LDM protocol for estimating plasmid conjugation rates v3

This is a general protocol to implement the LDM approach for estimating plasmid conjugation rates. This version of the protocol was originally submitted with the accompanying manuscript. The recommended version (i.e., the most up-to-date) of the LDM protocol will be linked in the corresponding GitHub repository. To get a conceptual overview of the approach, please read the accompanying manuscript. The protocol is intentionally designed to facilitate proper implementation by explicitly verifying that the modeling assumptions are met. We provide 5 streamlined phases, where the last phase involves executing the LDM conjugation assay. The initial 4 phases are to gather the necessary information for the proper execution of the LDM conjugation assay (i.e., phase 5). Here, we provide a brief description and goals for the initial 4 phases. Phase 1: Minimum inhibitory concentration assay with the transconjugant-selecting medium. The goal is to find a concentration of selective agents (e.g., antibiotics) in the transconjugant-selecting medium that inhibits donors and recipients while simultaneously permitting the growth of transconjugants. Phase 2: Extinction probability assays for selective liquid and agar media. The goal is to check the assumption that the focal cell type will successfully establish a lineage under the appropriate selective conditions. Phase 3: Growth rate assay. The goal is to determine the donor- and recipient-specific incubation times that will ensure the cultures enter exponential growth before initiating the conjugation assay. Phase 4: Finding an incubation time and initial densities for executing the LDM conjugation assay. The goal is to find a combination of incubation time and initial densities such that the probability of a conjugation event occurring is between 0 and 1 (i.e., where there will be a mixture of turbid and non-turbid co-cultures after incubation with transconjugant-selecting medium). Phase 5: LDM conjugation assay. The goal is to gather experimental estimates for the variables in the LDM equation to calculate an estimate of the conjugation rate.