (left) or with (right) aTc, which induces expression of the CRISPRi system. Positive controls were designed by .

Article Snippet: The sgRNA oligos were cloned into the BsmBI-digested CRISPRi backbone (plJR962 - Addgene plasmid #115162).

Techniques: Plasmid Preparation, Expressing

Journal: bioRxiv

Article Title: Phase separation of mycobacterial Rho factor is associated with acid stress

doi: 10.1101/2025.08.28.672908

Figure Lengend Snippet: Localization patterns for Msm Rho and its derivatives after 4-hour exposure to TSBT or pH 4 (citrate/phosphate buffer): (A) Wild-type Rho; (B) ΔIDR; (C) Δinitial; (D) Δinsertion. All cultures were supplemented with aTc to repress the native copy of rho through the CRISPRi system. Demographs on the right display the normalized Dendra intensity as heatmaps along the medial axis of single cells (n = 75) which are sorted by length. All analyses were carried out from three independent experiments, and representative images are shown. All scale bars, 2 µm.

Article Snippet: The sgRNA oligos were cloned into the BsmBI-digested CRISPRi backbone (plJR962 - Addgene plasmid #115162).

Techniques: