Singleplex Assay for Fitness Measurements v1

This protocol outlines an assay for the singleplex measurement the fitness of plasmid variants in E.coli. This protocol is used during onboarding of a new protein function for the Pooled, Growth-Based Assay. In this protocol, clonal bacterial stocks are measured instead of a pooled mixture, and the end-point OD measured after each growth cycle is used as a proxy for the DNA barcode count data that is produced by the Pooled, Growth-Based Assay. The fitness estimates from the end-point OD measurements are used to fine-tune the plasmid circuit and the assay conditions in advance of running the Pooled, Growth-Based Assay. 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), with optical density (OD) and fluorescent measurements being taken every 5 minutes and at the end of each growth plate's incubation. After this point, the cultures are ready to act as a inputs for subsequent growth cycles. The four subsequent growth cycles (i.e., timepoints 1-4) are all ~3 hours long, so that cells stay in mid-log phase. Small amount of the plated cultures at the end of each cycle acting as input for the subsequent growth cycle. The OD measured at the end of each growth cycle (i.e., the end-point OD) will be used in the downstream fitness calculation. Contents of each growth plate for timepoints 1-4 (growth plates 2-5): Timepoint 1: This growth plate contains additives to initiate gene expression, but no selection antibiotic. Timepoint 2: This growth plate contains additives to initiate gene expression and the selection antibiotic. Timepoint 3: This growth plate contains additives to initiate gene expression and the selection antibiotic. Timepoint 4: This growth plate contains additives to initiate gene expression and the selection antibiotic.