FIGURE 1.

Selection of mutants based on the ability to survive in the presence of alafosfalin. Cells expressing individual clones from each mutant library were grown in the absence (A) and presence (B) of alafosfalin and protein expression was induced at 2.5 h. A₆₀₀ was measured at 1 h and 4.5 h after induction. Representative primary data are shown for the native transporter (WT), the Y156A mutant (Y156A), and the clones later identified as mutants N306I and L324V. Reported A₆₀₀ values are from measurements in 96-well plates, and are ∼2.5 times lower than A₆₀₀ determined using a 1-cm path length. The means of triplicate measurements of A₆₀₀ for the native transporter and the Y156A loss-of-function mutant in the presence of alafosfalin from three individual experiments were compared using a paired t test within the Graphpad Prism 5.0 software, and were found to have a p value <0.0024, indicating that the difference is statistically significant.