2.5 Tracking transfer in sewage communities
The sewage community originating from the three WWTPs were supplemented with the E. coli donor harboring R27 or pB10 in a conjugation assay. To each donor/plasmid combination, six biological replicates were made and three controls of each donor and sewage community incubated individually. The dissemination of the plasmid was quantified by FACS, counting transconjugants (GFP positive), donors (mCherry positive) and recipients (colorless). To evaluate the host ranges, and the transconjugants donor potential of R27::gfp and pB10::gfp in influent sewage of Ellinge WWTP, 2 x 10500 transconjugants in 4 replicates, of each plasmid, were sorted out by FACS: one replicate for direct 16S rRNA gene sequencing, a second replicate for enrichment of transconjugants. The sorted sewage community/R27 and sewage community/pB10 were enriched in test tubes with 10% (10x diluted) LB supplemented with antibiotics, 30 ug kanamycin/ml for sewage community/R27 and 10 ug tetracycline/ml for sewage community/pB10, for 24 hours at 30°C with 250 RPM. Prior to conjugation assays, cell counts and purity were assessed by FACS ensuring GFP only from the donors. Three out of four biological replicates for each enriched transconjugant sewage community was found to be applicable. Next, enriched transconjugant sewage community samples underwent conjugation assays with the E. coli recipient in three technical replicates per biological replicate, in order to investigate transfer using FACS. In this scenario transconjugants are characterized by bi-fluorescence events of mCherry and GFP, while donors are exclusively GFP positive and recipients are mCherry positive. In one biological replicate of each mating per plasmid the enriched transconjugant sewage community had outcompeted the recipient and the results were not used. Additionally, control experiments with theE. coli donor and an untagged strain of the E. colirecipient were conducted: Six replicates were made per donor/plasmid combination. See figure 1 a) and b) for an overview of how plasmid transfer was determined.
Plasmid transfer was measured as transconjugants per recipient (T/R), calculated as the number of transconjugant (T) cells divided by the number of recipient cells \(\frac{T}{R}\)