plasmid pet 20b  (Azenta)

Journal: Cell reports

Article Title: Pseudomonas aeruginosa clinical isolates can encode plastic-degrading enzymes that allow survival on plastic and augment biofilm formation.

doi: 10.1016/j.celrep.2025.115650

Figure Lengend Snippet: Figure 1. Phylogenetic tree analysis of clinically relevant BLAST protein hits A neighbor-joining tree was constructed using the p distance of the protein sequences. Hits are color coded by bacteria. Pap1 is indicated by a star.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial strains E. coli strain BL21 StarTM (DE3) pLysS Invitrogen Cat number: C602003 P. aeruginosa strain PA-W23 Dr Stephan Heeb’s collection, University of Nottingham GCF_003833705.1 P. aeruginosa strain PA14 Laboratory collection GCF_000014625.1 P. aeruginosa strain PAO1 Prof A. Filloux’s collection, Imperial College London GCF_000001405.40 P. aeruginosa strain PAO1 ΔxcpA Durand et al.77 N/A P. aeruginosa strain PAO1 ΔpscN Soscia et al.78 N/A P. aeruginosa strain PAO1 ΔpilAΔfliCΔtpsB4 Garnett et al.79 N/A P. aeruginosa strain PA-W23 Δpap1 This work N/A P. aeruginosa strain PA14/miniTn7-GmlacIq-Ptac-Pap1_noSP This work N/A P. aeruginosa strain PA14/miniTn7-GmlacIq-Ptac This work N/A P. aeruginosa strain PA-W23/miniTn7-GmlacIq-Ptac-Pap1_noSP This work N/A P. aeruginosa strain PA-W23/miniTn7-GmlacIq-Ptac This work N/A P. aeruginosa strain PA-W23 Δpap1/ miniTn7-Gm-lacIq-Ptac-Pap1_noSP This work N/A P. aeruginosa strain PA-W23 Δpap1/ miniTn7-Gm-lacIq-Ptac This work N/A Chemicals, peptides, and recombinant proteins LB broth Miller (Luria-Bertani) Difco, Fisher Cat number: BD 244620 Agar-agar Sigma-Aldrich Cat number: 05039 Polycaprolactone Mn 80,000 Sigma-Aldrich Cat number: 440744 M9 minimal medium salts MP Biomedicals Cat number: 3037032 Pseudomonas isolation agar Merck Cat Number: 17208-500G PageRuler Plus Prestained Protein Lader Fisher Scientific Cat Number: 26619 EZBlue Coomassie Brilliant Blue Merck Cet Number: G1041-500ML Deposited data P. aeruginosa PA-W23 RNA-Seq data Gene Expression Omnibus GSE275972 Oligonucleotides pap1 fw PstI Integrated DNA Technologies N/A pap1 rv HindIII Integrated DNA Technologies N/A pap1 up fw Integrated DNA Technologies N/A pap1 up rv Integrated DNA Technologies N/A pap1 down fw Integrated DNA Technologies N/A pap1 down rv Integrated DNA Technologies N/A Recombinant DNA pET-20b(+)-pap1_SP GenScript Gene accession RPM52859.1 pET-20b(+)-pap1_noSP GenScript, His Tag added N/A pET-20b(+)-pap1_SP_Y172W GenScript N/A pUC18T-miniTn7T-Gm-lacIq-Ptac (pJM101) AddGene #110558 pJM101-pap1_noSP This work, His Tag added.

Techniques: Construct, Bacteria

Journal: Cell reports

Article Title: Pseudomonas aeruginosa clinical isolates can encode plastic-degrading enzymes that allow survival on plastic and augment biofilm formation.

