crispri plasmid backbone  (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