Singleplex Assay for Function Measurements v1

This protocol outlines an assay for measuring the function of plasmid variants in singleplex. The inputs include separate E. coli glycerol stocks for each variant. The protocol begins with several growths which convert the separate glycerol stocks into cultures that have reach stationary phase in a 96-well plate. The glycerol stocks are first grown overnight in separate tubes. The next morning, the optical density (OD) of each culture is measured, and then each culture is distributed into a 96-well growth plate. This plate is placed in a plate reader/incubator to grow to stationary phase (~12 hours) without antibiotics or additives (except those required for plasmid maintenance). After this point, the cultures are ready to act as an inputs for next growth cycle. Throughout the subsequent growths, optical density (OD) and fluorescent measurements are recommended to be taken every 5 minutes and at the end of each growth plate's incubation. The growth cycles are all ~3 hours long, so that cells stay in mid-log phase. At the end of the last growth cycle, a small amount of the plated cultures act as input for quantification using a flow-cytometer and fluorescent beads. Note that unlike the related fitness assays (e.g., https://www.protocols.io/view/singleplex-assay-for-fitness-measurements-c9vdz626), this assay does not use a selection antibiotic. Contents of each growth plates 2-3: Growth plate 2: This growth plate contains additives to initiate gene expression, but no selection antibiotic. Growth plate 3: This growth plate contains additives to initiate gene expression, but no selection antibiotic.