doi: 10.1016/j.celrep.2025.115650

Figure Lengend Snippet: Figure 4. P. aeruginosa PA-W23 degrades PCL using Pap1 (A) PCL clear zone assay after 4 days testing P. aeruginosa PA-W23, its derivative Δpap1 mutant, and the complemented mutant (Δpap1 with pap1_noSP expressed from miniTn7 transposon); mean and SD of 3 repeats are represented; ****p < 0.0001. Empty vector (EV) controls can be seen in Figure S4. (B) PCL clear zone assay after 4 days testing PA14 WT, WT with EV, and overexpressor (WT with Pap1_noSP) and PAO1 WT, WT with EV, and overexpressor (WT with Pap1_noSP); mean and SD of 3 biological replicates are represented. Statistical analysis was done by two-way ANOVA with Tukey’s correction. ****p < 0.0001; ns, non-significant. (C) SDS-PAGE gel image of secreted protein fractions from P. aeruginosa PA14, PA-W23, and the complemented Δpap1 mutant, all carrying a miniTn7 insertion to induce the expression of pap1 with the lacIq-Ptac system. The secreted protein fractions were obtained from cell-free culture supernatants of these strains after growing in the presence or absence of 1 mM IPTG for 18 h (as detailed in the STAR Methods). The red arrow indicates the band corresponding to Pap1 (theoretical molecular weight of 30.7 kDa). One representative image out of 3 biological replicates is shown. (D) SDS-PAGE gel image of secreted protein fractions from P. aeruginosa PAO1 and its mutant derivative ΔxcpA (T2SS defective), both carrying a miniTn7 insertion to induce the expression of pap1 with the lacIq-Ptac system. The secreted protein fractions were obtained from cell-free culture supernatants of these strains after growing in the presence or absence of IPTG 1 mM for 18 h (as detailed in the STAR Methods). The red arrow indicates the band corresponding to Pap1. One representative image out of 3 biological replicates is shown.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial strains E. coli strain BL21 StarTM (DE3) pLysS Invitrogen Cat number: C602003 P. aeruginosa strain PA-W23 Dr Stephan Heeb’s collection, University of Nottingham GCF_003833705.1 P. aeruginosa strain PA14 Laboratory collection GCF_000014625.1 P. aeruginosa strain PAO1 Prof A. Filloux’s collection, Imperial College London GCF_000001405.40 P. aeruginosa strain PAO1 ΔxcpA Durand et al.77 N/A P. aeruginosa strain PAO1 ΔpscN Soscia et al.78 N/A P. aeruginosa strain PAO1 ΔpilAΔfliCΔtpsB4 Garnett et al.79 N/A P. aeruginosa strain PA-W23 Δpap1 This work N/A P. aeruginosa strain PA14/miniTn7-GmlacIq-Ptac-Pap1_noSP This work N/A P. aeruginosa strain PA14/miniTn7-GmlacIq-Ptac This work N/A P. aeruginosa strain PA-W23/miniTn7-GmlacIq-Ptac-Pap1_noSP This work N/A P. aeruginosa strain PA-W23/miniTn7-GmlacIq-Ptac This work N/A P. aeruginosa strain PA-W23 Δpap1/ miniTn7-Gm-lacIq-Ptac-Pap1_noSP This work N/A P. aeruginosa strain PA-W23 Δpap1/ miniTn7-Gm-lacIq-Ptac This work N/A Chemicals, peptides, and recombinant proteins LB broth Miller (Luria-Bertani) Difco, Fisher Cat number: BD 244620 Agar-agar Sigma-Aldrich Cat number: 05039 Polycaprolactone Mn 80,000 Sigma-Aldrich Cat number: 440744 M9 minimal medium salts MP Biomedicals Cat number: 3037032 Pseudomonas isolation agar Merck Cat Number: 17208-500G PageRuler Plus Prestained Protein Lader Fisher Scientific Cat Number: 26619 EZBlue Coomassie Brilliant Blue Merck Cet Number: G1041-500ML Deposited data P. aeruginosa PA-W23 RNA-Seq data Gene Expression Omnibus GSE275972 Oligonucleotides pap1 fw PstI Integrated DNA Technologies N/A pap1 rv HindIII Integrated DNA Technologies N/A pap1 up fw Integrated DNA Technologies N/A pap1 up rv Integrated DNA Technologies N/A pap1 down fw Integrated DNA Technologies N/A pap1 down rv Integrated DNA Technologies N/A Recombinant DNA pET-20b(+)-pap1_SP GenScript Gene accession RPM52859.1 pET-20b(+)-pap1_noSP GenScript, His Tag added N/A pET-20b(+)-pap1_SP_Y172W GenScript N/A pUC18T-miniTn7T-Gm-lacIq-Ptac (pJM101) AddGene #110558 pJM101-pap1_noSP This work, His Tag added.

