TRANSPOSON FOR BIDIRECTIONAL INTRAMOLECULAR GENOME DELETIONS, CONSTRUCTION OF MICROORGANISM MUTANT AND IDENTIFICATION OF NONESSENTIAL GENES USING THE SAME

摘要

PURPOSE: A transposon for bidirectional intramolecular genome deletions, a construction of a microorganism mutant and the identification of nonessential genes using the same transposon are provided, thereby effectively constructing the nonessential gene-removed Escherichia coli mutant. CONSTITUTION: Transposon TnRIBD sequentially comprises Tn5 transposase recognition site having the nucleotide sequence of SEQ ID NO: 2; tetR gene having the nucleotide sequence of SEQ ID NO: 4; Tnγδ transposase recognition site having the nucleotide sequence of SEQ ID NO: 3; CmR gene having the nucleotide sequence of SEQ ID NO: 5; Tnγδ transposase recognition site having the reverse complementary nucleotide sequence with SEQ ID NO: 3; sacB gene having the nucleotide sequence of SEQ ID NO: 6; and Tn5 transposase recognition site having the reverse complementary nucleotide sequence with SEQ ID NO: 2 in a direction of 5' to 3'. A construction method of a microorganism mutant comprises the steps of: constructing the transposon comprising the transposase recognition site and a selection marker; inserting the transposon into a certain site in the genome of a microorganism; and cloning a transposase expression vector into the genome of the microorganism to remove a portion of chromosome in both ends.