Extended Data Fig. 2: The composition of a 21-member synthetic community is independent of the initial complexity, storage method and number of passages.
The heatmap shows the relative abundances of strains within a synthetic microbial community. Each column depicts one replicate. For samples from fresh cultures, three biological replicates are depicted; for cultures from glycerol stocks, both biological and technical replicates are shown. The community was assembled in two different ways: in both cases, the bacterial strains were first grown individually, and then the community was mixed in mGAM according to Step 32A of this protocol. In the first case, the community was assembled from all 30 species (30-spp; see Extended Data Table 1); in the second case, species that were not consistently detectable within the 30-spp community were omitted (21-spp). The nondetectable species included Akkermansia muciniphila, Bifidobacterium adolescentis, Bifidobacterium longum subsp. longum, Bilophila wadsworthia, Lactobacillus paracasei, Blautia obeum, Parabacteroides distasonis and Odoribacter splanchnicus. We did not observe growth of Prevotella copri in the 21-member community. Alternatively, cultures were inoculated from frozen glycerol stocks. These glycerol stocks were prepared from freshly assembled communities after 24-h incubation at 37 °C (Step 32B(iv)). Community composition was determined either directly after 24 h of growth or after subculturing (1:50) for 48 h. In all cases, cell pellets of the cultures were harvested by centrifugation and further processed for DNA extraction and 16S rRNA amplicon sequencing. In the example of the 30-spp community, a stable community of 21 species is formed within the first 24 h. A community with similar composition can be obtained from glycerol stocks or by omitting the strains that cannot grow in the community. For this community, it is therefore possible to work from cryopreserved, pre-assembled communities, which can be tested directly after a single O/N culture, without further passages. However, it is strongly recommended that similar pilot tests are carried out for each community to be tested and that the stability of the cryo-stocks is checked over the duration of the test.