Techniques: Mutagenesis, Plasmid Preparation, SDS Page, Expressing, Molecular Weight

Journal: Cell reports

Article Title: Pseudomonas aeruginosa clinical isolates can encode plastic-degrading enzymes that allow survival on plastic and augment biofilm formation.

doi: 10.1016/j.celrep.2025.115650

Figure Lengend Snippet: Figure 5. Transcriptional response to PCL exposure (A) Volcano plot representing dRNA-seq results from comparing global transcription in the presence of PCL in starvation media (10% LB). According to the dRNA- seq results, 65 genes were upregulated (red) and 36 were downregulated (blue) more than 1 logFC in the presence of PCL. Genes linked to fatty acid metabolism are highlighted in green. (B) Expression of pap1 in the presence and absence of PCL. Comparison of constitutively expressed housekeeping gene rpoB also shown. ns, non-significant.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial strains E. coli strain BL21 StarTM (DE3) pLysS Invitrogen Cat number: C602003 P. aeruginosa strain PA-W23 Dr Stephan Heeb’s collection, University of Nottingham GCF_003833705.1 P. aeruginosa strain PA14 Laboratory collection GCF_000014625.1 P. aeruginosa strain PAO1 Prof A. Filloux’s collection, Imperial College London GCF_000001405.40 P. aeruginosa strain PAO1 ΔxcpA Durand et al.77 N/A P. aeruginosa strain PAO1 ΔpscN Soscia et al.78 N/A P. aeruginosa strain PAO1 ΔpilAΔfliCΔtpsB4 Garnett et al.79 N/A P. aeruginosa strain PA-W23 Δpap1 This work N/A P. aeruginosa strain PA14/miniTn7-GmlacIq-Ptac-Pap1_noSP This work N/A P. aeruginosa strain PA14/miniTn7-GmlacIq-Ptac This work N/A P. aeruginosa strain PA-W23/miniTn7-GmlacIq-Ptac-Pap1_noSP This work N/A P. aeruginosa strain PA-W23/miniTn7-GmlacIq-Ptac This work N/A P. aeruginosa strain PA-W23 Δpap1/ miniTn7-Gm-lacIq-Ptac-Pap1_noSP This work N/A P. aeruginosa strain PA-W23 Δpap1/ miniTn7-Gm-lacIq-Ptac This work N/A Chemicals, peptides, and recombinant proteins LB broth Miller (Luria-Bertani) Difco, Fisher Cat number: BD 244620 Agar-agar Sigma-Aldrich Cat number: 05039 Polycaprolactone Mn 80,000 Sigma-Aldrich Cat number: 440744 M9 minimal medium salts MP Biomedicals Cat number: 3037032 Pseudomonas isolation agar Merck Cat Number: 17208-500G PageRuler Plus Prestained Protein Lader Fisher Scientific Cat Number: 26619 EZBlue Coomassie Brilliant Blue Merck Cet Number: G1041-500ML Deposited data P. aeruginosa PA-W23 RNA-Seq data Gene Expression Omnibus GSE275972 Oligonucleotides pap1 fw PstI Integrated DNA Technologies N/A pap1 rv HindIII Integrated DNA Technologies N/A pap1 up fw Integrated DNA Technologies N/A pap1 up rv Integrated DNA Technologies N/A pap1 down fw Integrated DNA Technologies N/A pap1 down rv Integrated DNA Technologies N/A Recombinant DNA pET-20b(+)-pap1_SP GenScript Gene accession RPM52859.1 pET-20b(+)-pap1_noSP GenScript, His Tag added N/A pET-20b(+)-pap1_SP_Y172W GenScript N/A pUC18T-miniTn7T-Gm-lacIq-Ptac (pJM101) AddGene #110558 pJM101-pap1_noSP This work, His Tag added.

