(a) Implementation of FACS enrichment of in vitro conjugations. (b) Conjugations between EcGT2 harboring vector libraries pGT-L3, pGT-L7, and pGT-L8 and mouse fecal bacteria were performed aerobically overnight. A mock conjugation using EcGT2 with no vector and a negative control conjugation using the pGT-NT nontransferable vector were also performed. 20,000 FACS-sorted events from Q3 (mCherry–GFP–) and Q4 (mCherry–GFP+) populations were plated on selective and nonselective media and grown aerobically to select for transconjugants. Cultivable aerobic transconjugants of pGT-L3 and pGT-L7 vectors were successfully enriched by FACS, although GFP+ CFUs may appear dim against the autofluorescent media. This experiment was performed independently twice, with similar results.