Supplementary Figure 9: Identification of FACS-enriched in situ transconjugants of multi-vector libraries. | Nature Methods

Supplementary Figure 9: Identification of FACS-enriched in situ transconjugants of multi-vector libraries.

From: Metagenomic engineering of the mammalian gut microbiome in situ

Supplementary Figure 9

(a) Flow cytometric quantification of in situ transconjugants in the total bacterial population after gavage of EcGT2 donors containing pGT-L4 (green; n = 4 mice) or pGT-L5 (blue; n = 4 mice) vector libraries. Control groups gavaged with PBS (black; n = 2 mice) or donors containing a nontransferable pGT-NT vector (red; n = 2 mice) produced no detectable transconjugants. Black bars indicate means. (b) Longitudinal analysis of mouse fecal microbiome by flow cytometry for presence of transconjugants after gavage of EcGT2 donors containing pGT-L4 (green; n = 6 mice) or pGT-L5 (blue; n = 6 mice). Donor cells of these libraries (orange; n = 12 mice) were lost within 48 h, whereas transconjugants were observed up to 72 h post-gavage. The dotted line indicates the detection limit of flow cytometry. Error bars indicate s.d. (c) 16S taxonomic classification of transconjugants (GFP+mCh) enriched by FACS of pGT-L4 and pGT-L5 recipient groups at 6 h post-gavage. Relative abundance of each OTU in the unsorted population is shown in the grayscale heat map on the left, and fold enrichment for transconjugants of each OTU is shown in the orange heat map on the right, with annotated taxonomic identities. Bracketed values indicate confidence of taxonomic assignment by RDP Classifier. Each column represents data from 6 mice from 2 independent cohorts whose fecal samples were combined for analysis. Genera with successfully cultivated isolates are denoted by stars.

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