Journal: mSystems

Article Title: A Reproducible and Tunable Synthetic Soil Microbial Community Provides New Insights into Microbial Ecology

doi: 10.1128/msystems.00951-22

Figure Lengend Snippet: Schematic of synthetic rhizosphere community generation using a liquid-handling machine with a piezo dispense capillary (PDC) device. (A) Isolates were grown for 3 to 4 days in liquid R2A medium, OD 600 -normalized to 0.025, and loaded into individual wells in the probe plate. The PDC drew liquid up from one well of the probe plate and dispensed a programmed number of drops in the desired wells of the target plate. This process was repeated for each isolate to result in a final mixed community. The communities were grown aerobically for the desired amount of time and then analyzed for composition and diversity via 16S rRNA sequencing. (B–D) Individual cells visualized within the nozzle of the PDC prior to dispensing. The machine identifies cells matching a tunable selection criterion (red circles), such as circularity, elongation, and maximum diameter. An initial test prior to generating the communities is shown here, using the machine’s CellenONE module. Droplet generation using various dilutions of the community members was tested to ensure that only 2 to 3 cells were repeatedly detected in the ejection zone of the nozzle (left of the green line) Examples using the organism Lysobacter are shown in panels B and C. An OD 600 dilution to 0.025 of each community member ensured 2 to 3 cells per droplet (droplet volume 390 to 420 pL). (D) Image of the PDC when dispensing sterile PBS. The black dots on the left edge of the ejection zone are background noise on the surface of the nozzle and are not cells.

Article Snippet: Individual isolates were grown in 3 to 5 mL liquid cultures of 1× R2A medium (Teknova, cat number R0005) in 14 mL culture tubes in aerobic conditions at 30°C without shaking.

Techniques: Sequencing, Selection, Sterility

Journal: mSystems

Article Title: A Reproducible and Tunable Synthetic Soil Microbial Community Provides New Insights into Microbial Ecology

doi: 10.1128/msystems.00951-22

Figure Lengend Snippet: Community diversity with hand-assembly and media dilutions. (A) OD 600 values of human-assembled (human) and machine-assembled communities (machine) over 3 days (72 h) of growth ( n = 6 to 8 each). (B) Bray-Curtis distances of 16S rRNA gene amplicon sequencing between replicates of the hand-assembled and machine-assembled communities. The hand-assembled communities are shown combined and broken into the four individuals. (One-way ANOVA with the Benjamini-Hochberg [BH] FDR correction; *, P < 0.05; ****, P < 0.001) (C) OD 600 values of the machine-assembled, equally mixed communities in 1×, 0.2×, and 0.1× R2A media over 3 days ( n = 8 each). (D) Observed OTUs (left) and Shannon diversity index (right) of media dilution communities (Student’s t test; *, P < 0.05; ***, P < 0.005). (E) Heat map of taxonomy relative abundance (genus level) of media dilution communities from 16S sequencing, with the genera differentially abundant from the 1× condition being marked with asterisks (DESeq2 Wald test; fitType = “parametric” with the BH FDR; *, P -adj < 0.06; ***, P -adj < 0.0001). Taxonomic order was determined via PCoA clustering of the Bray-Curtis distance. Replicates for each condition were merged for the heat map with the Phyloseq command merge_samples (group = “Media_dilution”).

Article Snippet: Individual isolates were grown in 3 to 5 mL liquid cultures of 1× R2A medium (Teknova, cat number R0005) in 14 mL culture tubes in aerobic conditions at 30°C without shaking.

Techniques: Amplification, Sequencing

Journal: mSystems

Article Title: A Reproducible and Tunable Synthetic Soil Microbial Community Provides New Insights into Microbial Ecology

doi: 10.1128/msystems.00951-22

Figure Lengend Snippet: Community growth and composition with plant colonization and following cryopreservation. (A and B) An equally mixed community was inoculated onto 12-day-old Brachypodium plants, with or without Pseudomonas , and was allowed to grow for 7 days. (A) PCoA of the Bray-Curtis distances between the rhizosphere communities grown on plants for 7 days ( n = 5 each) and the original inoculant ( n = 1). (B) Relative abundance heat map of the starting inoculum and the rhizosphere communities grown on plants for 7 days. The rug plot indicates the presence (black) or absence (red) of Pseudomonas in the inoculant. Differential abundances between communities with and without Pseudomonas are marked with asterisks (DESeq2, Wald test, fitType = “parametric” with the BH FDR; * , P < 0.05). (C and D) An equally mixed community was preserved with 20% glycerol, 20% DMSO, or lyophilization and was regrown in R2A medium (left panel), MS medium (middle panel), or PBS with 10% sucrose (right panel). The growth and composition were compared to a community that was not frozen. (C) Community growth measured through the OD 600 . The unfrozen control community is shown in red. ( n = 1) (D) A comparison of the log 10 (relative abundance) from the 16S sequencing between the frozen and unfrozen communities for each cryopreservation method. Each point represents the log 10 (relative abundance) of an individual genus in the frozen versus unfrozen community. Pearson’s correlation coefficient is reported for each comparison.

Article Snippet: Individual isolates were grown in 3 to 5 mL liquid cultures of 1× R2A medium (Teknova, cat number R0005) in 14 mL culture tubes in aerobic conditions at 30°C without shaking.

Techniques: Lyophilization, Comparison, Sequencing