Thermophilic site-specific recombination system for rapid insertion of heterologous dna

A system for stable insertion of a heterologous DNA, the system comprising:

• a thermophile bacterial cell comprising at least one att site in its chromosome wherein the att site is one member of a pair of attB and attP recombination sites;
• and
• a cargo plasmid comprising the heterologous DNA and a corresponding att site, wherein the corresponding att site is the other member of the pair of attB and attP recombination sites;
• wherein the cargo plasmid is capable of insertion into the chromosome of the thermophile at the at least one att site via recombination with the corresponding att site on the cargo plasmid;
• and
• wherein the recombination is mediated by a thermophilic site-specific recombinase that recognizes the pair of attB and attP recombination sites, and results in the formation of an attL site and an attR site, wherein the attL and attR sites are not substrates for further recombination.

A method for thermostable insertion of a heterologous DNA into a chromosome of an organism, the method comprising:

• a) transfecting a cargo plasmid into a thermophile bacterial cell comprising at least one att site in its chromosome wherein the att site is one member of a pair of attB and attP recombination sites,
• wherein the thermophile bacterial cell expresses a thermophilic site-specific recombinase that recognizes the pair of attB and attP recombination sites;
• wherein the cargo plasmid comprising a corresponding att recombination site, wherein the corresponding att recombination site is the other member of the pair of attB and attP recombination sites;
• wherein the cargo plasmid comprises the heterologous DNA;
• wherein the cargo plasmid is capable of insertion into the chromosome of the thermophile bacterial cell at the at least one att site via recombination with the at least one corresponding att site on the cargo plasmid;
• and
• wherein the recombination results in the formation of an attL site and an attR site, wherein the attL and attR sites are not substrates for further recombination,
• wherein the insertion is a site-specific recombination at a temperature at or above 50° C. and is thermostable;
• and
• b) selecting a thermophile bacterial cell wherein the heterologous DNA is inserted into the chromosome of the thermophile bacterial cell.