Fig. 1

Workflow of the microfluidic picoliter droplet screening of a fosmid metagenomic library. (a) A library of metagenomic clones in E. coli was encapsulated in droplets under a Poisson distribution (λ = 0.35, i.e., 25% were singly compartmentalized) in the presence of a fluorogenic substrate. (b) After 24 to 72 h incubation, at least 6 million droplets for each condition were sorted using FACS. The droplets exceeding a set product fluorescence threshold (> 5-fold background) were pooled and demulsified (c) before plating the clones. On the 19,501 clones recovered from the six droplet pools after overnight growth on agar plates, a secondary screening was performed on 372 candidates per sorting condition, to quantify their hydrolytic activity at pH 8 and 9. Of 2232 assayed combinations, 144 clones exhibited 2 times the value of a negative clone and were selected for further analysis. (d) The metagenomic DNA inserts (ranging from 30-40 kb) were sequenced. Assignment of clonal and partial redundancies led to identification of novel human glycan utilization pathways. (e) The ability of the different clones to breakdown native, non-fluorogenic human glycans was finally demonstrated by HPAEC-PAD analysis