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The FRT-flanked kan gene was eliminated from strain JW1380-KC (Keio collection)42, using flipase-mediated recombination43. Strain JW1380-KC was transformed with plasmid pCP20, and ampicillin-resistant transformants were selected at 30 °C, after which a few were colony-purified once nonselectively at 42 °C and then tested for loss of all antibiotic resistances. The majority lost the kan resistance gene and the FLP helper plasmid simultaneously, resulting in strain BHYT in which the feaB gene was deleted. The deletion was verified by PCR.
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