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. Author manuscript; available in PMC: 2019 Jul 14.
Published in final edited form as: Nat Methods. 2019 Jan 14;16(2):167–170. doi: 10.1038/s41592-018-0301-y

Figure 2. Identification and isolation of genetically tractable bacteria from the murine gut using MAGIC.

Figure 2.

(a) Implementation of MAGIC in a murine model with fecal bacterial analysis by FACS, antibiotic selection, and sequencing. (b) FACS dot plots of fecal bacteria, pre- and post-gavage of EcGT2 donors containing pGT-L3 or pGT-L6 vector libraries. Green boxes define the sorted GFP+/mCherry- transconjugant populations. For each vector library, fecal samples from 3 co-housed mice were independently evaluated by flow cytometry with similar results. (c) Longitudinal analysis of fecal microbiome by flow cytometry for presence of EcGT2 pGT-NT donor cells (red triangles, n= 4 mice) and transconjugants of vector libraries pGT-L3 (purple circles, n=3 mice), pGT-L6 (maroon circles, n=3 mice), pGT-NT control (green circles, n= 4 mice), or PBS (no donor) control (orange circles, n=2 mice). Donor cells and transconjugants were lost within 48 hours. The dotted line shows the detection limit. (d) 16S taxonomic classification of transconjugants (GFP+/mCh−) enriched by FACS of pGT-L3 and pGT-L6 recipient groups. Each column represents transconjugants from one mouse. Each OTU’s relative abundance in the total bacterial population is shown in the grayscale heat-map, while each OTU’s fold enrichment among transconjugants is shown in the orange heat-map.. Bracketed values indicate confidence of taxonomic assignment by RDP classifier. Genera with successfully cultivated isolates are denoted by white stars. (e) PCR confirmed the presence of the antibiotic resistance/GFP payload cassette from pGT-L3 and pGT-L6 vectors in diverse isolates that were engineered in the murine gut and isolated by selective plating with carbenicillin or tetracycline. NA indicates 16S sequences that were not available.