Introduction of DNA into bacillus strains by conjugation

A method of introducing a DNA construct encoding a polypeptide of interest into a cell of a Bacillus sp., comprising mixing (a) a population of bacterial donor cells harboring i) a plasmid comprising a DNA construct encoding a polypeptide of interest and at least one cis-acting DNA sequence required for the transfer of said plasmid by conjugation in the presence of a trans-acting mobilizing element, and ii) at least one DNA sequence encoding said trans-acting mobilizing element, and (b) a population of Bacillus sp. recipient cells, under conditions allowing the plasmid to be transferred from the population of donor cells to population of recipient cells by conjugation, the Bacillus sp. being an alkalophilic Bacillus sp., an industrial Bacillus strain and/or a mutant Bacillus strain having been mutated to be capable of producing and secreting the polypeptide of interest in an increased yield as compared to the corresponding unmutated strain.

A method of constructing a cell of a Bacillus sp. harboring a DNA construct encoding a polypeptide of interest, comprising mixing (a) a population of auxotrophic bacterial donor cells harboring i) a plasmid comprising a DNA construct encoding a polypeptide of interest and at least one cis-acting DNA sequence required for transfer of said plasmid by conjugation in the presence of a trans-acting mobilizing element, and ii) at least one DNA sequence encoding said trans-acting mobilizing element, and (b) a population of unmarked Bacillus sp. recipient cells, under conditions allowing the plasmid to be transferred from the population of auxotrophic donor cells to the population of unmarked recipient cells by conjugation, and the auxotrophic property of the donor cell is exploited to select for recipient cells.

A method of introducing a DNA construct encoding a polypeptide of interest into a cell of a Bacillus sp., comprising mixing (a) a population of bacterial donor cells harboring i) a curable plasmid comprising a DNA construct encoding a polypeptide of interest and at least one cis-acting DNA sequence required for transfer of said plasmid by conjugation in the presence of a trans-acting mobilizing element, and ii) at least one DNA sequence encoding said trans-acting mobilizing element, and (b) a population of Bacillus sp. recipient cells, under conditions allowing the plasmid to be transferred from the population of donor cells to the population of recipient cells by conjugation.