Techniques: Expressing, Comparison

Journal: Cell reports

Article Title: Pseudomonas aeruginosa clinical isolates can encode plastic-degrading enzymes that allow survival on plastic and augment biofilm formation.

doi: 10.1016/j.celrep.2025.115650

Figure Lengend Snippet: Figure 6. Impact of PCL on P. aeruginosa PA-W23 biofilm and virulence (A) PA14 and PA-W23 biofilm formation via crystal violet staining with and without PCL bead added to culture. Mean and SD of 3 repeats; statistical analysis using one-way ANOVA with multiple comparisons; *p < 0.05. Normalized to strain without PCL as 100%. (B) P. aeruginosa PA-W23 biofilm assay with PCL beads in each well. P. aeruginosa PA-W23 Δpap1, Δpap1 with EV, and Δpap1 complemented with Pap1_noSP; mean and SD of three technical replicates and two biological replicates; statistical analysis using one-way ANOVA with multiple comparisons; **p < 0.01; ****p < 0.0001. Normalized to WT as 100%. (C) P. aeruginosa PA-W23 biofilm assay without PCL beads. P. aeruginosa PA-W23 Δpap1, Δpap1 with EV, and Δpap1 complemented with Pap1_noSP; mean and SD of three technical replicates and two biological replicates; statistical analysis using one-way ANOVA with multiple comparisons resulting in no significant differences. Normalized to WT as 100%. (D) Survival of G. mellonella with and without a PCL implant, injected with either phosphate-buffered saline (PBS) as a control or P. aeruginosa PA-W23. Three biological replicates were performed with a minimum of 10 larvae per condition per biological replicate (n = 30). Kaplan-Meier survival curves were used to visualize data, and statistical analysis was performed with a log rank test. *p < 0.05.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial strains E. coli strain BL21 StarTM (DE3) pLysS Invitrogen Cat number: C602003 P. aeruginosa strain PA-W23 Dr Stephan Heeb’s collection, University of Nottingham GCF_003833705.1 P. aeruginosa strain PA14 Laboratory collection GCF_000014625.1 P. aeruginosa strain PAO1 Prof A. Filloux’s collection, Imperial College London GCF_000001405.40 P. aeruginosa strain PAO1 ΔxcpA Durand et al.77 N/A P. aeruginosa strain PAO1 ΔpscN Soscia et al.78 N/A P. aeruginosa strain PAO1 ΔpilAΔfliCΔtpsB4 Garnett et al.79 N/A P. aeruginosa strain PA-W23 Δpap1 This work N/A P. aeruginosa strain PA14/miniTn7-GmlacIq-Ptac-Pap1_noSP This work N/A P. aeruginosa strain PA14/miniTn7-GmlacIq-Ptac This work N/A P. aeruginosa strain PA-W23/miniTn7-GmlacIq-Ptac-Pap1_noSP This work N/A P. aeruginosa strain PA-W23/miniTn7-GmlacIq-Ptac This work N/A P. aeruginosa strain PA-W23 Δpap1/ miniTn7-Gm-lacIq-Ptac-Pap1_noSP This work N/A P. aeruginosa strain PA-W23 Δpap1/ miniTn7-Gm-lacIq-Ptac This work N/A Chemicals, peptides, and recombinant proteins LB broth Miller (Luria-Bertani) Difco, Fisher Cat number: BD 244620 Agar-agar Sigma-Aldrich Cat number: 05039 Polycaprolactone Mn 80,000 Sigma-Aldrich Cat number: 440744 M9 minimal medium salts MP Biomedicals Cat number: 3037032 Pseudomonas isolation agar Merck Cat Number: 17208-500G PageRuler Plus Prestained Protein Lader Fisher Scientific Cat Number: 26619 EZBlue Coomassie Brilliant Blue Merck Cet Number: G1041-500ML Deposited data P. aeruginosa PA-W23 RNA-Seq data Gene Expression Omnibus GSE275972 Oligonucleotides pap1 fw PstI Integrated DNA Technologies N/A pap1 rv HindIII Integrated DNA Technologies N/A pap1 up fw Integrated DNA Technologies N/A pap1 up rv Integrated DNA Technologies N/A pap1 down fw Integrated DNA Technologies N/A pap1 down rv Integrated DNA Technologies N/A Recombinant DNA pET-20b(+)-pap1_SP GenScript Gene accession RPM52859.1 pET-20b(+)-pap1_noSP GenScript, His Tag added N/A pET-20b(+)-pap1_SP_Y172W GenScript N/A pUC18T-miniTn7T-Gm-lacIq-Ptac (pJM101) AddGene #110558 pJM101-pap1_noSP This work, His Tag added.

