Computational protocol: Autoinducer-2 Quorum Sensing Contributes to Regulation of Microcin PDI in Escherichia coli

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Protocol publication

[…] Isolated colonies were inoculated into 5-mL LB media with appropriate antibiotic and grown as described. Overnight culture was diluted (1:100) into fresh M9 media (10 mL) and grown overnight at 37°C with 200 rpm shaking until OD600 ~ 0.6 at which point 0.02% (w/v) L-arabinose was added for 24 h at room temperature with shaking at 200 rpm. Total proteins were collected by centrifugation at 18,000 × g at 4°C for 5 min. Cell pellets were resuspended in 1x laemmli sample buffer (Bio-Rad) and boiled for 10 min. Any kD Tris-glycine precast gels (Bio-Rad) were used for SDS-PAGE protein separation. A Trans-Blot turbo transfer starter system (Bio-Rad) was used to transfer proteins onto a low-fluorescence polyvinylidene fluoride membrane (Bio-Rad) and Ponceau S stain was used to verify protein transfer prior to addition of antibodies for specific protein detection. Primary antibody anti-His-tag (1:1,000; Thermo Scientific) was used with secondary goat anti-mouse antibody (1:5,000; DyLight 650, conjugate). A ChemiDoc MP Imaging System (Bio-Rad) was used to detect fluorescent signal and band intensity was quantified with ImageJ software (Schneider et al., ). A ratio of McpM value to DnaK value served to normalize and quantify and are represented by arbitrary unit (AU). [...] Where appropriate, a one-way analysis of variance (ANOVA) was used to compare experimental results with a Dunnett's one-way multiple pairwise comparison test. Depending on the experimental design, a two-way ANOVA was used in conjunction with a Tukey's all pairwise multiple comparison test (SigmaPlot version 12.5; Systat Software, Inc., San Jose, CA). […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Escherichia coli