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BD), 2 mg/L mycobactin J (Allied Monitor Inc., Fayette, MO, USA) and 0.4% glycerol (Fisher Scientific, Fair Lawn, NJ, USA) was used to culture MAP A1-157, which is associated to a major clade accounting for over 80% of all MAP isolates found in Canada ( 2.2 Creation of MAP mutants 2.2.1 Construction of allelic exchange substrates Cosmid pYUB854 (kindly supplied by the Jacobs lab, Albert Einstein College of Medicine) was employed to generate the allelic exchange substrates (AESs), as previously described ( 2.2.2 Creating the specialized transducing phage Temperature-sensitive shuttle phasmid phAE159 (45 kb), which contain an ampicillin resistance cassette and replicates as a lytic phage at 30C, was employed to generate the specialized transducing mycobacteriophage ( 2 , 2 mM CaCl 2 )