US4963487A
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Method for deletion of a gene from a bacteria
Assignee: Massachusetts Institute of Technology
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Filing: 1987-09-14
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Granted: 1990-10-16
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Expires: 2007-09-14
Abstract
Abstract Disclosed is a method and linear DNA fragments for use in the deletion of a gene from a bacteria with a single step procedure that is applicable to any essential or nonessential gene which has been cloned. Chromosomal deletions are constructed by transformation of a cell strain with linear DNA fragments containing a locus for resistance to an antibiotic, or any other gene allowing for rapid phenotypic selection, flanked by sequences homologous to closely spaced regions on the cell chromosome on either side of the gene to be deleted, in combination with the immediate subsequent deletion or inactivation of the recA gene. By selecting for a double-crossover event between the homologous sequences, shown by the antibiotic resistance or other detectable phenotype, a chromosome disruption can be selected for which has effectively deleted an entire gene. Inactivation or deletion of the recA gene prevents recombination or incorporation of extrachromosomal elements from occurring, thereby resulting in a bacterial strain which is useful for screening for functional activity or production of genetically engineered proteins in the absence of specific contaminants.
Inventors
- 👤 Paul R. Schimmel
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-31 23:22
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/11; C12N15/62; C12N15/52; C12N15/87; C12N15/90; C12N15/902; C12N9/00; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/822; Y10S435/848; Y10S435/849
Claims Analysis
Total Claims: 13
Claim 1
Independent Claim
155 words
A method for deletion of a gene from a bacteria comprising: (a) constructing a linear DNA fragment wherein the fragment has sequences sufficiently homologous to regions on the cell chromosome flanking the gene to be deleted; to permit base pairing therebetween (b) placing a second gene whose expression results in a rapidly detectable phenotype between the hybridizing sequences in the DNA fragment; (c) introducing the linear DNA fragment into bacterial cells; (d) selecting for a double reciprocal recombination mediated by the RecA gene by culturing the cells containing the linear DNA under conditions wherein the phenotype encoded by the second nucleotide sequence is detected and cells with a double reciprocal recombination express the detectable phenotype; and (e) altering the cell strain phenotype from recA+ to reca- immediately after the linear DNA fragment is incorporated into the cell chromosome by introduction of a second linear DNA fragment containing sequences homologous to regions flanking the recA gene.
Claim 2
Independent Claim
155 words
A method for deletion of a gene from a bacteria comprising: (a) constructing a linear DNA fragment wherein the fragment has sequences sufficiently homologous to regions on the cell chromosome flanking the gene to be deleted; to permit base pairing therebetween (b) placing a second gene whose expression results in a rapidly detectable phenotype between the hybridizing sequences in the DNA fragment; (c) introducing the linear DNA fragment into bacterial cells; (d) selecting for a double reciprocal recombination mediated by the RecA gene by culturing the cells containing the linear DNA under conditions wherein the phenotype encoded by the second nucleotide sequence is detected and cells with a double reciprocal recombination express the detectable phenotype; and (e) altering the cell strain phenotype from recA+ to reca- immediately after the linear DNA fragment is incorporated into the cell chromosome by introduction of a second linear DNA fragment containing sequences homologous to regions flanking the recA gene.
Claim 3
Dependent Claim
31 words
The method of claim 1 further comprising selecting the host bacterial strain from the group of bacteria consisting of Proteus, Erwinia, Shigella, Haemophilus, Streptococcus, Salmonella, Pseudomonas, Rhizobium, Agrobacterium, Vibrio, and Bacillus.
Claim 4
Dependent Claim
41 words
The method of claim 1 wherein the cell strain phenotype is altered from RecA+ to RecA- immediately by incorporating into the linear fragment containing the sequences flanking the gene to be deleted a sequence homologous to and deleting the RecA region.
Claim 5
Dependent Claim
36 words
The method of claim 1 wherein the cell strain phenotype is altered from RecA+ to RecA- by transduction of the second linear fragment into the cell strain immediately following the double reciprocal recombination deleting the gene.
Claim 6
Dependent Claim
28 words
The method of claim 1 wherein the cell strain phenotype is altered from RecA+ to RecA- immediately by mating the RecA+ strain with a second appropriate RecA- strain.
Claim 7
Dependent Claim
32 words
The method of claim 1 for deleting a gene for a first host protein, wherein the detectable phenotype for the gene deletion is the absence of the second protein in the host.
Claim 8
Dependent Claim
43 words
An isolated DNA fragment consisting essentially of between approximately 80 to 100 nucleotides homologous to nucleotide sequences located adjacent to the recA gene in bacteria, wherein binding between the nucleotide sequences and the isolated fragment results in deletion of the rec a gene.
Claim 9
Dependent Claim
43 words
An isolated DNA fragment consisting essentially of between approximately 80 to 100 nucleotides homologous to nucleotide sequences located adjacent to the recA gene in bacteria, wherein binding between the nucleotide sequences and the isolated fragment results in deletion of the rec a gene.
Claim 10
Dependent Claim
14 words
The linear DNA fragment of claim 7 further comprising sequences encoding the recA gene.
Claim 11
Dependent Claim
21 words
The linear DNA fragment of claim 7 further comprising a gene encoding a detectable phenotype when expressed in a bacterial host.
Claim 12
Dependent Claim
113 words
A linear DNA fragment for deleting a gene from a bacterial chromosome consisting essentially of between approximately 80 to 100 nucleotides homologous to sequences 5' and 3' to the gene to be deleted and a gene selected from the group consisting of genes whose expression results in a detectable phenotype of temperature sensitivity, ultraviolet radiation sensitivity, auxotrophism for a sugar, auxotrophism for an amino acid, auxotrophism for a protein, auxotrophism for a nucleotide, and which encode for a specific cellular component detectable by chemical, radioactive, or immunological screening techniques, wherein binding between the nucleotide sequences and bacterial chromosome results in insertion of the selected gene in place of the gene to be deleted.
Claim 13
Dependent Claim
113 words
A linear DNA fragment for deleting a gene from a bacterial chromosome consisting essentially of between approximately 80 to 100 nucleotides homologous to sequences 5' and 3' to the gene to be deleted and a gene selected from the group consisting of genes whose expression results in a detectable phenotype of temperature sensitivity, ultraviolet radiation sensitivity, auxotrophism for a sugar, auxotrophism for an amino acid, auxotrophism for a protein, auxotrophism for a nucleotide, and which encode for a specific cellular component detectable by chemical, radioactive, or immunological screening techniques, wherein binding between the nucleotide sequences and bacterial chromosome results in insertion of the selected gene in place of the gene to be deleted.
References Cited (Backward Citations)
No citation records indexed.
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