Rapid identification of Escherichia coli transformed by pBR322 carrying inserts at the PstI Site

An iodometric assay for β-lactamase has been employed for identifying colonies of Escherichia coli transformed to tetracycline resistance (Tc^r) by pBR322 carrying inserts at the PstI site. This assay is based upon the ability of β-lactamase produced by ampicillin-resistant (Ap^r) cells to convert penicillin to penicilloic acid which in turn binds iodine. Growth and selection of E. coli transformed to Ap^rTc^r or Ap^sT^r are obtained on Luria agar plates containing soluble starch and tetracycline. When indicator solution containing penicillin and iodine is added to the colonized plates, β-lactamase-producing (Ap^r) colonies rapidly clear the overlying indicator solution whereas non-β-lactamase-producing (Ap^s) colonies exhibit no clearing effect. This reaction persists and substantial numbers of viable cells remain well beyond the end of the 15-min observation period. In post-test assessment of phenotype, all nonclearing colonies exhibited the Ap^sTc^r phenotype while those that cleared the indicating solution exhibited the Ap^rTc^r phenotype. Application of this assay to an actual transformation experiment permitted rapid and unambiguous identification of the Ap^sTc^r phenotype.