Patent Document ID: 20030215799
Application ID: 09808658
Patent Flag: 0

Claim One:
1. A method for the identification of an essential gene responsible for the presence of a specific phenotype in a recombination proficient microorganism comprising: a) contacting In vitro: (i) a transposable element comprising at least one first genetic marker; (ii) a transposase for the insertion of the transposable element into the essential gene; and (iii) target DNA containing the essential gene, said gene having a homolog in the genome of the recombination proficient microorganism; under suitable conditions whereby the transposable element inserts within the essential gene to form a transposon disrupted gene; b) cloning the transposon disrupted gene into a suitable vector to form a chromosomal integration vector, said vector comprising at least one second genetic marker; c) transforming a recombination proficient microorganism which is not naturally transformable with the chromosomal integration vector of step (b) to create transformants; d) selecting the transformants of step (c) under conditions whereby no chromosomal integration occurs between the chromosomal integration vector and the genome of the transformant, by identifying transformants expressing the first genetic marker; e) culturing the identified transformants of step (d) under conditions whereby chromosomal integration occurs between the chromosomal integration vector and the genome of the transformant; f) selecting transformants of step (e) which express either the first genetic marker or both the first and second genetic markers by which transformants having undergone either a single or double crossover event are identified; g) screening the transformants of step (f) which have undergone either a single or double crossover event, for the presence of a specific phenotype wherein the transformants which are positive for the specific phenotype contain a transposon disrupted gene; and h) isolating the transposon disrupted gene from the transformant of step (g) which is positive for the specific phenotype.