WO2001009351A1 - Novel vectors and system for selectable targeted integration of transgenes into a chromosome without antibiotic resistance markers - Google Patents

Abstract

A method for making a transgenic cell comprising the steps of introducing a linear vector into the cell wherein said vector contains a cassette consisting of sequentially from 5' to 3': a 5' flanking sequence, a polylinker site containing a nucleic acid sequence of interest, and a 3' flanking sequence wherein the 5' and 3' flanking sequences are homologous to a sequence in a chromosome in the cell where the nucleic acid sequence of interest is to be inserted; integration of said vector into the chromosome through recombination between the flanking sequences and the homologous DNA sequences wherein said homologous DNA sequences flank a conditional killing module in a chromosome; and negatively selecting against cells retaining said conditional killing module and deficient for integration of said linear vector.

Description

Novel Vectors and System for Selectable Targeted

Integration of Transgenes into a Chromosome

Without Antibiotic Resistance Markers

FIELD OF THE INVENTION

The invention relates to vectors and a method for creating a transgenic cell by negatively selecting cells deficient in integration of a transgene.

BACKGROUND OF THE INVENTION

Gene expression and cloning in bacteria is almost exclusively done utilizing plasmids, and most of them are derived from multicopy ColE 1 plasmids such as pBR322 and its relatives. Expression of genes from plasmids causes multiple significant problems for in vivo experimental work. For instance, nearly all plasmids are maintained in cells using selection for antibiotic resistance, and for in vivo studies in which multiple mutations are also generated in host cells, one can exhaust the available resistance markers. Expression or overexpression of some genes can cause toxicity to the cell, or subsequent slow growth of the cell, and thus create a selection for the faster growing cells that lose the plasmid or for cells that accumulate plasmids containing a mutation that inactivates the gene. Also, expression or overexpression of some genes can cause toxicity to the plasmid (genes such as helicases that can disrupt plasmid replication machinery components) and subsequent slow growth of the cell, thus creating a selection for the faster growing cells that lose the plasmid or for cells which accumulate plasmids containing a mutation which inactivates the gene. Plasmids containing certain sequences such as chi sites that promote rolling circle replication of plasmids, direct or inverted repeats can render the plasmid unstable and subsequently lost from the cell. Furthermore, antibiotics used for selection will interfere with purification and recovery of desirable protein products. Another problem with antibiotic selection is that the cost of antibiotics for selection of plasmids can be significant. Also, the frequent use of common antibiotics will facilitate the spread of resistant bacteria, which is causing a crisis in the treatment of infectious diseases (Neu, 1992; Davies, 1994). The use of antibiotic resistance for plasmid selection is particularly troublesome with plasmids whose selectable marker is ampicillin resistance (most pBR322 derivatives); because the resistance is diffusable, most cells in a culture can survive without the plasmid because the medium is detoxified by a few plasmid-bearing cells.

Finally, plasmids replicated by rolling circle replication in particular desirable bacterial strains can result in loss of sequences for two reasons: (i) rolling circle replication can be allowed across inverted repeats present in hairpin structures; and (ii) long linear plasmid multimers made during rolling circle replication are unstable, which promotes plasmid loss even in the presence of an antibiotic.

Many of the complications associated with cloning and expression in plasmids can be avoided by single-copy expression of the gene from the bacterial chromosome. A variety of methods for integrating a gene into the chromosome have been developed, including use of bacteriophage lambda (Boyd et al., 2000), different recombination-proficient backgrounds (ElKaroui etal., 1999; Murphy, 1998; Russell et al, 1989; Shevell et al, 1988; Datsenko and Wanner, 2000; Yu et al, 2000) and homologous and site-specific recombination (Hamilton et al, 1989; Atlung et al, 1991 ; LeBorgne et /., 1998; Martinez-Morales etal, 1999; Yu and Court, 1998). All of these methods make use of antibiotic resistance markers for selection of the integrated DNA.

Because most gene expression and cloning in Escherichia coli utilizes plasmids, there are only a few available integration systems for inserting transgenes. Integration avoids potential problems with overexpression and escape from repression of a transgene on a multicopy plasmid and subsequent toxicity to the plasmid or cell. Furthermore, another advantage to integration of a transgene is to avoid producing aberrant phenotypes or phenotypic differences. For instance, differences in phenotypes can occur in response to expression of a single copy and expression of multiple copies of a transgene (e.g. Ow and Ausubel, 1983). Most integration vectors use selectable antibiotic-resistance markers to select integration events, including kanamycin-resistance (Hasan et al, 1994; Atlung et al, 1991), ampicillin-resistance (Diedrich et al, 1992), or chloramphenicol- or other antibiotic-resistance (Balbas et al, 1996; Alexeyev et al, 1995). For the aforementioned reasons concerning antibiotic selection, these systems are less than desirable, and the present invention describes new vectors for integration which are selectable without using antibiotic-resistance markers. Although a plasmid may alternatively be maintained in a cell through complementation of an auxotrophic mutation on the chromosome, this approach severely restricts the composition of the fermentation medium to one lacking the required nutrient of the host bacteria. Moreover, syntrophism may allow cells to continue growth after loss of the plasmid.

Although examples exist of integration events occurring under temperature sensitive conditions, these events reflect the temperature sensitivity of replication of the plasmid vector (U.S. Pat. No. 5,733,753; Perez-CasaL etα/., 1993; Prozorov, etα/., 1985; Lagos and Goldstein, 1984). That is, cells are selected for by integration of a plasmid- borne selectable marker, and in those cells where the plasmid has not integrated, the plasmid is lost at high temperature and the cell can no longer live under the selective conditions. Growth at permissive temperatures following integration allows excision of the integrated DNA.

Cloning vectors for integration of a plasmid into prophage sequences at the phage lambda origin of replication have been utilized (Boyd and Sherratt, 1995). Again, antibiotic resistance is the means for selection for integration.

