Fig. 1: Workflow of scRACS-Seq/Culture for revealing organic-PSM in the sewage that links genotype to phenotype (metabolic activity). | ISME Communications

Fig. 1: Workflow of scRACS-Seq/Culture for revealing organic-PSM in the sewage that links genotype to phenotype (metabolic activity).

From: Single-cell Raman-activated sorting and cultivation (scRACS-Culture) for assessing and mining in situ phosphate-solubilizing microbes from nature

Fig. 1

① Cells in the sewage were extracted by Nycodenz density gradient separation (NDGS). ② For sorting organic-PSM, the sewage extract was incubated under the conditions of adding lecithin as organic phosphorus in dephosphorization sewage in 50%-D2O for 24 h. ③ Organic-PSM were identified based on the preset C-D ratio threshold (metabolically active) in SCRS, and then sorted out in the RAGE chip, as a one-cell-encapsulated droplet, in an one-cell-one-tube manner. ④ For scRACS-Culture, pL-volume culture was employed for seven days, following by μL volumes for another seven days in broth medium. ⑤ Single-cell-derived organic-PSM cultures were obtained for further validation. ⑥ For scRAGE-Seq, the RAGE-sorted cells were lysed, and then the genomic DNA was amplified by MDA and then processed for 16S rRNA sequencing and whole-genome shotgun sequencing. ⑦ Assembly and annotation of the single-cell shotgun sequencing reads, and then the metabolic pathways were reconstructed to establish the link between genotype (sequencing based) and the metabolic phenotype (SCRS based) at precisely one-cell resolution.

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