growth medium  (Gold Biotechnology Inc)

Journal: bioRxiv

Article Title: Exploiting Endogenous Cas9-Based Genome Editing of Lacticaseibacillus rhamnosus GG (LGG) for Precision Food Fermentation and Live Biotherapeutics

doi: 10.1101/2025.01.21.634116

Figure Lengend Snippet: (A) Architecture of the CRISPR-Cas locus in LGG. The type II-A CRISPR-Cas9 system contains four Cas protein-encoding genes ( cas1, cas2, csn2 , and the critical cas9 ). The CRISPR array includes 24 spacers (30 bp, blue rectangles) interspaced with repeats (black diamonds). (B) PAM prediction visualized using a frequency plot generated using WebLogo. (C) Schematic of the plasmid interference assay. (D) Interference plasmids with a protospacer (underlined) matching the first spacer of the native CRISPR array, with or without an adjacent PAM (5’-NGAAA-3’), were designed for plasmid interference assays. (E) Transformation efficiencies of interference plasmids compared to the control plasmid pTRK870. CFU: colony forming unit. Distinct letters were employed to denote significant differences ( p < 0.05 ). (F) Structure of the SgRNA, designed by linking the crRNA and tracrRNA with an artificial AAUC loop. (G) Schematic of targeting and editing plasmids. repA and repC , plasmid replication gene; erm , erythromycin resistance gene; HR1 and HR2, the homologous repair templates; Ppgm, the promoter of phosphoglycerate mutase ( Ppgm ) from L. acidophilus NCFM.

Article Snippet: Antibiotic was added to the growth medium when applicable: erythromycin (Em; Gold Biotechnology, St. Louis, MO, USA) 5 µg/ml for LGG and 150 µg/ml for E. coli .

Techniques: CRISPR, Generated, Plasmid Preparation, Transformation Assay, Control