There are numerous examples of site-specific integration at attB sites in bacteria (e.g. U.S. Pat. No. 5,736,367, 5,733,753 or 5,364,779).

In U.S. Pat. Nos. 4,506,013 and 4,650,761 vectors containing the temperature sensitive cl repressor and a gene of interest are introduced into the cell and provide in trans repression of chromosome-borne lethal genes.

In U.S. Pat. No. 4,673,640 a lambda phage site specific recombination enzyme system is utilized in which the int and xis gene products of lambda phage are repressed by a temperature sensitive repressor until a shift in temperature causes loss of repression. As a consequence, rearrangement of two segments of DNA separated by lambda phage attachment sites, borne on a plasmid in a preferred embodiment, occurs to allow production of a functional polypeptide.

Finally, in Jones and Errington (1987) vectors are described which incorporate a temperature sensitive repressor introduced on a plasmid in which integration is identified by resistance to chloramphenicol. Elevation of temperature promotes induction of the lysogenic phage, which the authors exploit to construct genomic libraries

(Errington and Jones, 1987).

It is noteworthy that although the repressor/phage system has been described in the related art of U.S. Pat. Nos.4,506,013, 4,650,761, 4,673,640 and Jones andErrington

(1987) well over a decade ago, it has not been used for the innovative purpose of integration and negative selection for a transgene, suggesting such an invention is clearly nonobvious.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a method for creating a transgenic cell comprising the steps of: (1) introducing a linear vector into the cell wherein said vector contains a cassette comprising sequentially from 5' to 3': a 5' flanking sequence, a polylinker site containing a nucleic acid sequence of interest, and a 3' flanking sequence wherein said 5' and 3' flanking sequences are homologous to a sequence in a chromosome in the cell where the nucleic acid sequence of interest is to be inserted; and (2) integrating said cassette into the chromosome through recombination between the flanking sequences and the homologous DNA sequences wherein said homologous DNA sequences flank a conditional killing module in a chromosome; and (3) negatively selecting against cells retaining said conditional killing module and deficient for integration of said linear vector.

In a specific embodiment of the present invention the cassette further includes a 5' rare-cutter restriction site which is 5' to the 5' flanking sequence and a 3' rare-cutter restriction site which is 3' to the 3' flanking sequence wherein said 5' and 3' rare-cutter restriction sites have one or more sequences which are cut by rare cutter restriction enzymes. In a specific embodiment the rare-cutter restriction sites are selected from the group consisting of Srfi, Ndel, Sfil, Avrll and Ascl. In another specific embodiment the 5' and 3' flanking sequences comprise nucleic acid sequences which flank αttB in Escherichia coli. In another embodiment of the present invention there is a method for creating a transgenic cell comprising the steps of: (1) introducing a linear vector into the cell wherein said vector contains a cassette comprising sequentially from 5' to 3': a 5' flanking sequence, a nucleic acid sequence of interest, and a 3' flanking sequence wherein said 5' and 3' flanking sequences are homologous to a sequence in a chromosome in the cell where the nucleic acid sequence of interest is to be inserted; and (2) integrating said cassette into the chromosome through recombination between the flanking sequences and the homologous DNA sequences wherein said homologous DNA sequences flank a conditional killing module in a chromosome; and (3) negatively selecting against cells retaining said conditional killing module and deficient for integration of said linear vector. In a specific embodiment the cassette is generated by polymerase chain reaction.

In specific embodiments said conditional killing module includes a conditional repressor. In a preferred embodiment said conditional killing module contains temperate prophage sequences including a temperature sensitive conditional repressor. In a further embodiment said cell is Escherichia coli and said conditional killing module is the lambda xisl clts857 prophage. In other embodiments said cell is Escherichia coli and said conditional killing module is the lambda wi clts857 prophage, the defective lambda prophage lambda clts857 A(cro-bioA), the defective lambda prophage lambda clts857 clind A(cro-bioA), the defective lambda prophage lambdaxisl clts857 A(cro-bioA), the defective lambda prophage lambdaxisl clts857 clind A(cro-bioA), the defective lambda prophage lambda clts857 clind A(cro-αttR), or the defective lambda prophage lambda clts<°57 clind P_BAD A(cro-αttR).

In an additional embodiment the integrated cassette is transduced by recombination between cells. In a specific embodiment the integrated cassette is transduced between cells with the assistance of a helper phage. In an additional embodiment said cell is Escherichia coli and said integrated cassette is transduced between Escherichia coli cells which contain the prophage lambda xisl clts857 with the assistance of a PI helper phage. In additional embodiments said cell is a proficient host for linear replacement using homologous recombination and is selected from the group consisting of recD, recBC sbcBC, recBC sbcA, recB carrying λ red" genes and rec⁺ carrying λ red" and gam⁺ genes.

Another specific embodiment is a vector selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

An additional embodiment is the vector for introduction of a sequence of interest into a cell wherein said vector includes a cassette, said cassette comprising sequentially from 5' to 3': a 5' rare-cutter restriction site which is 5' to a 5' flanking sequence, a polylinker site containing the nucleic acid sequence of interest, and a 3' rare cutter restriction site which is 3' to the 3' flanking sequence wherein said 5' and 3' rare cutter restriction sites have one or more sequences which are cut by rare-cutter restriction enzymes and wherein said 5' and 3' flanking sequences are homologous to a sequence in a chromosome in the cell where the nucleic acid sequence of interest is to be inserted.

In preferred embodiments of the method and the vector the 5' and 3' flanking sequences of said cassette are sequences which flank attB in Escherichia coli. In a preferred embodiment the 5' and 3' rare cutter restriction sites are selected from the group consisting of Srfi, Ndel, Sfil, Avrll, and Ascl.

