Recombinant bacteriophage for heterologous cloning of bacillus microorganisms and method for its production

Claim 1

A method for producing and selecting a recombinant bacteriophage containing a desired genetic trait, suitable for use in heterologous transformation of a host Bacillus microorganism which is characterized in (a) obtaining genetic fragments encoding said desired genetic trait; (b) isolating DNA fragments from a bacteriophage; (c) ligating the genetic fragments obtained in step (a) with the DNA obtained in step (b) to produce a mixture containing a series of recombinant molecules; (d) incubating the mixture of step (c) with DNA isolated from a second Bacillus microorganism which does not contain said genetic fragments obtained in step (a), said second Bacillus microorganism having substantial genetic homology with said host Bacicclus microorganism and being prototrophic for a growth requirement; (e) incubating the mixture of step (d) with said host Bacillus microorganism, said host Bacillus being lysogenic for said bacteriophage of step (b) and auxotrophic for said growth requirement to produce a mixture containing transformed host Bacillus which are prototrophic for said growth requirement, lysogenic for said desired recombinant bacteriophage and which contain said desired genetic trait within said bacteriophage; (f) selecting said transformed host Bacillus by growing said mixture on a growth medium which does not contain said growth requirement and determining the presence of said desired - genetic trait; and (g) obtaining therefrom said recombinant bacteriophage containing said desired genetic trait by inducing said prototrophic host Bacillus.

Claim 2

A method for producing and selecting a recombinant bacteriophage containing a desired genetic trait, suitable for use in heterologous transformation of a host Bacillus microorganism which is characterized in (a) obtaining genetic fragments encoding said desired genetic trait; (b) isolating DNA fragments from a bacteriophage; (c) ligating the genetic fragments obtained in step (a) with the DNA obtained in step (b) to produce a mixture containing a series of recombinant molecules; (d) incubating the mixture of step (c) with DNA isolated from a second Bacillus microorganism which does not contain said genetic fragments obtained in step (a), said second Bacillus microorganism having substantial genetic homology with said host Bacicclus microorganism and being prototrophic for a growth requirement; (e) incubating the mixture of step (d) with said host Bacillus microorganism, said host Bacillus being lysogenic for said bacteriophage of step (b) and auxotrophic for said growth requirement to produce a mixture containing transformed host Bacillus which are prototrophic for said growth requirement, lysogenic for said desired recombinant bacteriophage and which contain said desired genetic trait within said bacteriophage; (f) selecting said transformed host Bacillus by growing said mixture on a growth medium which does not contain said growth requirement and determining the presence of said desired - genetic trait; and (g) obtaining therefrom said recombinant bacteriophage containing said desired genetic trait by inducing said prototrophic host Bacillus.

Claim 7

A method for producing and selecting B. subtiZis recombinant bacteriophage 03T (ATCC 31595-Bl) containing the gene encoding biosynthesis of a-amylase suitable for use in heterologous transformation of a host Bacillus microorganism which is characterized in: (a) obtaining genetic fragments encoding a-amylase from B. amyloliquefaciensH RUB 500 (ATCC 31592); (b) isolating DNA fragments from a ø3T bacteriophage; (c) ligating the genetic fragments obtained in step (a) with the DNA obtained in step (b) to produce a mixture containing a series of recombinant molecules; (d) incubating the mixture of step (c) with DNA isolated from a B. subtiZis RUB 200 (ATCC 31593) microorganism which does not contain said genetic fragments obtained in step (a), said RUB 200 microorganism having substantial genetic homology with a host B. subtilis RUB 201 (ATCC 31594) and being prototrophic for threonine; (e) incubating the mixture of step (d) with said host B. subtilis RUB 201 said host B. subtilis RUB 201 being lysogenic for said bacteriophage of step (b) and auxotrophic for thr onine to produce a mixture containing host B. subtilis RUB 201 transformed into B. subtilis RUB 204 (ATCC 31595) which are prototrophic for threonine, lysogenic for said B. subtiZis recombinant bacteriophage ø3T and which contain the gene encoding biosynthesis of a-amylase within said bacteriophage; (f) selecting said host B. subtiZis RUB 204 by growing said mixture on a growth medium which does not contain threonine and determining the presence of α-amylase; and (g) obtaining therefrom B. subtilis recombinant bacteriophage ø3T (ATCC 31595-Bl) containing the gene encoding biosynthesis of α-amylase by inducing said host B. subtilis RUB 204.

Claim 8

A method for producing and selecting B. subtiZis recombinant bacteriophage 03T (ATCC 31595-Bl) containing the gene encoding biosynthesis of a-amylase suitable for use in heterologous transformation of a host Bacillus microorganism which is characterized in: (a) obtaining genetic fragments encoding a-amylase from B. amyloliquefaciensH RUB 500 (ATCC 31592); (b) isolating DNA fragments from a ø3T bacteriophage; (c) ligating the genetic fragments obtained in step (a) with the DNA obtained in step (b) to produce a mixture containing a series of recombinant molecules; (d) incubating the mixture of step (c) with DNA isolated from a B. subtiZis RUB 200 (ATCC 31593) microorganism which does not contain said genetic fragments obtained in step (a), said RUB 200 microorganism having substantial genetic homology with a host B. subtilis RUB 201 (ATCC 31594) and being prototrophic for threonine; (e) incubating the mixture of step (d) with said host B. subtilis RUB 201 said host B. subtilis RUB 201 being lysogenic for said bacteriophage of step (b) and auxotrophic for thr onine to produce a mixture containing host B. subtilis RUB 201 transformed into B. subtilis RUB 204 (ATCC 31595) which are prototrophic for threonine, lysogenic for said B. subtiZis recombinant bacteriophage ø3T and which contain the gene encoding biosynthesis of a-amylase within said bacteriophage; (f) selecting said host B. subtiZis RUB 204 by growing said mixture on a growth medium which does not contain threonine and determining the presence of α-amylase; and (g) obtaining therefrom B. subtilis recombinant bacteriophage ø3T (ATCC 31595-Bl) containing the gene encoding biosynthesis of α-amylase by inducing said host B. subtilis RUB 204.