biolog tm phenotype microarray plates 18c  (Biolog Inc)

Journal: NPJ Systems Biology and Applications

Article Title: Addressing genome scale design tradeoffs in Pseudomonas putida for bioconversion of an aromatic carbon source

doi: 10.1038/s41540-024-00480-z

Figure Lengend Snippet: A Serial dilution plating for growth on solid agar medium supplemented with rich (LB) or p -CA minimal medium. Abbreviated genotypes are indicated to the left of each image. B The native PP_0897 promoter was replaced with two different low abundance promoters and the impact on viability in rich medium or M9 p- CA minimal medium was quantified. C P. putida strains assayed for indigoidine production in M9 60 mM p -CA medium at the indicated time points following induction with 1.5% arabinose (w/v). Control is WT P. putida harboring genome integrated indigoidine pathway cassette and ∆PP_0897 is the single gene deletion strain. Strain D1b_gf ∆PP_0897 did not grow nor produced in M9 p- CA medium. D P. putida strains with promoter-varied PP_0897 expression assayed for indigoidine production. Specific indigoidine yield (mg/CFU) was calculated by normalizing the indigoidine with the number of total viable cells at 24 h. E P. putida strains with promoter-varied PP_0897 expression with or without induction of the indigoidine pathway in M9 60 mM p- CA, sampled at 24 h and 48 h for glutamate and indigoidine (Supplementary Fig. ). F , G Strains D1b_gf with or without ∆PP_0897 were transformed with an empty vector plasmid or a plasmid containing a P BAD -PP_0897 construct. F Growth in M9 60 mM p- CA medium with basal BAD promoter induction without inducer ( F ) or with 1.5% (w/v) arabinose ( G ) in 24 well microtiter dishes. Strain genotypes are indicated above each panel. Measurements are average of at least three independent replicates. Error bars represent mean ± S.D. ( n = 4) in ( C – E ). The shaded area represents mean ± S.D. ( n = 3) in ( F , G ).

Article Snippet: A BIOLOG TM phenotype microarray assay (Cat. No. 12111, & Cat. No. 12112, Hayward CA) was conducted using strain D1b_gf ΔPP_0897.

Techniques: Serial Dilution, Control, Produced, Expressing, Transformation Assay, Plasmid Preparation, Construct

Journal: NPJ Systems Biology and Applications

Article Title: Addressing genome scale design tradeoffs in Pseudomonas putida for bioconversion of an aromatic carbon source

doi: 10.1038/s41540-024-00480-z

Figure Lengend Snippet: A Overall workflow of the simulations performed to model the permissible flux space limiting growth coupled production. Proteomics data, the D1b_gf reduced model, and the iMAT algorithm are used to develop context-specific models, which are then used for sensitivity analysis. A double robustness analysis is applied to determine the tradeoff between growth represented as biomass formation flux, fumarase flux, and glutamine exchange reaction flux, removing glutamine from the system represented as glutamine flux. Double robustness analysis 3D plots of the genome-scale metabolic model (GSMM) of P. putida WT (iJN1462) ( B ), context-specific D1b_gf reduced model ( C ) and context-specific D1b_gf P PP_0415 -PP_0897 model (iMAT_PP_0415p_PP_0897) ( D ). A red box indicates the region of interest replotted in the detailed view. The units for fumarase and glutamine flux are mmol/gDCW/h, and the unit for biomass is h −1 . Infinity (Inf) represents unconstrained flux for the reaction and may have a variable value based on the definition of bounds for a GSMM.

Article Snippet: A BIOLOG TM phenotype microarray assay (Cat. No. 12111, & Cat. No. 12112, Hayward CA) was conducted using strain D1b_gf ΔPP_0897.

Techniques:

Journal: NPJ Systems Biology and Applications

Article Title: Addressing genome scale design tradeoffs in Pseudomonas putida for bioconversion of an aromatic carbon source

doi: 10.1038/s41540-024-00480-z

Figure Lengend Snippet: A Initial auxotrophy analysis and complementation via ectopic plasmid expression of the Design 1 cutset strain, D1b_gf ∆PP_0897 transformed with P BAD - PP_0897 Refer to Fig. for complementation of the PP_0897 deletion mutant on M9 p -CA medium. B Heatmap showing selected BIOLOG TM metabolite sources that rescued growth under different media conditions including basal media supplemented with 50 mM p -CA and/or 100 mM l -malate.