Another embodiment is the vector for introduction into a cell wherein said vector includes a cassette comprising sequentially from 5' to 3': a 5' rare cutter restriction site selected from the group consisting of Srfi, Ndel, Sfil, Avrll, and Ascl, a 5' flanking sequence, a polylinker site containing a nucleic acid sequence of interest, a 3' flanking sequence and a 3' rare cutter restriction site selected from the group consisting of Srfi, Ndel, Sfil, Avrll, and Ascl. In a specific embodiment the 5' and 3' flanking sequences consist of nucleic acid sequences which flank attB in Escherichia coli. In another embodiment the nucleic acid sequence of interest is regulated by nucleic acid sequence selected from the group consisting of promoter sequence and bacterial translation- initiation sequence. In a further embodiment the promoter is an inducible promoter. In an additional specific embodiment the promoter is the P_BAD promoter. In another specific embodiment the bacterial translation-initiation sequence is a Shine Dalgarno sequence.

Other and further objects, features and advantages would be apparent and eventually more readily understood by reading the following specification and by reference to the company drawing forming a part thereof, or any examples of the presently preferred embodiments of the invention which are given for the purpose of the disclosure.

DESCRIPTION OF THE DRAWINGS

Figure 1 depicts a strategy for linear replacement using the TGV system. Figure 2 depicts the TGV-Light plasmid having SEQ ID NO: 1. Figure 3 depicts the TGV-Cat plasmid having SEQ ID NO:2.

Figure 4 depicts the TGV-Kan plasmid having SEQ ID NO:3. Figure 5 depicts the TGV-Tet plasmid having SEQ ID NO:4. Figure 6 depicts the TGV-ProkExpress plasmid having SEQ ID NO:5. Figure 7 depicts the TGV-Express plasmid having SEQ ID NO:6. Figure 8 depicts the TGV-Express Cat plasmid having SEQ ID NO:7.

Figure 9 depicts the TGV-Express Kan plasmid having SEQ ID NO: 8. Figure 10 depicts the TGV-Express Tet plasmid having SEQ ID NO:9. Figures 11A and 11B demonstrate a comparison of efficiency of linear replacement TGV in different E. coli rec mutant genotypes. Figure 11A shows a comparison of mono- versus multi-lysogens with and without the addition of pTGV-Light DNA. Figure 1 IB shows a comparison of isogenic strains with and without the addition of pTGV-Light DNA.

Figures 12A and 12B demonstrate verification of linear replacement by colony PCR. In Figure 12A the presence of a PCR product was assayed. In Figure 12B the verification of PCR products was confirmed. DESCRIPTION OF THE INVENTION

It is readily apparent to one skilled in the art that various embodiments and modifications may be made to the invention disclosed in this Application without departing from the scope and spirit of the invention.

The term "blunt-cutter restriction enzyme site" as used herein is defined as a restriction enzyme site at which said enzyme cleaves one phosphodiester bond of each DNA strand between the same two specific hydrogen-bonded base pairs. Said cleavage produces DNA fragments with ends which contain no single stranded protrusion. Said sites may be of any size or form, including methylated form, and some examples include Smal, EcoKV, or Xmnl.

The term "cell" as used herein is defined as a structural unit of an organism, surrounded by a membrane and composed of cytoplasm and at least one nucleus or nucleoid. In some cells there may be a cell wall outside the membrane. Cells as used herein can mean any kind of cells, including prokaryotic microorganisms, such as bacteria, and eukaryotic microorganisms, such as fungi, yeasts, algae, etc. Cells may also be of vegetable or animal (including human) origin.

The term "conditional killing module" as used herein is defined as DNA sequence which contains (i) sequence encoding a gene product that will kill the cell under some conditions but not others. One example is a sequence encoding a conditional repressor and a sequence whose expression is controlled by said conditional repressor and whose expression is lethal to the cell. One skilled in the art would know that any sequences whose expression is lethal to the cell and is subject to a conditional repressor would work in the present invention. Any sequence encoding a product constitutively expressed that is toxic to the cell only under some condition, for example, when cells are exposed to a particular chemical, nutrient, nucleic acid, protein, sugar, lipid, carbohydrate, drug, temperature, light, pressure, or sound. A specific example is sacB which is not subject to conditional expression and kills only in the presence of sucrose. In a preferred embodiment said conditional killing module contains prophage sequences which encode a gene product required for cell lysis and sequence which encodes a temperature sensitive repressor which represses expression of said cellular lysis gene product. Illustrative examples exist of other sequences which are lethal to the cell such as CcdB or SacB, or which may contain mutations lethal to the cell including mutations in chromosomal DNA replication, cell wall synthesis, ribosome function, RNApolymerase, tRNA synthesis and modification, aminoacyl tRNA synthetase, DNA restriction and modification, and cell division. Other sequences which would be lethal in any form to the cell would be apparent to those skilled in the art.

The term "DNA" as used herein is defined as deoxyribonucleic acid. The term " 5' " as used herein refers to a reference point for an entity such as a nucleotide or nucleotides which are in a position relative to a specific nucleotide or nucleotides through which there is linkage with the fifth carbon of 2-deoxyribose.

The term "5' flanking sequence" as used herein is defined as a sequence which is 5' relative to a polylinker site which contains a nucleic acid sequence of interest.

The term "5' rare cutter restriction site" as used herein is defined as a restriction enzyme site which is 5' relative to both a polylinker site which contains a nucleic acid sequence of interest and a 5' flanking sequence.

The term "homologous recombination" as used herein is defined as the replacement of one sequence with a homologous sequence.

The term "linear replacement" as used herein is defined as homologous recombination between a linear DNA fragment and a chromosomal site. The term "lysogen" as used herein is defined as a bacteria which contains viral genetic material within its host genome.

The term "negatively selecting" as used herein is defined as the act of using a specific condition to kill, or prevent from growing, cells with a particular phenotype while permitting survival and growth of cells without said particular phenotype. The phenotype of the preferred embodiment of the invention is the presence of prophage sequences containing a temperature sensitive conditional repressor which represses gene products required for cell lysis.