Techniques: Staining, Biofilm Production Assay, Injection, Saline, Control

Journal: Cell reports

Article Title: Pseudomonas aeruginosa clinical isolates can encode plastic-degrading enzymes that allow survival on plastic and augment biofilm formation.

doi: 10.1016/j.celrep.2025.115650

Figure Lengend Snippet: Figure 7. The role of 6OH-HA in PA-W23 biofilm formation (A) Extracted ion chromatogram of 6OH-HA (quantifier ion) in biofilms isolated from PA-W23 (blue), PA-W23 Δpap1 (green), and com- plemented strain (orange), magnified peak of in- terest: 1.82 min (inset). (B) Quantification of 6OH-HA in biofilms isolated from PA-W23, PA-W23 Δpap1, and com- plemented strain. Mean and SD of four biological replicates are represented. Statistical analysis was performed using one-way ANOVA with mul- tiple comparisons; *p < 0.05. (C) Quantification of 6OH-HA in supernatant iso- lated from cultures of PA-W23, PA-W23 Δpap1, and complemented strain and uninoculated LB that was incubated with a PCL bead. Mean and SD of three biological replicates are repre- sented. Statistical analysis was performed using one-way ANOVA with multiple comparisons; ****p < 0.0001. (D) Exogenous supplementation of media with 2.17 mg/mL 6OH-HA enhanced PA-W23 biofilm formation. Mean and SD of six biological repli- cates are represented; **p < 0.01 with Student’s t test.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial strains E. coli strain BL21 StarTM (DE3) pLysS Invitrogen Cat number: C602003 P. aeruginosa strain PA-W23 Dr Stephan Heeb’s collection, University of Nottingham GCF_003833705.1 P. aeruginosa strain PA14 Laboratory collection GCF_000014625.1 P. aeruginosa strain PAO1 Prof A. Filloux’s collection, Imperial College London GCF_000001405.40 P. aeruginosa strain PAO1 ΔxcpA Durand et al.77 N/A P. aeruginosa strain PAO1 ΔpscN Soscia et al.78 N/A P. aeruginosa strain PAO1 ΔpilAΔfliCΔtpsB4 Garnett et al.79 N/A P. aeruginosa strain PA-W23 Δpap1 This work N/A P. aeruginosa strain PA14/miniTn7-GmlacIq-Ptac-Pap1_noSP This work N/A P. aeruginosa strain PA14/miniTn7-GmlacIq-Ptac This work N/A P. aeruginosa strain PA-W23/miniTn7-GmlacIq-Ptac-Pap1_noSP This work N/A P. aeruginosa strain PA-W23/miniTn7-GmlacIq-Ptac This work N/A P. aeruginosa strain PA-W23 Δpap1/ miniTn7-Gm-lacIq-Ptac-Pap1_noSP This work N/A P. aeruginosa strain PA-W23 Δpap1/ miniTn7-Gm-lacIq-Ptac This work N/A Chemicals, peptides, and recombinant proteins LB broth Miller (Luria-Bertani) Difco, Fisher Cat number: BD 244620 Agar-agar Sigma-Aldrich Cat number: 05039 Polycaprolactone Mn 80,000 Sigma-Aldrich Cat number: 440744 M9 minimal medium salts MP Biomedicals Cat number: 3037032 Pseudomonas isolation agar Merck Cat Number: 17208-500G PageRuler Plus Prestained Protein Lader Fisher Scientific Cat Number: 26619 EZBlue Coomassie Brilliant Blue Merck Cet Number: G1041-500ML Deposited data P. aeruginosa PA-W23 RNA-Seq data Gene Expression Omnibus GSE275972 Oligonucleotides pap1 fw PstI Integrated DNA Technologies N/A pap1 rv HindIII Integrated DNA Technologies N/A pap1 up fw Integrated DNA Technologies N/A pap1 up rv Integrated DNA Technologies N/A pap1 down fw Integrated DNA Technologies N/A pap1 down rv Integrated DNA Technologies N/A Recombinant DNA pET-20b(+)-pap1_SP GenScript Gene accession RPM52859.1 pET-20b(+)-pap1_noSP GenScript, His Tag added N/A pET-20b(+)-pap1_SP_Y172W GenScript N/A pUC18T-miniTn7T-Gm-lacIq-Ptac (pJM101) AddGene #110558 pJM101-pap1_noSP This work, His Tag added.

Techniques: Isolation, Incubation