Journal: bioRxiv

Article Title: A whole organism screening platform identifies gut microbiome microproteins that modulate host metabolism

doi: 10.64898/2026.02.11.705328

Figure Lengend Snippet: A . A diverse 236-member library of gut-derived putative secreted microproteins was shortlisted via bioinformatics. From an existing dataset of microbial smORFs , those derived from gut metagenomic samples and predicted to be secreted were selected. Removal of redundant sequences resulted in a list of 2,412 total proteins. Representatives from each microprotein family were chosen to maximise protein diversity B . The smORF library was synthesised as an oligo pool, cloned into a pET9a expression vector using pooled SapI-mediated Golden Gate Assembly, then transformed into E. coli Lemo21. Positive control strains expressing the nematicidal toxin Cry6A and a reduced-function mutation, Cry6Am, were also produced. The resulting cloned expression library was arrayed into 96-well microplates, which included strains expressing 126 unique microproteins. To screen, bacteria were grown as lawns and Day 1 adult C. elegans N2 worms were allowed to crawl freely across the lawn. After 4 hours of exposure, worm behaviour was recorded for ∼16 minutes and quantified using a semi-automated worm tracking and statistical analysis pipeline. C . Hierarchical clustering of worm behavioural fingerprints in response to control, Cry6A, or Cry6Am exposure. Number of replicate wells screened: Cry6A_a: 43; Cry6A_b: 159; Cry6Am_a: 49; Cry6Am_b: 104; Control_a: 47; Control_b: 264. Each column corresponds to an individual feature from the Tierpsy 48 feature set, and each row shows the average Z-normalised feature value for that feature across all replicate wells of given condition. Rows were clustered using average linkage hierarchical clustering with Euclidian distance computed across all features of the matrix. Columns are coloured based on whether the feature name contains one of the key words listed below the heatmap. Rows are coloured according to the experimental condition.

Article Snippet: The purified PCR products (Monarch PCR and DNA Cleanup Kit, New England Biolabs) were cloned into pET9a_linker using SapI-mediated Golden Gate Assembly (New England Biolabs), and the resulting plasmids were transformed into E. coli Lemo21 and plated onto chloramphenicol/kanamycin selection plates.

Techniques: Derivative Assay, Clone Assay, Expressing, Plasmid Preparation, Transformation Assay, Positive Control, Mutagenesis, Produced, Bacteria, Control

Journal: bioRxiv

Article Title: A whole organism screening platform identifies gut microbiome microproteins that modulate host metabolism

doi: 10.64898/2026.02.11.705328

Figure Lengend Snippet: A . A diverse 236-member library of gut-derived putative secreted microproteins was shortlisted via bioinformatics. From an existing dataset of microbial smORFs , those derived from gut metagenomic samples and predicted to be secreted were selected. Removal of redundant sequences resulted in a list of 2,412 total proteins. Representatives from each microprotein family were chosen to maximise protein diversity B . The smORF library was synthesised as an oligo pool, cloned into a pET9a expression vector using pooled SapI-mediated Golden Gate Assembly, then transformed into E. coli Lemo21. Positive control strains expressing the nematicidal toxin Cry6A and a reduced-function mutation, Cry6Am, were also produced. The resulting cloned expression library was arrayed into 96-well microplates, which included strains expressing 126 unique microproteins. To screen, bacteria were grown as lawns and Day 1 adult C. elegans N2 worms were allowed to crawl freely across the lawn. After 4 hours of exposure, worm behaviour was recorded for ∼16 minutes and quantified using a semi-automated worm tracking and statistical analysis pipeline. C . Hierarchical clustering of worm behavioural fingerprints in response to control, Cry6A, or Cry6Am exposure. Number of replicate wells screened: Cry6A_a: 43; Cry6A_b: 159; Cry6Am_a: 49; Cry6Am_b: 104; Control_a: 47; Control_b: 264. Each column corresponds to an individual feature from the Tierpsy 48 feature set, and each row shows the average Z-normalised feature value for that feature across all replicate wells of given condition. Rows were clustered using average linkage hierarchical clustering with Euclidian distance computed across all features of the matrix. Columns are coloured based on whether the feature name contains one of the key words listed below the heatmap. Rows are coloured according to the experimental condition.

Article Snippet: After linearising the pET9a_SC_mut using primers NS20 and NS21 to introduce necessary overhangs, a linker sequence containing two SapI sites was introduced by HiFi Assembly (New England Biolabs), forming pET9a_linker.

Techniques: Derivative Assay, Clone Assay, Expressing, Plasmid Preparation, Transformation Assay, Positive Control, Mutagenesis, Produced, Bacteria, Control