The term "non blunt-cutter restriction enzyme site" as used herein is defined as a restriction enzyme site at which said enzyme cleaves one phosphodiester bond of each DNA strand between two different specific hydrogen-bonded base pairs. Said cleavage produces DNA fragments with ends which contain either a 5' or 3' single stranded protrusion. Said sites may be of any size or form, including methylated form, and some examples include EcoRI, Sac I, or Sal I.

The term "P_BAD" ^{as use}d herein is defined as the arabinose inducible promoter. The term "PCR" as used herein is defined as polymerase chain reaction. Methods regarding all aspects of PCR, including reaction conditions and primer design, are well known in the art.

The term "polylinker site" as used herein is defined as a site containing at least one restriction enzyme site for the purpose of inserting a nucleotide sequence of interest. A skilled artisan is aware which restriction enzyme sites would be useful for such a purpose. One skilled in the art is aware that the restriction sites themselves can be of any size or form, including a methylated form. Said restriction enzyme site can include any DNA sequence which is recognized by a specific restriction enzyme and could be a blunt- cutter restriction enzyme site or a non blunt-cutter restriction enzyme site.

The term "prophage" as used herein is defined as a virus which has incorporated its genetic material into the cell or genome of a host.

The term "rare cutter restriction enzyme"as used herein is defined as a restriction enzyme which cuts DNA at a rare cutter restriction site. Some examples include Srfi, Ndel, Sfil, Avrll, and Ascl.

The term "rare cutter restriction site" as used herein is defined as a restriction site which is present in the DNA of the cell used at a frequency no greater than one in every

10,000 base pairs such that it is likely to be useful in cloning any nucleic acid sequence of interest. One skilled in the art is aware that the restriction sites themselves can be of any size or form, including a methylated form or a blunt cut site. Some examples include

Srfi, Ndel, Sfil, Avrll, and Ascl. The term "rec" as used herein is defined as the recombination defective mutant.

The term "reef' as used herein is defined as the bacteriophage λ homologous recombination system.

The term "repressor" as used herein is defined as an agent which represses expression of a particular DNA sequence. The term "RNA" as used herein is defined as ribonucleic acid.

The term "sbc" as used herein is defined as supressor of RecBCD. The term "syntrophism" as used herein is defined as the act of two entities (proteins, nucleic acids, cells, etc.) which can not perform a function independently, but acting together can perform the function.

The term "temperate prophage" as used herein is defined as a virus which infects a bacteria cell and whose genetic material can become integrated into the host genome, therein duplicated along with the host material upon replication. The viral DNA has the capacity to educt from the host genome which subsequently leads to cellular lysis.

The term " 3' " as used herein refers to a reference point for an entity such as a nucleotide or nucleotides which are in a position relative to a specific nucleotide or nucleotides through which there is linkage with the third carbon of 2-deoxyribose.

The term "3' flanking sequence" as used herein is defined as a sequence which is 3' relative to a polylinker site which contains a nucleic acid sequence of interest.

The term "3' rare cutter restriction site" as used herein is defined as a restriction enzyme site which is 3' relative to both a polylinker site which contains a nucleic acid sequence of interest and a 3' flanking sequence.

The term "transgene" as used herein is defined as a foreign gene introduced into the genome of the host or a native gene of the host introduced into a new position in the host. In a specific embodiment, the term "transgene" is used herein interchangeably with the term "nucleic acid sequence of interest." The term "vector" as used herein is defined as any vehicle which delivers a nucleic acid into a cell. In a preferred embodiment, said vector is a linear DNA fragment.

Examples include viral vectors, PCR products, or fragments and derivatives thereof. One skilled in the art would be cognizant of kinds of linear DNA fragments and means to obtain them. In a specific embodiment the linear DNA fragment is a restriction fragment. One embodiment of the present invention is a method for creating a transgenic cell comprising the steps of: (1) introducing a linear vector into the cell wherein said vector contains a cassette consisting of sequentially from 5' to 3': a 5' flanking sequence, a polylinker site containing a nucleic acid sequence of interest, and a 3' flanking sequence wherein said 5' and 3' flanking sequences are homologous to a sequence in a chromosome in the cell at a location where the nucleic acid sequence of interest is to be inserted; and

(2) allowing integration of said vector into the chromosome through recombination between the flanking sequences and the homologous DNA sequences wherein said homologous DNA sequences which flank a conditional killing module in a chromosome; and (3) negatively selecting against cells retaining said conditional killing module and deficient for integration of said linear vector. In a specific embodiment of the present invention the cassette can further include a 5' rare cutter restriction site which is 5' to the 5' flanking sequence and a 3' rare cutter restriction site which is 3' to the 3' flanking sequence wherein said 5' and 3' rare cutter restriction sites have one or more sequences which are cut by rare cutter restriction enzymes. In specific embodiments said conditional killing module includes a conditional repressor. In a preferred embodiment said conditional killing module contains temperate prophage sequences including a temperature sensitive conditional repressor. In a further embodiment said cell is Escherichia coli and said conditional killing module is the lambda xisl clts857 prophage. In other embodiments said cell is Escherichia coli and said conditional killing module is the lambdaxisl clts857 prophage, the defective lambda prophage lambda clts857 A(cro-bioA), the defective lambda prophage lambda clts857 clind A(cro-bioA), the defective lambda prophage lambda xisl clts857 A(cro-bioA), the defective lambda prophage lambdaxisl clts857 clind^' A(cro-bioA), the defective lambda prophage lambda clts857 clind A(cro-αttR), or the defective lambda prophage lambda clts857 clind P_BAD A(cro-αttR).

