Computational protocol: Insights on Osmotic Tolerance Mechanisms in Escherichia coli Gained from an rpoC Mutation

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Protocol publication

[…] Perturbations to transcriptional regulation in the presence of 0.6 M NaCl were determined using microarray analysis using strains BW25113, EJW3, MG1655, and EYG1 with two biological replicates each. Samples were cultivated using the same condition as in metabolite analysis with 25 mL cultures and an initial OD600 of ~0.05. When OD600 reached ~0.5, cells were quickly chilled to ≤4 °C on dry ice/isopropanol bath, then harvested by centrifugation (4470× g) at 4 °C followed by immediate re-suspension in 5 mL of RNAlater (Sigma-Aldrich, St. Louis, MO, USA). Total RNA was extracted by using the ZR Fungal/Bacterial RNA MicroPrep™ (Zymo Research, Irvine, CA, USA) kit. A total of 10 μg of the isolated total RNA was mixed with 1.5 μg random primers (Promega, Madison, WI, USA), incubated at 70 °C for 10 min and then cooled on ice (4 °C). The cDNA was synthesized by combining the total RNA mixture with 10 U SuperScript® III reverse transcriptase, 1× first strand buffer, 0.01 M DTT (Invitrogen, Carlsbad, CA, USA), and nucleotides (0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dCTP, 0.2 mM dTTP (Promega, Madison, WI, USA) and 0.3 mM amino-allyl dUTP (Thermo Fisher Scientific, Waltham, MA, USA)), and incubated at 42 °C for 3 h. The cDNA was recovered with ice-cold ethanol precipitation, labeled with either Cy3- or Cy5- mono-reactive dye (GE Healthcare, Little Chalfont, Buckinghamshire, UK), and then hybridized to the E. coli Gene Expression Microarray (Agilent Technologies). The arrays were scanned using the GenePix 4100A Microarray Scanner and the images were analyzed using GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA, USA). The MIDAS software package (TM4) [] was used to normalize the data using LOWESS based normalization algorithm []. The rank product method with a critical p-value < 0.01 was used to identify the differentially expressed genes using MeV (TM4) microarray analysis software []. Gene ontology analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) [,]. […]

Pipeline specifications

Software tools GenePix Pro, TM4, DAVID
Application Gene expression microarray analysis
Organisms Escherichia coli
Chemicals Amino Acids, Methionine, Sodium Chloride, Acetic Acid