Last year, we encountered some issues with the validity of using a plating assay to characterize the growth of our operon. As we measured the dynamic range of growth of each promoter-riboswitch pair, we realized that the reliability of simply counting circles of growth and examining the density of the growth by eye was not sufficient. Due to the need for more quantitative data, we decided to transition from serial dilutions to a growth-based viability assay to allow for high throughput characterization of our operon. To carry out our growth-based viability assay, bacteria were first incubated in variations of the inhibitory condition, which included a standard level of chloramphenicol and varying levels of fluoride. After overnight growth, we diluted the bacteria into fresh mediums to promote unrestricted growth. Next, we measured the Optical Density (OD) to determine the relative differences between the growth of bacteria based on the initial inhibited condition. OD is measured by a spectrophotometer, which measures the amount of visible light scattered by a cell suspension, and thus demonstrates cell growth and concentration. We expected to see peak growth in a small range of concentrations of fluoride, and our data mostly supported this, although not conclusively. A growth curve was used to plot OD vs. time in order to better view the change in growth (first increasing as the bacteria used the available resources, then decreasing once the bacteria reached carrying capacity) for the various initial conditions. OD was measured in a decimal concentration out of 1, at 600nm. To further quantify the data, we can assign each inhibitory condition with the Standard Growth Time (SGT) of the bacteria with in it, which describes the time it takes for bacteria within each condition for reach a pre-specified Optical Density. Thus, the SGT assay also allows for better reproducibility, and better predictions for future usage. As we established SGT values are established for varying concentrations of fluoride in the initial conditions, future serums can be adjusted so that SGT values found can be matched with our established values.