In an additional embodiment the integrated cassette is transduced between cells. In a specific embodiment the integrated cassette is transduced between cells with the assistance of a helper phage. In an additional embodiment said cell is Escherichia coli and said conditional killing module is transduced between Escherichia coli cells which contain the prophage lambda xisl clts857 with the assistance of a PI helper phage. In additional embodiments said cell is a proficient host for linear replacement using homologous recombination and has a genotype selected from the group consisting of recD, recBC sbcBC, recBC sbcA, recB carrying λ red" genes and rec⁺ carrying λ red" and gam⁺ genes. Another specific embodiment is a vector selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

An additional embodiment is the vector for introduction of a sequence of interest into a cell wherein said vector includes a cassette, said cassette comprising sequentially from 5' to 3': a 5' rare-cutter restriction site which is 5' to a 5' flanking sequence, a polylinker site containing the nucleic acid sequence of interest, and a 3' rare-cutter restriction site which is 3' to the 3' flanking sequence wherein said 5' and 3' rare-cutter restriction sites have one or more sequences which are cut by rare restriction enzymes and wherein said 5' and 3' flanking sequences are homologous to a sequence in a chromosome in the cell where the nucleic acid sequence of interest is to be inserted.

In preferred embodiments of the methods and the vectors provided herein, the 5' and 3' flanking sequences of said cassette are sequences which flank attB in E. coli. A skilled artisan is aware of standard sequence repositories which contain such sequences and would know how to routinely search such databases to obtain them, including the following: http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/framik?db=Genome&gi=l 15 ; http ://bsw3.aist-nara.ac.jp/GTC/mori/research/dbservice/ecoli-e.html; http : //www . ncb i . nlm . n i h . gov/C o mp lete_Genome s/Eco l i/ ; and/or http://www.genetics.wisc.edu/. In addition, a skilled artisan is aware of and could utilize other repositories such as the American Type Culture Collection (ATCC), available at http://phage.atcc.org/searchengine/all.html, to obtain bacteria and phages which may be utilized for the present invention. A skilled artisan is also aware that sequences and strains utilized in published articles in the art are available upon request to the authors.

In a preferred embodiment the 5' and 3' rare-cutter restriction sites are selected from the group consisting of Srfi, Ndel, Sfil, Avrll, and Ascl.

Another embodiment is the vector for introduction into a cell wherein said vector includes a cassette comprising sequentially from 5' to 3': a 5' rare-cutter restriction site selected from the group consisting of Srfi, Ndel, Sfil, Avrll, an Ascl, a 5' attB flanking sequence, a polylinker site containing a nucleic acid sequence of interest, a 3' attB flanking sequence and a 3' rare-cutter restriction site selected from the group consisting of Srfi, Ndel, Sfil, Avrll, and Ascl.

A skilled artisan would understand that said 5' flanking sequences and 3' flanking sequences incorporated in the vector for homologous recombination would have to be homologous to sequences present within the host genome and each flanking sequence must be at least about 23 base pairs of DNA. One skilled in the art would also know that the identity of the sequence could be any sequence so long as loss of the interstitial sequence beween said 5' and 3' flanking sequences upon its removal through integration of the transgene would not be lethal to the cell. One skilled in the art recognizes that increasing the size of sequence utilized in the vector for homologous recombination would increase the likelihood of recombination. This, however, is balanced by the increased likelihood of introducing an undesired restriction enzyme site already present within the cassette and rendering the vector more difficult to work with. Thus, a skilled artisan would take into consideration these factors in determining the size of flanking sequence to use for homologous recombination. In a preferred embodiment, the sequences which flank either side of attB in the E. coli genome are utilized in the present invention as the 5' flanking sequences and 3' flanking sequences. These sequences are provided herein as SEQ ID NO:34 and SEQ ID NO:35. A skilled artisan is aware that any region within these sequences may be utilized for homologous recombination so long as they meet the conditions mentioned above. Furthermore, in the specific embodiment wherein the linear vector is a PCR-generated nucleic acid, any sequence within these sequences may be utilized to design primers for polymerization, and a skilled artisan would be aware how to design the primers and what parameters should be considered.

The vector backbone which contains the cassette of the invention may be any nucleic acid sequence which would allow liberation of said cassette upon restriction enzyme digestion with a rare-cutter restriction site of said cassette or upon PCR or other means of liberation of the cassette. Alternatively, a linear fragment for introduction into a cell is generated by polymerase chain reaction by methods well known in the art. The primers utilized for the polymerase chain reaction include sequence which derives from either 5' or 3' flanking sequences, such as those which flank attB in the Escherichia coli genome, and sequence which derives from the nucleic acid sequence of interest. The nature of the transgene to be integrated can be any DNA fragment which would be useful to make a transgenic organism. A skilled artisan would be fully aware how to generate the vector constructs using standard molecular biology methods well known in the art and could use discretion regarding what sequences would be useful to insert into the genome of the host organism. A skilled artisan would also know that in addition to inserting DNA sequences which produce a useful protein as a final gene product that DNA sequences which produce a useful RNA as a final gene product may be inserted. Furthermore, one skilled in the art would know that control sequences required for expression of said transgene, including promoters, enhancers, or any cis- acting elements required for regulation of expression may be employed. Also, any sequences necessary for production of the final gene product, for instance Shine- Dalgarno sequences or AUG initiator codons, may be included in the cassette. A skilled artisan is aware that a Shine-Dalgarno sequence is, in a specific embodiment, part or all of the polypurine sequence AGGAGG and is present just prior to an AUG initiation codon. The sequence is associated with binding of a ribosome to mRNA and in a specific embodiment is complementary to a sequence at the 3' end of 16S rRNA. Based on the gene of interest, one skilled in the art would know which vectors and regulatory elements would be useful.

The present invention provides a conditional repressor to repress genes whose expression would be lethal to the cell. In a preferred embodiment the nature of the activity of the conditional repressor is temperature sensitive. In specific embodiments a cl gene product serves as the conditional repressor. One skilled in the art would know that in addition to λclts857, any other λcl gene that produces a functional repressor can be used. Other repressor genes, such as, for example, the λcro gene can also be used. It would be known to those skilled in the art that repressors responsive to other conditions, for instance changes in light, chemicals, osmolarity, pressure, touch, sound, stress, or concentration or nature of an associated protein may be used. In a further specific embodiment said repressor is a nonsense repressor. A nonsense repressor is a gene product which allows the insertion of an amino acid into an extending polypeptide chain in response to a nonsense codon. A nonsense codon is a chain-terminating codon introduced as a result of a nonsense mutation such as a base substitution or frameshift mutation. In this embodiment the sequence of said conditional killing module is a sequence containing a lethal nonsense mutation and wherein said nonsense repressor is a conditional nonsense repressor.

