SIMPLE METHOD TO EVALUATE STERILIZATION O F RECOMBINANT PSEUDOMONAS CARRYING INSECTICIDAL PROTEIN GENE

A recombinant strain of Pseudomonas fluorescens carrying plasmid pKBR 60, constructed with an insecticidal toxin gene from Bacillus thuringi ensis and a marker gene for tetracycline resistance, was treated with acetic acid or a mixture of iodine and acetic acid to kill it. We esta blished a method to evaluate whether the sterilization of the recombin ant was complete using the different sensitivity of the bacteria again st several antibiotics. Wild-type B. thuringiensis could not grow in a medium containing 500 U/ml penicillin, but the wild-type host P. fluo rescens KPF30 did grow in this medium. P. fluorescens KPF30 could not grow in a medium containing 30 mug/ml tetracycline but the recombinant KPR800 could. The recombinant bacteria treated with acetic acid or th e mixture did not generate any colonies on medium containing either pe nicillin or tetracycline. Thus the sterilization seemed to be complete . Bacteria, like P. aeruginosa, which does not carry the toxin gene or resistance against these two antibiotics, were preferentially segrega ted from bacteria having the gene by colony hybridization.