Patent ID: 8778683

Claim:
A method of making a recombinant temperature sensitive (TS) bacteria comprising an essential peptide, comprising: screening a psychrophilic microbial genome for detection therein of an essential polynucleotide that confers temperature sensitivity to a bacterium upon expression of a protein from the essential polynucleotide that is inactivated at a temperature greater than 30° C.; isolating said essential polynucleotide; constructing a nucleic acid construct comprising the essential polynucleotide and one or more control sequences operably linked to the essential polynucleotide; inserting the nucleic acid construct into the genome of a selected mesophilic bacterial host cell thereby functionally replacing the selected mesophilic bacterial host cell's homologue of the essential polynucleotide whereby the essential polynucleotide encodes a protein that is operable at a temperature less than 30° C. and inoperable at a temperature greater than 30° C.; culturing the mesophilic bacterial host cell comprising the essential polynucleotide at a temperature less than 30° C. to confirm the viability of the mesophilic bacterial host cell comprising the essential polynucleotide; further culturing the mesophilic bacterial host cell comprising the essential polynucleotide at a temperature greater than 30° C. to determine if the mesophilic bacterial host cell comprising the essential polynucleotide is killed; and if the mesophilic bacterial host cell comprising the essential polynucleotide is killed, inserting the nucleic acid construct into the genome of a selected destination mesophilic bacterial host cell, thereby functionally replacing the selected destination mesophilic bacterial host cell's homologue of the essential polynucleotide, thereby generating a recombinant TS bacteria.