Although a preferred embodiment of the present invention utilizes the arabinose- inducible P_BAD promoter in the vectors described in Examples 9, 10 and 11, a skilled artisan would be aware of other promoter or promoter derivatives or fragments thereof which would have the same function of providing an element for inducibility of expression of a nucleotide sequence. Other examples of inducible promoters are the galactose promoter, the lac, tac, or mac promoters and the Pspac promoter. A variety of methods for introducing vectors into host cells are known in the art, including but not limited to electroporation; transformation employing calcium chloride, rubidium chloride calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (where the vector is an infectious agent, such as a retro viral genome). In an embodiment of the present invention, a linear fragment generated by polymerase chain reaction is introduced into a cell, wherein the fragment comprises a 5' flanking sequence, a nucleic acid sequence of interest, and a 3' flanking sequence, wherein the 5' and 3' flanking sequences are homologous to a sequence in a chromosome in the cell at a location where the nucleic acid sequence of interest is to be inserted; integration of the cassette into the chromosome through recombination occurs between the flanking sequences and the homologous sequences, wherein the homologous DNA sequences flank a conditional killing module in the chromosome; and negatively selecting against cells retaining the conditional killing module and deficient for integration of the cassette. In a specific embodiment the linear DNA for introduction is a polymerase chain reaction product generated with one primer containing approximately

30-40 nt of 5' flanking sequence and approximately 20-25 nt of sequence from a gene of interest and another primer having 20-25 nt of sequence from a gene of interest and approximately 30-40 nt of 3' flanking sequence by methods and in orientations for polymerization well known to a skilled artisan. However, a skilled artisan is aware that other lengths of sequences would work in the invention and would know how to determine such lengths and identify optimal sequences for the primers for polymerase chain reaction. In another embodiment, the primer contains flanking sequence anywhere in the range from about 15 to about 30 nt and the primer contains sequence from a gene of interest in the range from about 10 to about 20, although other lengths would also work. A skilled artisan is aware that in addition to the length of the primer, other parameters are important for design including the content of G's and C's, the melting temperature of the primer and the presence of any sequence which may facilitate undesirable secondary structure within the primer or with another primer.

In another embodiment of the present invention the vector comprises a 5' flanking sequence, a nucleic acid sequence of interest, an antibiotic resistance gene, and a 3' flanking sequence. In this embodiment, the prophage/conditional killing module system of the present invention is not required. Instead, the cassette integrates into the sequences which correspond to the 5' and 3' flanking sequence, and the antibiotic resistance is selected for to identify transgenic cells which have incorporated the nucleic acid sequence of interest.

The following discussion describes the nature of the system involved in the invention and helps to illustrate specific embodiments. Bacteriophage λ is a temperate bacteriophage which upon infection of Escherichia coli follows either of two mutually exclusive cycles. In the lytic phase the bacteriophage DNA replicates autonomously, directs synthesis and assembly of bacteriophage components, and kills the cells concomitant with the release of mature bacteriophage. Alternatively, in the lysogenic phase the bacteriophage becomes integrated into the host chromosome as a prophage, replicates on the chromosome with the endogenous host DNA, and blocks synthesis of bacteriophage components. A bacteriophage gene, λ cl, codes for a repressor that maintains the lysogenic state and blocks expression of genes for bacteriophage components and maturation. If the repressor is inactivated or is no longer present in the cell the prophage educts from the chromosome, enters the lytic cycle, and subsequently kills the cell. Bacteriophage with a defective λ cl gene can not maintain the lysogenic state and are lethal to the cell. A temperature sensitive allele of the λ cl gene, λ clts857, allows repression at lower permissive temperatures but loses repressor activity at higher non-permissive temperatures. A temperature shift to higher temperatures lyses the cells by inducing the lytic cycle of the lambda prophage which, in accordance with the present invention, has been incorporated into the host cell strain. As is readily apparent, when a temperature sensitive repressor which represses a lethal sequence that causes host cell death is used and when the host cells are cultured at a temperature which inactiviates the repressor and, at a temperature which is not within the range for permissive culture, the present invention provides cellular death. In a specific embodiment of the invention the prophage is the temperate bacteriophage λ residing within the host chromosome of Escherichia coli, the temperature sensitive repressor repressing expression of λ prophage sequences is λclts857, and the temperature shift to greater than 34° C inactivates said repressor.

In specific embodiments said conditional killing module includes a conditional repressor. In a preferred embodiment said conditional killing module contains temperate prophage sequences including a temperature sensitive conditional repressor. In a further embodiment said cell is Escherichia coli and said conditional killing module is the lambda xisl clts857 prophage. In other embodiments said cell is Escherichia coli and said conditional killing module is the lambdaxzsi clts857 prophage, the defective lambda prophage lambda clts<°57 A(cro-bioA), the defective lambda prophage lambda clts<°57 clind^' A(cro-bioA), the defective lambda prophage lambdaj ύJ clts857 A(cro-bioA), the defective lambda prophage lambdaxisl clts857 clind A(cro-bioA), the defective lambda prophage lambda clts857 clind^' A(cro-αttR), or the defective lambda prophage lambda clts<°57 clind P_BAD A(cro-αttR).

The present invention provides significant improvements over the present art by describing vectors in which transgenes and flanking DNA are inserted in the chromosome by linear replacement via homologous recombination in a cell carrying a prophage with a conditional allele of a repressor. In a preferred embodiment, said condition is an increase in temperature. The present invention uses as a mode of negative selection the system of repression of lethal gene products by said conditional repressor, in marked contrast to the presence of a marker, such as an antibiotic resistance marker, which is one of the significant improvements the present invention has over the related art. Upon shifting the lysogenic cells to high temperature, all lysogens are killed and only those cells which have lost their prophage due to homologous replacement by the linear vector fragment survive.

