Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-04-30
2000-04-11
Yucel, Remy
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435471, 435476, 435479, 435485, 435486, 435487, 4353201, 4352523, 43525231, 43525235, C12Q 168, C12N 1574, C12N 1576, C12N 1500, C12N 120
Patent
active
060486945
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
The present invention relates to the field of molecular biology and the use of selection vectors for the identification of recombinant bacteria containing heterologous DNA. More specifically a B. amyloliquifaciens signal sequence mutant gene has been discovered that conveys lethality to Bacillus sp. and may be used in a method for the direct selection of recombinant Bacillus host cells containing cloned, foreign DNA.
BACKGROUND
A method for readily isolating bacteria with cloned DNA is essential for successfully cloning DNA from any source. Identification of bacteria containing cloned DNA is most easily accomplished whenever the cloned DNA encodes a function that can be subjected to direct genetic selection, i.e., whenever survival of recombinant bacteria depends upon acquiring and expressing a function encoded by the DNA that is to be cloned. Identifying recombinant bacteria with cloned DNA is significantly more difficult when the cloned DNA does not encode a genetically selectable function because recombinant bacteria with cloned DNA must be identified against a potentially high background of recombinant bacteria that contain the cloning vector but lack DNA.
One approach to identifying recombinant bacteria with cloned inserts is to screen bacterial colonies for insertional inactivation of a reporter gene such as lacZ, the structural gene for .beta.-galactosidase (Sambrook et al., Molecular Cloning, A Laboratory Manual, p. 1.85-p. 1.86 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). .beta.-galactosidase can not be made when DNA is cloned into a restriction site that separates the coding portion of lacZ from its promoter. As a result, on medium with the chromogenic substrate X-gal, recombinants with cloned DNA appear as white colonies against a background of blue recombinant colonies that contain religated vector alone. Screening methods such as insertional inactivation of lacZ facilitate identification of bacteria with cloned DNA by flagging the appropriate colonies in some way. However, these methods do not favor or select for growth of bacteria with cloned DNA. Hence, whenever a screening procedure is used, it is usually necessary to look through large numbers of colonies to find a few with cloned DNA. A common strategy for reducing the number of background colonies is to prevent self-ligation of the vectors by using alkaline phosphatase to remove the terminal 5' phosphates from vector molecules because DNA ligase requires a terminal 5' phosphate to join the ends of DNA molecules (Sambrook et al. supra, p. 1.56-p. 1.58). This method generally reduces, but does not eliminate, background colonies lacking cloned DNA.
An approach that minimizes the number of bacteria lacking cloned inserts uses a vector that enables direct selection for recombinant bacteria with cloned DNA. The advantage of a selection method over a screening method is that growth of bacteria with cloned DNA is greatly favored over bacteria lacking cloned DNA.
Direct or positive selection vectors containing genes that convey lethality to the host are well known. For example, expression of the B. subtilis or the B. amyloliquefaciens sacB genes in the presence of sucrose is lethal to E. coli and a variety of other Gram-negative and Gram-positive bacteria [Cai et al., J. Bacteriol. 172, 3138, (1990); Gay et al., J. Bacteriol. 164, 918, (1985); Jager et al., FEMS Microbiol. Let. 126,1 (1995); Jager et al., J. Bacteriol. 174, 5462, (1992); Kamoun et al., Mol. Microbiol. 6,809, (1992); Kaniga et al., Gene 109,137 (1991); Ried et al., Gene 57,239, (1987); Simon et al., J. Bacteriol. 173,1502, (1991)]. The sacB gene encodes levansucrase [Gay et al., J. Bacteriol. 153,1424 (1983); Lepesant et al., Mol. Gen Genet. 128,213 (1974)]. Levansucrase catalyzes both the hydrolysis of sucrose and the polymerization of sucrose to form levan. The basis for the lethality of levansucrase in the presence of sucrose is not fully understood. However, the inability of E. coli and certain other bacteria to grow if sacB is express
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Bramucci Michael Gene
Nagarajan Vasantha
E. I. Du Pont de Nemours and Company
Yucel Remy
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