Pooled, Growth-Based Assays v1

This protocol outlines a pooled, growth-based assay for measuring the fitness of a library of variants in E.Coli. This protocol defines paths for either: only measuring a pool of control variants or an entire library of variants pooled in addition to the controls. The inputs for this protocol are: 1) barcoded normalization variants, 2) barcoded calibration variants, and depending on the branch taken 3) a pooled library of barcoded variants containing 100,000-500,000 members as well. The protocol begins with several growths which convert the separate glycerol stocks into pooled cultures that have reach stationary phase in a 96-well plate. The glycerol stocks are first grown overnight in separate tubes and flasks. The next morning, the optical density (OD) of each culture is measured, after which all cultures are pooled into a single flask and grown for 4-5 hours to achieve one doubling. The OD of this pooled culture is then measured again before distributing it 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). These cultures are then used as an input for the next growth cycle (i.e., timepoint 1), explained in further detail in the next section. The 4 subsequent growth cycles (i.e., timepoints 1-4) will produce culture samples that will eventually be sequenced and used in the fitness calculation. These growth cycles (i.e., timepoints 1-4) are all ~3 hours long, so that cells stay in mid-log phase. At the end of each growth cycle, a small amount of each culture acts as input for the subsequent growth cycle, while the remaining culture is processed for downstream measurement (see the Notes section below for details on subsequent steps). The media for the first of these 4 growth cycles (i.e., timepoint 1) contains only additives to initiate gene expression (i.e., inducers), but no selection antibiotic. The media for the following 3 growth cycles contain both the additives and the selection antibiotic. Notes: Throughout all growths involving 96-well plates, OD and fluorescent measurements are recommended to be taken every 5 minutes and at the end of each growth plate's incubation. The OD measured at the end of each ~3 hour growth should be constant or just slightly decreasing across timepoints 1-4. Immediately after each growth plate is done incubating and a sample from each well has been transferred to the next growth plate, perform a DNA extraction protocol on the remaining culture in each well. After DNA extraction is complete for each timepoint, you can proceed to the Automated Bar-Seq Library Preparation and Pooling protocol