The following examples are offered by way of example, and are not intended to limit the scope of the invention in any manner.

EXAMPLE 1 General Strategy

The TGV system employs a simple two-step method of cloning and homologous replacement. Each vector contains a multiple-cloning site (MCS) flanked by approximately 2 kb of sequence from the attB region of the E. coli chromosome (Fig. 1). The multiple cloning site replaces 23 bp of the attB core sequence (Weisberg and Landy, 1983) with eight restriction endonuclease sites for cloning transgenes (Fig . 1 ) . The gene being transferred (nucleic acid sequence of interest) is cloned into this multiple cloning site. The vector, now containing the transgene (nucleic acid sequence of interest), is linearized using one of the five infrequently cutting restriction enzymes that cut in the rare-cutter sites (RCS) at the outside ends of the entire region of homology (Fig. 1). This linear fragment is then transformed into recombination-proficient E. coli carrying a lambda prophage, which is excision-defective (xisl) and carries a temperature-sensitive Cl repressor (clts857) such that the prophage is quiescent at 34°C or lower, but is induced and kills the cell at high temperature (42°C) . Transformants are selected by plating at the restrictive temperature (42°C). Recombinants in which the prophage has been replaced survive this selection, whereas lysogens are killed. Linear replacement is confirmed by PCR across the inserted gene. In Figure 1, MCS stands for multiple cloning site (such as Sph I, Xbal, Xhol, BgHl, Hindlϊl, CM, Sna l, and Agel) and RCS stands for rare cutter sites (such as Ascl, Avrll, Sfil, Ndel, and Srfi).

Nine different vectors have been developed (Table 1 and the following

Examples). pTGV-Light is the simplest version containing the regions of homology, Table 1. Vectors

Unique cloning sites Prokaryotic translation Selection for integrati available in multi- Size (bp) Promoter initiation signal provided Drug^κ transgene cloning site³ provided on plasmid

Name

pTGV-Light 8 6275 None No Amp Temperature^ (against

pTGV-ProkExpress 7 6583 ^pBAD No Amp Temperature ^R (against

pTGV-Express 7 6610 ^PBAD Yes Amp Temperature^ (against

pTGV-CAT 8 7240 None No Amp/Cam Chloramphenicol^R/Tem

pTGV-Kan 5 7157 None No Amp/Kan Kanamycin^R/Temp^R

pTGV-Tet 7 7515 None No Amp/Tet Tetracycl ine ^R/Temp ^R

pTGV-Express CAT 7 7323 ^PBAD Yes Amp/Cam Chloramphenicol^R/Tem

pTGV-Express Kan 7492 ^PBAD Yes Amp/Kan Kanamycin^R/Temp^p

pTGV-Express Tet 7850 ^PBAD Yes Amp/Tet Tetracycline^R/Temp^R

flanked by rare-cutter sites, and the multiple cloning site for cloning genes with their own promoters. pTGV-ProkExpress is pTGV-Light with an arabinose-inducible promoter (P_BAD) (Guzman et al, 1995) added just upstream of the multiple cloning site, to allow regulatable expression of the transgene. The AraC regulatory protein is expressed from its normal chromosomal location, although in a specific embodiment it may be provided on a TGV vector or another vector. pTGV-Express, like pTGV-ProkExpress, contains P_BAD and also includes the Shine-Dalgarno prokaryotic translation-initiation sequence added between the P_BAD promoter and the multiple cloning site for expression of heterologous genes in E. coli. The ATG sequence in the recognition site of Sphl, in the multiple cloning site, functions as the initiating methionine. Versions of pTGV-Light and pTGV-Express were also constructed with genes encoding chloramphenicol-resistance, kanamycin-resistance, or tetracycline-resistance so that selection for antibiotic resistance may be used if desired. No prophage is necessary when antibiotic selection is used. Sites not available in these vectors are as follows: Agel in pTGV-ProkExpress, pTGV-Express, pTGV-Express CAT, and pTGV-Express Tet; Htndlll, CM, and Xhol in pTGV-Kan; Sphl in pTGV-Tet; and Agel, CM and Xhol in pTGV-Express Kan. All sites are separated by one or two nucleotides. Plasmid sequences are available.

EXAMPLE 2 The TGV System: TransGenic Escherichia coli Sectors for chromosomal gene expression

An approximately 2 kilobase pair region of Escherichia coli chromosomal DNA that flanks the bacterial chromosome site into which phage λ integrates upon lysogeny, attB, has been cloned into a standard ColEl plasmid. The λ attB insertion site is a small precise sequence and was replaced in the vector with a polycloning site for insertion of transgenes. At the ends of this sequence, synthetic sequences encoding the different rare restriction sites Srfi, Ndel, Sfil, Avrll, and Ascl were inserted. These allow liberation of the linear attB flanking DNA for subsequent integration by homologous recombination into the bacterial chromosome. The rare-cutter restriction site is selected such that digestion with the rare-cutting enzymes will not cut whatever transgene(s) of interest will be inserted into a polycloning site that was engineered into the center of the bacterial homology, replacing the attB site itself.

