Journal: Nucleic Acids Research

Article Title: PE-STAR: prime editing with SOS-triggered and RecJ-augmented repair enables high-efficiency editing in Escherichia coli

doi: 10.1093/nar/gkag285

Figure Lengend Snippet: Large-fragment genome integration enabled by a PASTE-like two-step attB – attP recombination workflow using PE-STAR. ( A ) Schematic overview of the two-stage integration strategy. In phase I, PE-STAR–mediated prime editing installs a 46 bp attB site at the chromosomal xylB locus. In phase II, Bxb1 integrase catalyzes site-specific recombination between the genomic attB site and a donor plasmid–borne attP site, enabling targeted chromosomal integration of large DNA payloads, including a 3.2 kb GFP cassette or an 8.0 kb riboflavin biosynthetic pathway. ( B ) Transformation efficiencies (CFU per μg DNA) following attB–attP recombination for GFP and riboflavin donor constructs. ( C ) Phenotypic validation of large-fragment integration. Left, fluorescence microscopy confirming robust GFP expression in GFP integration strains. Right, colony morphology and liquid culture appearance of riboflavin-producing strains compared with non-integrated controls, showing characteristic yellow pigmentation indicative of riboflavin accumulation. ( D ) Growth profiles (OD 600 ) of the riboflavin-integrated strain and the control strain during 96 h fermentation. ( E ) Time-course quantification of riboflavin production by the PASTE-like/riboflavin strain during 96 h fermentation. Data are presented as mean ± s.d. ( n = 3 independent biological replicates).

Article Snippet: The column temperature was maintained at 30°C, the injection volume was 15 μl, and the total run time was 40 min. Riboflavin was detected at 370 nm [ ], with a retention time of ~5 min. For calibration, 20 mg of riboflavin standard (Product B21290-20 mg; Shanghai Yuanye Bio-Technology Co., Ltd., China) was dissolved in 0.01 mol/l hydrochloric acid and diluted to a final volume of 200 ml, yielding a stock solution with a concentration of 0.1 mg/ml.

Techniques: Plasmid Preparation, Transformation Assay, Construct, Biomarker Discovery, Fluorescence, Microscopy, Expressing, Control