empty sgrna (triple- bsai ) expression cassette  (Addgene inc)

Journal: Archives of Microbiology

Article Title: Generation and validation of a versatile inducible multiplex CRISPRi system to examine bacterial regulation in the Euprymna-Vibrio fischeri symbiosis

doi: 10.1007/s00203-025-04354-8

Figure Lengend Snippet: SgRNA spacer cloning. A the triple BsaI TIIS cloning site allows for efficient cloning of 20-nt spacer sequences in-frame with the adjacent sgRNA framework sequence. B Ligation of annealed 20-nt targeting oligos into the resulting empty BsaI-cut cloning site. The spacer shown is the (RR1) spacer used in this study to target the mRFP gene

Article Snippet: For the single (no targeting spacer sequence) sgRNA plasmid psgRNA, the empty sgRNA (triple- BsaI ) expression cassette from plasmid pJMP1339 (Addgene #119271) was PCR amplified with tail primers that provided ~ 30 bp homologous end overlap to the pVSV105 backbone PCR amplicon (See Table S2 for sequences).

Techniques: Cloning, Sequencing, Ligation

Journal: Archives of Microbiology

Article Title: Generation and validation of a versatile inducible multiplex CRISPRi system to examine bacterial regulation in the Euprymna-Vibrio fischeri symbiosis

doi: 10.1007/s00203-025-04354-8

Figure Lengend Snippet: Multiplexed sgRNA repression of mRFP fluorescence. A Cultures grown with no IPTG; B Cultures grown with 2 mM IPTG induction of repression to an OD 600 of 0.4, with triplicate 200 mL aliquots assayed for fluorescence in a 96-well multimodal plate-reader. Specific fluorescence (relative fluorescence units/OD 600 ; excitation 584 nm, emission 607 nm) of the single and multiplexed sgRNA plasmid carrying strains was normalized to the control strain ES114:JMP1189 (mRFP + , no sgRNA). Columns indicate mean values of three independent experiments (biological replicates). Different letters on the abscissa denotate significant differences between groups according to the Tukey post-hoc comparison

Article Snippet: For the single (no targeting spacer sequence) sgRNA plasmid psgRNA, the empty sgRNA (triple- BsaI ) expression cassette from plasmid pJMP1339 (Addgene #119271) was PCR amplified with tail primers that provided ~ 30 bp homologous end overlap to the pVSV105 backbone PCR amplicon (See Table S2 for sequences).

Techniques: Fluorescence, Plasmid Preparation, Control, Comparison

Journal: Archives of Microbiology

Article Title: Generation and validation of a versatile inducible multiplex CRISPRi system to examine bacterial regulation in the Euprymna-Vibrio fischeri symbiosis

doi: 10.1007/s00203-025-04354-8

Figure Lengend Snippet: Multiplexed sgRNA repression of luminescence. ES114:JMP1189/pMM(RR1, LC1, FA1) cells were sub-cultured in SWT with the indicated levels of IPTG inducer and grown to an OD600 of 2.0, with triplicate 200 mL aliquots assayed for luminescence in a 96-well multimodal plate-reader. The specific luminescence (relative luminescence units/OD 600 ) of the single and multiplexed sgRNA plasmid carrying strains was normalized to the control strain ES114. Columns reflect mean values; error bars indicate standard deviations. Different letters on the abscissa denotate significant differences between groups according to the Tukey post-hoc comparison

Article Snippet: For the single (no targeting spacer sequence) sgRNA plasmid psgRNA, the empty sgRNA (triple- BsaI ) expression cassette from plasmid pJMP1339 (Addgene #119271) was PCR amplified with tail primers that provided ~ 30 bp homologous end overlap to the pVSV105 backbone PCR amplicon (See Table S2 for sequences).

Techniques: Cell Culture, Plasmid Preparation, Control, Comparison