Journal: Mlife
Article Title: Optimizing a high‐sensitivity NanoLuc‐based bioluminescence system for in vivo evaluation of antimicrobial treatment
doi: 10.1002/mlf2.12091
Figure Lengend Snippet: Construction of stable and functional Nluc‐based luciferase‐producing Staphylococcus aureus reporter strains. (A) Western blot analysis of the expression of PBP2a, Eno, CobB, and SarA in S. aureus USA300 over time. The protein gel served as a loading control (Lc), and the molecular weights of the protein markers (M) are indicated on the left. (B) Semi‐quantitative analysis of the gray values of the indicated bands in each lane of (A) using ImageJ software. Data are presented as mean ± SEM. Statistical significance was calculated by two‐way analysis of variance (ANOVA); ns indicates no significance, ** p < 0.01, and *** p < 0.001. (C) Western blot analysis of Eno fusion proteins in the total cell lysates using mouse anti‐Eno polyclonal antibodies. (D) Bioluminescence (BL) photographs of different S. aureus reporter strain and substrate combinations. (E) Representative BL images of 50 µl of S. aureus reporter strains (1 × 10 7 CFU/ml) mixed with 50 µl of different doses of HFZ, FUR, or DTZ (3.125–100 µM). (F) Quantification of total flux produced from S. aureus reporter strains with different concentrations of substrates. Data are presented as mean ± SEM. Statistical significance was analyzed by two‐way ANOVA; ns indicates no significance, * p < 0.05, ** p < 0.01, and *** p < 0.001. (G) Images of BL signals from USA300/Eno‐Nluc, USA300/Eno‐Teluc, and USA300/Eno‐Antares2 over a range of bacterial loads with 50 µl of HFZ (100 µM) in the black 96‐well plates. (H) Good correlation between the BL intensity and bacterial number of S. aureus USA300/Eno‐Nluc, USA300/Eno‐Teluc, or USA300/Eno‐Antares2. The experiment was repeated at least three times in triplicate. Data are presented as mean ± SEM. x , bacterial number; y , BL intensity.
Article Snippet: The nucleotide sequences of nluc, teluc , and antares2 according to the codon usage bias of S. aureus USA300 were chemically synthesized by BGI‐Shenzhen (China) and cloned into the pUC18 plasmid to achieve pUC‐ nluc , pUC‐ teluc , and pUC‐ antares2 , respectively (Table ).
Techniques: Functional Assay, Luciferase, Western Blot, Expressing, Control, Software, Produced
