(a) Illustration of FACS enrichment method to isolate transconjugant cells from complex recipient populations. GFP and mCherry fluorescence are used to gate cell populations consisting of E. coli donors and diverse recipients. Quadrants Q1 and Q2 correspond to donor cells (mCh+), and unmanipulated recipients are in quadrant Q3. Quadrant Q4 contains transconjugants that received the GFP gene cargo and are not naturally mCherry-fluorescent (GFP+mCh–). Q4 cells are isolated and further analyzed. This gating was used to analyze fecal samples from each individual mouse in each in situ experiment, as well as every in vitro conjugation in this study by flow cytometry. (b) To validate the FACS enrichment method, we mixed GFP+ E. coli with a natural mouse fecal bacterial community at given levels (1–100% of population) and retrieved the E. coli by FACS. 16S sequencing of the samples showed that the fluorescent E. coli were efficiently and specifically enriched by FACS. Although the raw Q4 population contained some autofluorescent cells, the only remaining OTU in Q4 after application of an enrichment filter (see Methods) was E. coli.