EP0316677A2
Expired / Lapsed
EP
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Method for cloning in lactic acid bacteria
Assignee: Bayer Corp
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Filing: 1988-11-04
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Granted: —
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Expires: 2008-11-04
Abstract
Abstract A plasmid-borne gene from a first lactic acid bacterium is cloned in a second lactic acid bacterium by means of a cloning vector that contains a gene for a phenotypic marker and that is devoid of an origin of replication for the second bacterium. The plasmid from the first lactic acid bacterium is combined with the cloning vector to produce a recombinant plasmid that is easily selectable in the second lactic acid bacterium and that contains both the gene to be cloned and an origin of replication for the second lactic acid bacterium. The recombinant plasmid or plasmid-borne functional gene segment so identified can be inserted into a lactic acid bacterial host by conjugation or transformation to provide an improved starter culture bacterial strain.
Inventors
- 👤 Mary Ellen Sanders
- 👤 John William Shultz
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-09 20:29
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/63; C12N15/65; C07; C07K; C07K14/00; C07K14/195; C07K14/315; C12N15/74; C12N15/746
Claims Analysis
Total Claims: 13
Claim 1
Independent Claim
159 words
A method for producing a transformed lactic acid bacterium from a lactic acid bacterial host and isolating said transformed lactic acid bacterium, wherein the transformed bacterium contains a plasmid-borne functional gene segment capable of conferring a phenotype not previously present in the lactic acid bacterial host, the method comprising the steps of: (a) forming a recombinant plasmid by joining, under DNA-ligating conditions, (i) a first plasmid from a lactic acid bacterium, said plasmid having a gene of interest and an origin of replication which is functional in the lactic acid bacterial host with (ii) a vector which contains a selectable marker but does not contain an origin of replication which is functional in the lactic acid bacterial host; (b) directly transforming the lactic acid bacterial host with the recombinant plasmid under transforming conditions; (c) cultivating the transformants resulting from step (b) and selecting and isolating a transformant which exhibits the phenotype conferred to the host by the selectable marker.
Claim 2
Independent Claim
159 words
A method for producing a transformed lactic acid bacterium from a lactic acid bacterial host and isolating said transformed lactic acid bacterium, wherein the transformed bacterium contains a plasmid-borne functional gene segment capable of conferring a phenotype not previously present in the lactic acid bacterial host, the method comprising the steps of: (a) forming a recombinant plasmid by joining, under DNA-ligating conditions, (i) a first plasmid from a lactic acid bacterium, said plasmid having a gene of interest and an origin of replication which is functional in the lactic acid bacterial host with (ii) a vector which contains a selectable marker but does not contain an origin of replication which is functional in the lactic acid bacterial host; (b) directly transforming the lactic acid bacterial host with the recombinant plasmid under transforming conditions; (c) cultivating the transformants resulting from step (b) and selecting and isolating a transformant which exhibits the phenotype conferred to the host by the selectable marker.
Claim 3
Dependent Claim
23 words
The method of claim 1 in which the plasmid containing lactic acid bacterium and the host lactic acid bacterium are both lactic streptococci.
Claim 4
Independent Claim
21 words
The method of any of claims 1 and 2 in which the host streptococcus lactic acid bacterium is S. lactis .
Claim 5
Dependent Claim
23 words
Use of a streptococcus lactic acid bacterium produced by the method of any of claims 1 to 3 as a starter culture bacterium.
Claim 6
Dependent Claim
37 words
A vector comprising a covalently closed recombinant plasmid deficient in an origin of replication for lactic acid bacterium but containing a marker selectable in lactic acid bacterium, preferably an antibiotic resistance marker selectable in lactic acid bacterium.
Claim 7
Dependent Claim
37 words
A vector comprising a covalently closed recombinant plasmid deficient in an origin of replication for lactic acid bacterium but containing a marker selectable in lactic acid bacterium, preferably an antibiotic resistance marker selectable in lactic acid bacterium.
Claim 8
Dependent Claim
23 words
The vector of claim 5 additionally comprising an origin of replication for a secondary host cell and optionally a restriction enzyme cleavage site.
Claim 9
Dependent Claim
10 words
The vector of claim 6 which is identified as pSA34.
Claim 10
Dependent Claim
88 words
A transformant obtained by the method of claim 1, comprising; a lactic acid bacterium host cell containing a recombinant plasmid, which plasmid is composed of: (a) a marker selectable in the host; (b) a gene encoding a gene product not previously produced by the host cell, preferably the gene product results in a phenotype, such as phage resistance, not previously observed in the host; and (c) an origin of replication functional in the host cell; and optionally additionally comprising an origin of replication functional in a secondary host.
Claim 11
Independent Claim
127 words
A method for identifying a functional gene segment capable of conferring a phenotype to a lactic acid bacterium not previously exhibiting such phenotype, comprising the steps of: (a) combining a plasmid from a first lactic acid bacterium with a vector identified as pSA34, the lactic acid bacterium plasmid including a gene capable of conferring a phenotype not previously exhibited by a host lactic acid bacterium and an origin of replication functional in the host; (b) transforming, directly, the recombinant plasmid into the lactic acid bacterium host to form a transformant; (c) culturing the transformant on an agar containing erythromycin; and d) screening the cultured transformant for the phenotype; wherein the phenotype is preferably phage resistance and the screening is for the absence or reduction of plaque formation.
Claim 12
Independent Claim
127 words
A method for identifying a functional gene segment capable of conferring a phenotype to a lactic acid bacterium not previously exhibiting such phenotype, comprising the steps of: (a) combining a plasmid from a first lactic acid bacterium with a vector identified as pSA34, the lactic acid bacterium plasmid including a gene capable of conferring a phenotype not previously exhibited by a host lactic acid bacterium and an origin of replication functional in the host; (b) transforming, directly, the recombinant plasmid into the lactic acid bacterium host to form a transformant; (c) culturing the transformant on an agar containing erythromycin; and d) screening the cultured transformant for the phenotype; wherein the phenotype is preferably phage resistance and the screening is for the absence or reduction of plaque formation.
Claim 13
Dependent Claim
42 words
A starter culture, comprising: a lactic acid bacterium which has the ability to ferment milk, containing the recombinant plasmid obtained by the method of any of claims 1 ot 3 or a function gene segment of interest found in the recombinant plasmid.
References Cited (Backward Citations)
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