Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification
Reexamination Certificate
2000-11-28
2002-11-19
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
C435S091200, C435S252300, C435S325000, C435S463000
Reexamination Certificate
active
06482647
ABSTRACT:
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A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
FIELD OF THE INVENTION
The invention applies the fields of classical and molecular genetics to the evolution of DNA sequences for facilitating cellular DNA uptake by a variety of mechanisms.
BACKGROUND OF THE INVENTION
Most procedures in molecular genetics require means for introducing nucleic acids into cells. This is usually accomplished by chemical transformation (e.g., CaCl
2
treatment), electroporation or, for
E. coli,
in vitro packaging of phage lambda. All of these methods are somewhat labor-intensive and time consuming, particularly, if a procedure requires many cycles of isolating, manipulating and transforming DNA. Furthermore, the efficiency of the procedures is relatively low. For example, even when transforming purified supercoiled DNA, at best, about {fraction (1/100)} molecules become stably established in a cell. For ligation mixtures, the efficiencies are 2-3 orders of magnitude lower. Even these efficiencies are applicable to only a relatively small number of preferred cell types commonly used in genetic engineering. It would be desirable to be able to obtain high transfection efficiency in any cell of interest.
A few bacterial isolates are naturally competent (i.e., are capable of taking up DNA from their medium). Reports exist for Bacillus, Neisseria (Rudel et al.,
PNAS
92, 7986-7990 (1995); Facius & Meyer,
Mol. Microbiol.
10, 699-712 (1993)); Haemophilus (Williams et al.,
J. Bacteriol.
176, 6789-6794 (1994)), Helicobacter (Haas et al.,
Mol. Microbiol.
8, 753-760 (1993)), Acinetobacter (Lorenz et al.,
Arch. Microbiol.,
157, 355-360 (1992)), Streptococcus (Lopez et al.,
J. Gen. Microbiol.
135, 2189-2197 (1989)), Campylobacter (Nedenskov-Sorensen,
J. Infect. Dis.
161, 356-366 (1990)), Synechocystis (Barten & Lill,
FEMS Microbiol. Lett.
129, 83-88 (1995)), Lactobacillus and Amycolatopis (Vrijbloed et al., Plasmid 34, 96-104 (1995)).
Some information has emerged concerning the genetic basis of natural competence in bacteria. Some genes have been identified and correlated with a role in mediating DNA uptake. In Neisseria, two proteins, PilC and PilE, having roles in phase variation, have been shown to be essential for natural competence (Rudel et al.,
Proc. Natl. Acad. Sci. USA
92, 7986-7990 (1995)). PilE is the major pilus subunit protein, and PilC functions in assembly and adherence of gonococcal pili. Both genes serve to convert linearized plasmid DNA into a DNase-resistant form. DNA uptake requires a Neisseria-specific uptake signal on the DNA and a functional RecA protein. DNA is taken up in linear form. Transformation with non-episomal DNA fragments requires homology to the chromosomal DNA to allow integration by homologous recombination. Other genes required for DNA uptake, called dud, and for transformation uptake, called ntr, have been identified (Biswas et al.,
J. Bact.,
171, 657-664 (1989)). In Haemophilus, the sxy gene has been reported to be essential for competence. Overexpression of the sxy gene product confers constitutive competence on wildtype Haemophilus cells, (Williams et al.,
J. Bact.,
176, 6789-6794 (1994)). In
E. coli,
the comA gene has been reported to be involved in natural competence (Facius & Meyer,
Mol. Microbiol.,
10, 699-712 (1993)). Regulatory genes involved in competence are the homologs of the
E. coli
cya gene, encoding adenylate cyclase, and
E. coli
crp genes, encoding the cAMP receptor protein.
The present invention is generally directed to transferring genes conferring DNA-uptake capacity in one species to another and evolving the genes so that they also confer comparable or better DNA-uptake capacity in the second species and/or the original species. Genes are evolved by a process termed recursive sequence recombination which entails performing iterative cycles of recombination and screening/selection. Cells expressing the evolved genes can be transfected without undertaking the time consuming preparatory steps of prior methods and/or with greater efficiency than the cells of prior methods.
SUMMARY OF THE INVENTION
In a first embodiment, the invention provides methods of enhancing competence of a cell by iterative cycles of recombination and screening/selection. In the first cycle, at least first and second DNA segments from at least one gene conferring DNA competence are recombined. The segments differ from each other in at least two nucleotides. Recombination produces a library of recombinant genes. At least one recombinant gene is screened from the library that confers enhanced competence in the cell relative to a wildtype form of the gene. In the second cycle, at least a segment from one or more of the recombinant genes identified by screening is recombined with a further DNA segment from the gene conferring competence to produce a further library of recombinant genes. At least one further recombinant gene is screened from the further library of recombinant genes that confers enhanced competence in the cell relative to a previous recombinant gene. Further cycles of recombination and screening/selection are performed until a recombinant gene is produced that confers a desired level of enhanced competence in the cell.
Diversity between the first and second segments in the first cycle of recombination can result from generation of the the second segment by error-prone PCR replication of the first segment or propagation of the first segment in a mutator strain. Alternatively, the second segment can be the same as the first segment except that a portion of the first is substituted with a mutagenic cassette.
In some methods, at least one recombining step is performed in vitro, and the resulting library of recombinants is introduced into the cell whose competence is to be enhanced generating a library of cells containing different recombinants. A typical in vitro recombining step entails: cleaving the first and second segments into fragments; mixing and denaturing the fragments; and incubating the denatured fragments with a polymerase under conditions which result in annealing of the denatured fragments and formation of the library of recombinant genes.
Often screening/selection identifies a pool of cells comprising recombinant genes conferring enhanced competence from the library. For example, selection can be achieved by transfecting a vector encoding a selective marker into the library of cells containing different recombinants, and selecting for cells expressing the selective marker. In some methods, the vector encoding the selective marker is a suicide vector.
In some methods, the further DNA segment in the second or subsequent round of recombination is a recombinant gene or library of such genes produced in a previous step. For example, the second or subsequent round of recombination can be performed by dividing the pool of cells surviving screening/selection into first and second pools. Recombinant genes are isolated from the first pool, and transfected into the second pool where the recombinant genes from the first and second pools recombine to produce the further library of recombinant genes.
In some methods, at least one recombining step is performed in vivo, for example, by homologous recombination or by site-specific recombination. In vivo recombination can be performed, for example, by propagating a collection of cells, each cell containing a vector comprising an origin of transfer and a member of a recombinant gene library, and each cell expressing tra genes whose expression products conjugally transfer the vector between cells.
In some methods at least one of the DNA segments comprises a substantially complete genome. In some methods, each of the DNA segments comprises a cluster of genes collectively con
Ketter James
Kruse Norman J.
Loeb Bronwen M.
Maxygen Inc.
Powers Margaret A.
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