Method of isolating restriction fragment deletions in vibrio cholerae and products thereof
AssigneeSwiss Serum and Vaccine Institute Berne
InventorsJames B. Kaper, Myron M. Levine
Life of this patent
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Abstract
This invention relates to a method of isolating deletion mutants of Vibrio cholerae, wherein the deletion is predetermined by digestion with restriction endonucleases of known specificity. The deletions are inserted into the Vibrio cholerae chromosome by in vivo recombination betweem a plasmid carrying the desired deletion, with adjacent flanking sequences, and the Vibrio cholerae chromosome. The invention includes the isolation and characterization of a new Vibrio cholerae strain having a deletion in the tox gene, as defined by Acc I restriction endonuclease sites.
Claims60
A culture of Vibrio cholerae comprising a Vibrio cholerae strain having a restriction endonuclease fragment of DNA coding for Vibrio cholerae toxin or the A subunit thereof deleted to confer avirulence and having the capacity to colonize the intestine of a host animal, said strain constructed by selection of an in vivo recombinant between a virulent Vibrio chloerae and a first plasmid, said first plasmid comprising Vibrio cholerae flanking sequences of the deleted DNA and a gene for a first selectable marker of foreign origin ligated to said flanking sequences to substitute for and to be in the place of said deleted DNA, said flanking sequences being of sufficient length to promote detectable in vivo recombination, said gene for said first selectable marker being incorporated into the chromosome of Vibrio cholerae, said selection being performed by selecting for said first selectable marker and for a second selectable marker to isolate an in vivo recombinant in the presence of a second plasmid carrying the second selectable marker, said second plasmid being incompatible with the first plasmid.
A method of isolating deletion mutants of Vibrio cholerae comprising the steps of
- (a) constructing a first plasmid comprising Vibrio cholerae flanking sequences of a deleted restriction endonuclease fragment and a gene for a first selectable marker of foreign origin ligated to said flanking sequences to substitute for and to be in the place of said deleted fragment, wherein said flanking sequences are of sufficient length to promote detectable in vivo recombination;
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- (b) mating a virulent strain of Vibrio cholerae with a first microorganism carrying the first plasmid;
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- (c) selecting for Vibrio cholerae expressing the first selectable marker;
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- (d) mating the selected product of step (c) with a second microorganism carrying a second plasmid with a second selectable marker, said second plasmid being incompatible with the first plasmid;
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- (e) selecting for Vibrio cholerae expressing both the first selectable marker and the second selectable marker to isolate an in vivo recombinant.
A culture of Vibrio cholerae comprising a Vibrio cholerae strain having a restriction endonuclease fragment of DNA coding for Vibrio cholerae toxin or the A subunit thereof deleted to confer avirulence and having the capacity to colonize the intestine of a host animal, said strain constructed by selection of an in vivo recombinant between virulent Vibrio cholerae N16961 of the Inaba serotype and a first plasmid, said first plasmid comprising Vibrio cholerae flanking sequences of the deleted DNA and a gene for a first selectable marker of foreign origin ligated to said flanking sequences to substitute for and to be in the place of said deleted DNA, said flanking sequences being of sufficient length to promote detectable in vivo recombination, said gene for said first selectable marker being incorporated into the chromosome of Vibrio cholerae, said selection being performed by selecting for said first selectable marker and for a second selectable marker to isolate an in vivo recombinant in the presence of a second plasmid carrying the second selectable marker, said second plasmid being incompatible with the first plasmid.
A culture of Vibrio cholerae comprising a Vibrio cholerae strain having a restriction endonuclease fragment of DNA coding for Vibrio cholerae toxin or the A subunit thereof deleted to confer a virulence and having the capacity to colonize the intestine of a host animal, said strain constructed by selection of in vivo recombinant JDK56 between a virulent Vibrio cholerae and a first plasmid, said first plasmid comprising Vibrio cholerae flanking sequences of the deleted DNA and a gene for a first selectable marker of foreign origin ligated to said flanking sequences to substitute for and to be in the place of said deleted DNA, said flanking sequences being of sufficient length to promote detectable in vivo recombination, said gene for said first selectable marker being incorporate into the chromosome of Vibrio cholerae, said selection being performed by selecting for said first selectable marker and for a second selectable marker to isolate in vivo recombinant JBK56 in the presence of a second plasmid carrying the second selectable marker, said second plasmid being incompatable with the first plasmid.
A method of isolating deletion mutants of Vibrio cholerae comprising the steps of
- (a) constructing a first plasmid comprising Vibrio cholerae flanking sequences of a deleted restriction endonuclease fragment and a gene for a first selectable marker of foreign origin ligated to said flanking sequences to substitute for and to be in the place of said deleted fragment, wherein said flanking sequences are of sufficient length to promote detectable in vivo recombination;
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- (b) mating a virulent strain of Vibrio cholerae with a first microorganism carrying the first plasmid;
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- (c) selecting for Vibrio cholerae expressing the first selectable marker;
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- (d) mating the selected product of step (c) with a second microorganism carrying a second plasmid with a second selectable marker, said second plasmid being incompatible with the first plasmid;
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- (e) selecting for Vibrio cholerae expressing both the first selectable marker and the second selectable marker to isolate an in vivo recombinant;
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- (f) mating the in vivo recombinant of step (e) with a third microorganism carrying a third plasmid, said third plasmid carrying a third selectable marker;
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- (g) selecting for Vibrio cholerae expressing the third selectable marker wherein the selected product is JBK70;
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- (h) curing the selected product of step (g) of plasmids.
