Method for constructing a transposon for identifying a DNA sequence coding for a signal peptide in lactic acid bacteria

摘要

A method for constructing a transposon derivative for identifying in a lactic acid bacterium a DNA sequence coding for a signal peptide (SP) comprises selecting a DNA molecule comprising a transposon including between its left terminus (LR) and its right terminus (RR) a sequence comprising a promoterless promoter reporter gene and a ribosome binding site (RBS). A region located between the LR and the promoterless reporter gene is deleted form the DNA molecule to obtain a modified DNA molecule that has retained its transposability and its RBS. A unique restriction is then inserted into the remaining region located between the LR and the promoterless gene of the modified DNA molecule. A DNA sequence coding for a secretion reporter molecule, which is without a sequence coding for a SP, is inserted into the unique restriction site. The obtained transposon derivative is without stop codons in-frame with the reporter molecule thus allowing upon transposition translational fusions from upstream the LR. Also described is a method for identifying in a lactic acid bacteria a DNA coding sequence coding for a SP using the transposon derivative and the isolation of the sequences.