Article Snippet: A BIOLOG TM phenotype microarray assay (Cat. No. 12111, & Cat. No. 12112, Hayward CA) was conducted using strain D1b_gf ΔPP_0897.

Techniques: Plasmid Preparation, Expressing, Transformation Assay, Mutagenesis

Journal: NPJ Systems Biology and Applications

Article Title: Addressing genome scale design tradeoffs in Pseudomonas putida for bioconversion of an aromatic carbon source

doi: 10.1038/s41540-024-00480-z

Figure Lengend Snippet: A Model validation using BIOLOG TM phenotyping. Blue dots indicate each of the 190 metabolites tested (refer to Supplementary Table for details), and the light blue shaded region shows the accurately predicted metabolites, including 16 true positives and 75 true negatives. Red dots indicate the subset of metabolites that successfully rescued growth when fed both p -CA and l -malate. B Double robustness analysis on mixed carbon source utilization for growth and production. C – E Strains D1b_gf and D1b_gf ∆PP_0897 were fed three carbon sources, 50 mM p -CA, 100 mM malate, and 100 mM alanine. Samples were analyzed at the indicated time points for growth and metabolic profiles. C Optical density (OD 600 ) was monitored every 4 h. D Glutamate concentrations for strains D1b_gf and D1b_gf ∆PP_0897, intracellular and extracellular values pooled together. E Consumption profiles of the three carbon sources (malate, alanine, and p -CA) from the culture medium were monitored at the indicated time points using LC–MS. Error bars represent mean ± S.D. ( n = 3) in ( C ). The shaded region represents mean ± S.D. ( n = 3) in ( D and E ).

Article Snippet: A BIOLOG TM phenotype microarray assay (Cat. No. 12111, & Cat. No. 12112, Hayward CA) was conducted using strain D1b_gf ΔPP_0897.

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: The Cell Surface

Article Title: Biochemical and phenotypic characterisation of the Mycobacterium smegmatis transporter UspABC

doi: 10.1016/j.tcsw.2021.100052

Figure Lengend Snippet: Carbon source utilisation profiles of M. smegmatis , M. smegmatis Δ uspC and M. smegmatis Δ uspAEC mutants. XY plot of M. smegmatis WT (black), M. smegmatis Δ uspC (red) and Δ uspAEC (blue) grown on Biolog Phenotype MicroArray TM PM01 (A) and PM02A (B) plates. x-axis is time in hours and y-axis is respiration signal. The respiration signal is used as a read-out for bacterial growth. The individual carbon source plate maps are shown in Supplementary . (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: XY plot of M. smegmatis WT (black), M. smegmatis Δ uspC (red) and Δ uspAEC (blue) grown on Biolog Phenotype MicroArray TM PM01 (A) and PM02A (B) plates. x-axis is time in hours and y-axis is respiration signal.

Techniques: Microarray

Journal: The Cell Surface

Article Title: Biochemical and phenotypic characterisation of the Mycobacterium smegmatis transporter UspABC

doi: 10.1016/j.tcsw.2021.100052

Figure Lengend Snippet: Heat map and hierarchical clustering depending on the carbon substrate utilisation of Δ uspC and Δ uspAEC mutant strains grown in Phenotype Microarray (Biolog) plates PM01 and PM02A . A + B: Δ uspC vs M. smegmatis WT (A: PM01, B: PM02A). C + D: Δ uspAEC vs M. smegmatis WT (C: PM01, D: PM02A). Red represents high substrate utilisation and blue represents low substrate utilisation compared to WT M. smegmatis over 96 h. Carbon sources in red text indicated increased growth and carbon sources in blue font indicate reduced growth of the mutant strains compared to WT. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: XY plot of M. smegmatis WT (black), M. smegmatis Δ uspC (red) and Δ uspAEC (blue) grown on Biolog Phenotype MicroArray TM PM01 (A) and PM02A (B) plates. x-axis is time in hours and y-axis is respiration signal.

Techniques: Mutagenesis, Microarray