Growth Assay:Article Title: Role of Ring6 in the Function of the E. coli MCE Protein LetB.
Article Snippet: .. LSB (Sigma-Aldrich, catalog #D0431) and cholate (Alfa Aesar, catalog #A17074-18) sensitivity of E. coli strains was based upon a previously described growth assay on LB agar plates.22,23 To improve discrimination between WT and mutant strains, we systematically explored two variables that we suspected may modulate the phenotype: (1) the preparation of the LB agar; and (2) the growth phase of the cultures used for dilutions and plating. .. The manner in which the LB Agar was prepared had a significant effect on the level of LSB and cholate sensitivity of E. coli strains, and consequently on the optimal concentrations of LSB and cholate required; similar observations have been previously reported for other bacterial growth phenotypes.27 We prepared LB agar from separate components (“LB Agar 1”: 10 g Tryptone (Gibco, catalog #211705, 10 g NaCl (SigmaAldrich, catalog #S3014), 5 g Yeast extract (Gibco, catalog #212750, 15 g Agar (BD Difco, catalog #214530) per 1L H2O); by combining pre-mixed LB broth (BD Difco, catalog #244620) with agar (“LB Agar 2”); or a complete LB + agar pre-mix (“LB Agar 3”; BD Difco, catalog # DF0445-07-6) and found that LB Agar 3 requires about 10x less of the additive lauryl LSB ( 0.1%) or 2 less cholate ( 4.5–5%) than LB Agar 1 to achieve the same degree of bacterial growth inhibition (Figure S1(F)).
Article Title: Role of Ring6 in the function of the E. coli MCE protein LetB
Article Snippet: .. LSB (Sigma-Aldrich, catalog #D0431) and cholate (Alfa Aesar, catalog #A17074-18) sensitivity of E. coli strains was based upon a previously described growth assay on LB agar plates ( , ). ..
Mutagenesis:Article Title: Role of Ring6 in the Function of the E. coli MCE Protein LetB.
Article Snippet: .. LSB (Sigma-Aldrich, catalog #D0431) and cholate (Alfa Aesar, catalog #A17074-18) sensitivity of E. coli strains was based upon a previously described growth assay on LB agar plates.22,23 To improve discrimination between WT and mutant strains, we systematically explored two variables that we suspected may modulate the phenotype: (1) the preparation of the LB agar; and (2) the growth phase of the cultures used for dilutions and plating. .. The manner in which the LB Agar was prepared had a significant effect on the level of LSB and cholate sensitivity of E. coli strains, and consequently on the optimal concentrations of LSB and cholate required; similar observations have been previously reported for other bacterial growth phenotypes.27 We prepared LB agar from separate components (“LB Agar 1”: 10 g Tryptone (Gibco, catalog #211705, 10 g NaCl (SigmaAldrich, catalog #S3014), 5 g Yeast extract (Gibco, catalog #212750, 15 g Agar (BD Difco, catalog #214530) per 1L H2O); by combining pre-mixed LB broth (BD Difco, catalog #244620) with agar (“LB Agar 2”); or a complete LB + agar pre-mix (“LB Agar 3”; BD Difco, catalog # DF0445-07-6) and found that LB Agar 3 requires about 10x less of the additive lauryl LSB ( 0.1%) or 2 less cholate ( 4.5–5%) than LB Agar 1 to achieve the same degree of bacterial growth inhibition (Figure S1(F)).
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