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( A ) Chemoreceptor genes were collected from 24 <t>Pseudomonas</t> strains and amplified by PCR to generate a library of 975 sequences. ( B ) The pooled chemoreceptor library was cloned under the control of an IPTG-inducible P lac UV5 promoter and chromosomally integrated into Pseudomonas putida <t>KT2440</t> ΔmcpP (strain MC105) via CRAGE-mediated recombination at a defined landing pad. ( C ) The resulting library population (LS18) was subjected to soft-agar assays for functional enrichment of chemoreceptors based on chemotactic expansion. Cells were collected from the leading edge of expanding populations, followed by DNA extraction and sequencing to quantify enrichment.
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( A ) Chemoreceptor genes were collected from 24 <t>Pseudomonas</t> strains and amplified by PCR to generate a library of 975 sequences. ( B ) The pooled chemoreceptor library was cloned under the control of an IPTG-inducible P lac UV5 promoter and chromosomally integrated into Pseudomonas putida <t>KT2440</t> ΔmcpP (strain MC105) via CRAGE-mediated recombination at a defined landing pad. ( C ) The resulting library population (LS18) was subjected to soft-agar assays for functional enrichment of chemoreceptors based on chemotactic expansion. Cells were collected from the leading edge of expanding populations, followed by DNA extraction and sequencing to quantify enrichment.
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( A ) Chemoreceptor genes were collected from 24 <t>Pseudomonas</t> strains and amplified by PCR to generate a library of 975 sequences. ( B ) The pooled chemoreceptor library was cloned under the control of an IPTG-inducible P lac UV5 promoter and chromosomally integrated into Pseudomonas putida <t>KT2440</t> ΔmcpP (strain MC105) via CRAGE-mediated recombination at a defined landing pad. ( C ) The resulting library population (LS18) was subjected to soft-agar assays for functional enrichment of chemoreceptors based on chemotactic expansion. Cells were collected from the leading edge of expanding populations, followed by DNA extraction and sequencing to quantify enrichment.
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( A ) Chemoreceptor genes were collected from 24 Pseudomonas strains and amplified by PCR to generate a library of 975 sequences. ( B ) The pooled chemoreceptor library was cloned under the control of an IPTG-inducible P lac UV5 promoter and chromosomally integrated into Pseudomonas putida KT2440 ΔmcpP (strain MC105) via CRAGE-mediated recombination at a defined landing pad. ( C ) The resulting library population (LS18) was subjected to soft-agar assays for functional enrichment of chemoreceptors based on chemotactic expansion. Cells were collected from the leading edge of expanding populations, followed by DNA extraction and sequencing to quantify enrichment.

Journal: bioRxiv

Article Title: A pooled screening approach reveals bacterial chemoreceptors for short-chain carboxylic acids

doi: 10.64898/2026.04.10.717710

Figure Lengend Snippet: ( A ) Chemoreceptor genes were collected from 24 Pseudomonas strains and amplified by PCR to generate a library of 975 sequences. ( B ) The pooled chemoreceptor library was cloned under the control of an IPTG-inducible P lac UV5 promoter and chromosomally integrated into Pseudomonas putida KT2440 ΔmcpP (strain MC105) via CRAGE-mediated recombination at a defined landing pad. ( C ) The resulting library population (LS18) was subjected to soft-agar assays for functional enrichment of chemoreceptors based on chemotactic expansion. Cells were collected from the leading edge of expanding populations, followed by DNA extraction and sequencing to quantify enrichment.

Article Snippet: We used a wild-type Pseudomonas putida KT2440 (ATCC 47054) as a host of a chemoreceptor gene library.

Techniques: Amplification, Clone Assay, Control, Functional Assay, DNA Extraction, Sequencing

( A ) Time-course of colony expansion for Pseudomonas putida KT2440 on soft-agar plates containing glycerol alone or glycerol supplemented with 1 mM lactate. ( B ) Quantification of expansion rates under glycerol-only and glycerol + lactate conditions. ( C ) Expansion rates of wild-type (WT), ΔmcpP (MC105), and ΔmcpP strains carrying the chemoreceptor library (LS18) without (−) or with (+) IPTG induction in the presence of lactate. ( D ) Representative images of colony expansion under the indicated conditions. ( E ) Expansion rates measured in the presence of propionate, showing similar trends as observed with lactate. Data are shown as mean ± s.d. (n = 3 biological replicates). Statistical significance was assessed using a two-tailed Welch’s t-test (B) and ordinary one-way ANOVA (C, E); ns, not significant; *P < 0.05; **P < 0.01.

Journal: bioRxiv

Article Title: A pooled screening approach reveals bacterial chemoreceptors for short-chain carboxylic acids

doi: 10.64898/2026.04.10.717710

Figure Lengend Snippet: ( A ) Time-course of colony expansion for Pseudomonas putida KT2440 on soft-agar plates containing glycerol alone or glycerol supplemented with 1 mM lactate. ( B ) Quantification of expansion rates under glycerol-only and glycerol + lactate conditions. ( C ) Expansion rates of wild-type (WT), ΔmcpP (MC105), and ΔmcpP strains carrying the chemoreceptor library (LS18) without (−) or with (+) IPTG induction in the presence of lactate. ( D ) Representative images of colony expansion under the indicated conditions. ( E ) Expansion rates measured in the presence of propionate, showing similar trends as observed with lactate. Data are shown as mean ± s.d. (n = 3 biological replicates). Statistical significance was assessed using a two-tailed Welch’s t-test (B) and ordinary one-way ANOVA (C, E); ns, not significant; *P < 0.05; **P < 0.01.

Article Snippet: We used a wild-type Pseudomonas putida KT2440 (ATCC 47054) as a host of a chemoreceptor gene library.

Techniques: Two Tailed Test