A method of isolating deletion mutants of Vibrio cholerae comprising the steps of
- (a) constructing a first plasmid comprising Vibrio cholerae flanking sequences of a deleted restriction endonuclease fragment and a gene for a first selectable marker of foreign origin ligated to said flanking sequences to substitute for and to be in the place of said deleted fragment, wherein said flanking sequences are of sufficient length to promote detectable in vivo recombination;
- and
- (b) mating a virulent strain of Vibrio cholerae with a first microorganism carrying the first plasmid;
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- (c) selecting for Vibrio cholerae expressing the first selectable marker;
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- (d) mating the selected product of step (c) with a second microorganism carrying a second plasmid with a second selectable marker, said second plasmid being incompatible with the first plasmid;
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- (e) selecting for Vibrio cholerae expressing both the first selectable marker and the second selectable marker to isolate an in vivo recombinant wherein the selected product is JBK56.
A method of isolating selection mutants of Vibrio cholerae comprising a the steps of
- (a) constructing a first plasmid comprising Vibrio cholerae flanking sequences of one or more deleted restriction endonuclease fragments and a gene for a first selectable marker of foreign origin ligated to said flanking sequences to substitute for and to be in the place of said deleted fragment, wherein said sequences are of sufficient length to promote detectable in vivo recombination;
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- (b) mating a virulent strain of Vibrio cholerae with a first microorganism carrying the first plasmid;
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- (c) selecting for Vibrio cholerae expressing the first selectable marker;
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- (d) mating the selected product of step (c) with a second microorganism carrying a second plasmid with a second selectable marker, said second plasmid being incompatable with the first plasmid;
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- (e) selecting for Vibrio cholerae expressing both the first selectable marker and the second selectable marker to isolate an in vivo recombination wherein the selected product is JBK56;
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- (f) mating the in vivo recombination of step (e) with a third microorganism carrying a third plasmid, said third plasmid carrying a third selectable marker;
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- (g) selecting for Vibrio cholerae expressing the third selectable marker;
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- (h) curing the selected product of step (g) of plasmids.
A culture of Vibrio cholerae comprising a Vibrio cholerae strain having a restriction endonuclease fragment of DNA coding for Vibrio cholerae toxin or the A subunit thereof deleted to confer avirulence and having the capacity of colonize the intestine of a host animal, said strain constructed by selection of an in vivo recombinant between a virulent Vibrio cholerae and a first plasmid, said first plasmid comprising Vibrio cholerae flanking sequences of the deleted DNA and a gene for a first selectable marker of foreign origin ligated to said flanking sequences to substitute for and to be in the place of said deleted DNA, said flanking sequences being of sufficient length to promote detectable in vivo recombination, said gene for said first selectable marker being incorporated into the chromosome of Vibrio cholerae, said selection being performed by selecting for said first selected marker and for a second selectable marker to isolate an in vivo recombinant in the presence of a second plasmid pR751 carrying the second selectable marker, said second plasmid pR751 being incompatable with the first plasmid.
A method of isolating deletion mutants of Vibrio cholerae comprising the steps of
- (a) constructing a first plasmid comprising Vibrio cholerae flanking sequences of a deleted restriction endonuclease fragment and a gene for a first selectable marker of foreign origin ligated to said flanking sequences to substitute for and to be in the place of said deleted fragment, wherein said flanking sequences are of sufficient length to promote detectable in vivo recombination;
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- (b) mating virulent strain Vibrio cholerae N16961 of the Inaba serotype with a first microorganism carrying the first plasmid;
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- (c) selecting for Vibrio cholerae the first selectable marker;
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- (d) mating the selected product of step (c) with a second microorganism carrying a second plasmid with a second selectable marker, said second plasmid being incompatable with the first plasmid;
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- (e) selecting for Vibrio cholerae expressing both the first selectable marker and the second selectable marker to isolate an in vivo recombinant.
A method of isolating deletion mutants of N16961 of the Inaba serotype of Vibrio cholerae comprising the steps of
- (a) constructing a first plasmid comprising Vibrio cholerae flanking sequences of a deleted restriction endonuclease fragment and a gene for a first selectable marker of foreign origin ligated to said flanking sequences to substitute for and to be in the place of said deleted fragment, wherein said flanking sequences are of sufficient length to promote detectable in vivo recombination;
- (b) mating N16961 of the Inaba serotype of Vibrio cholerae with a first microorganism carrying the first plasmid;
- (c) selecting a Vibrio cholerae expressing the first selectable marker;
- (d) mating the selected product of step (c) with a second microorganism carrying a second plasmid with a second selectable marker, said second plasmid being incompatable with the first plasmid;
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- (e) selecting for Vibrio cholerae expressing both the first selectable marker and the second selectable marker to isolate an in vivo recombinant.
Dependencies are inferred from the claim text.