pJB658-derived plasmids bearing the studied AlkB enzymes were prepared as described before [59 (link)]. Cultures of E. coli strain HK 82/F′ which were transformed with pJB658-derived plasmids bearing the studied AlkB enzymes were induced with 2 mM toluic acid (Sigma) at OD600 = 0.1, and further grown until they reached A600 = 0.5. Bacteria in 500 μL of the culture were pelleted, resus-pended in 250 μL of M9 minimum salt medium, and then mixed with an equal volume of minimum salt medium containing an appropriate concentration of chloroacetaldehyde (CAA). Bacteria were incubated with CAA for 30 min in 37 °C, subsequently diluted 10,000-fold and plated at LB plates. Colonies were counted after 24 h incubation at 37 °C.
Toluic acid
Manufactured by Merck Group
Toluic acid is a chemical compound with the molecular formula C6H5CH3COOH. It is a colorless crystalline solid that is insoluble in water but soluble in organic solvents. Toluic acid has a carboxylic acid group attached to a toluene ring structure.
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Lab products found in correlation
2 protocols using toluic acid
1
Alkylation Repair Activity Assay
2
Complementation of EcAlkB Repair by ALKBH8
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To test the ability of ALKBH8 proteins for complementation of the repair function of EcAlkB protein, pJB658-derived plasmids encoding these proteins were transformed into the F-pilus-expressing, alkB-deficient E. coli strain HK82/F’. Protein expression was induced by the addition of 2 mM toluic acid (Fluka/Sigma-Aldrich). To introduce methyl lesions or etheno adducts, ssDNA bacteriophage M13mp18, ssRNA bacteriophage MS2 or bacteria were treated with methyl methanesulphonate (MMS; Sigma-Aldrich) or chloroacetaldehyde (CAA; Sigma-Aldrich), respectively. Phage and bacteria survival was scored by counting the resulting plaques or bacterial colonies, respectively. The experiments were performed essentially as previously described [38] .
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