The transgenes and flanking DNA are placed in the Escherichia coli chromosome by homologous recombination as follows: the bacterial DNA region, of 3 kilobases plus inserted transgenes, is amplified by PCR with primers complementary to the ends of the bacterial segment to produce a linear fragment or alternatively is liberated by restriction enzyme digestion of a plasmid preparation. This linear fragment is elecfroporated into Escherichia coli cells which are recD, recBC sbcBC, recBC sbcA, recB strains carrying λ red" genes, or rec⁺ strains carrying λ red" and gam⁺ genes. Cells which are recD do not degrade linear DNA, and linear replacement-recombination reactions work efficiently in this background (Russell et α/.,1989). Linear replacement in recD cells has been used previously to construct several useful alleles (e.g. seeRazavy etal, 1996). The recD cell is a λ lysogen whose prophage carries a temperature-sensitive repressor allele, clts857. Such a prophage is lysogenic at 32° but becomes lytic at temperatures greater than 34° and kills the cell (Murray, 1983). Selection for linear replacements of the prophage occurs by plating the elecfroporated cells at 42° following a suitable low temperature incubation period to let the replaced chromosome segregate from prophage-bearing sister chromosomes. The cells may also carry a λ-resistance mutation so that they are not killed by any phage in the culture on loss of their prophage, and the prophage is also excision defective due to the xisl allele. Methods concerning similar lysogens and their selection are known in the art (e.g. Rosenberg et al, 1985; Rosenberg, 1985; Rosenberg et al, 1985 ; Rosenberg, 1987). Colonies that survive high temperature have incorporated the nucleic acid sequence of interest, which had been inserted into the polycloning site of the vector.

EXAMPLE 2 Construction of the TGV plasmids The TGV plasmids are derivatives of pLGR2, a pBR322-derived-ampicillin resistant plasmid that contains approximately 2 kilobases of DNA flanking both sides of the bacteriophage lambda attachment site, attB, (SEQ ID NO : 34 and SEQ ID NO : 35) and has sites for infrequently-cutting restriction enzymes flanking the entire 4kb of insert DNA. pLGR2 was constructed from pWRl 01 (a pBR322-based plasmid carrying the Escherichia coli attB site within a 1.7 kb EcoRI-Ztø/wHI insert) as below. All portions of the TGV plasmids that were constructed using PCR-generated DNA are sequenced. Methods well known in the art were utilized for cloning (Sambrook et al, 1989 The template for PCR was either the appropriate purified plasmid DNA (such as for resistance genes) or the bacterial chromosome (such as in colony suspensions). Bacterial strain MG1655 (Bachman, 1996) was used as chromosomal template in all cases, except in construction of pMJl in which W3110 (Bachman, 1996) was used, and in construction of the original TGV-Light polylinker.

EXAMPLE 3

Construction of pLGRl

Additional chromosomal flanking sequence was placed on the BamHl side of the pWRlOl insert by ligating an Ndel-BamHl fragment generated by PCR into Ndel- ifømHI-digested p WRl 01 to create pMJ 1. The PCR primers amplify the Escherichia coli bio region. One primer (5'- TCCGGTCTTCATATGCAGCAACGTGCT-3'; SEQ ID NO: 10) creates an Ndel site for cloning and the other (5'- AAGGCCGAATCCAGACA- 3'; SEQ ED NO:l 1) contains a natural BanϊΑl site. Sites for the rare-cutter restriction enzymes Srfi, Ndel, Sfil, Avrll, and Ascl were added by ligating annealed oligonucleotides (5'- TATGGCGCGCCTAGGCCAATTGGGCCCGGGCA-3' (SEQ ID NO: 12) and 5'-'TATGCCCGGGCCCAATTGGCCTAGGCGCGCCA-3' (SEQ ID NO: 13) containing those sites and Ndel-compatible overhangs into the Ndel site of pMJl , creating pLGRl .

EXAMPLE 4 Construction of pLGR2 Additional chromosomal flanking sequence and sites for the "rare-cutter" enzymes Srfi, Ndel, Sfil, Avrll, and Ascl were added to the EcøRI side of the insert by ligating an EcoRI-EcøO190I fragment generated by PCR using primers 5'- GTGAGTATCAGGGAACGGTA-3' (SΕQ ID NO: 14; t h e Ε c o R I s i t e i s a n a t u r a l s i t e ) a n d 5 ' - GAGCTGACAGAGGCCCTGGCGCGCCTAGGCCATATGGGCCCGGGCGAGC ATATTGATCCGCTGCAAACTGAA-3' (SΕQ ID NO:15; creating an EcoO190I site and the above rare-cutter sites) into EcoRI-EcoO190I digested pMJl . This plasmid was designated pLGR2.

EXAMPLE 5 Construction of TGV-Light

TGV-Light (Figure 2) was constructed by replacing the attB core sequence of pLGR2 with a polylinker. The polylinker was created by PCR with four primers : primer A 5'- GAGGTACCAGGCGCGGTTTG-3' (SEQ ID NO: 16); primer B 5'- GCACCGGTACGTATCGATAAGCTTAGATCTCTCGAGT-3' (SEQ ID NO: 17); primer C 5'- ACGTACCGGTGCGAAACGGGAAGGT-3' (SEQ ID NO: 18); and primer D 5'- GACGCGTACCGACTTTGG-3' (SEQ ID NO: 19) used in a two step PCR procedure with pMJl template DNA to create a final product with Kpnl and Mlul sites on the ends and a polylinker in place of the attB core sequence. Primers A (SEQ ID NO: 16) and B (SEQ ID NO: 17) were used in one PCR reaction and primers C (SEQ ID NO : 18) and D (SEQ ID NO : 19) in a separate reaction. The two resulting PCR products were used as template in a final reaction with primers A (SEQ ID NO: 16) and D (SEQ ID NO: 19). Sequence overlap between the two products allows generation of the final product which was digested with Kpnl and Mlul and ligated into Kp l-Mlul digested pLGR2. The resulting plasmid is identical to pLGR2 except that the attB core sequence is replaced with the polylinker sequence. This original polylinker was later replaced with anew one with additional nucleotides between some sites to enhance cleavage efficiency. The replacement polylinker was ligated into the outermost polylinker sites Sphl and Agel as annealed oligonucleotides 5'-GC ATGCGTCTAGAGCTCGAGGTAGATCTGAAAG CTTGAATCGATGTACGTACTATACCGGT-3' (SEQ ID NO:20) and 5'-ACCGGTAT AGTACGTACATCGATTCAAGCTTTCAGATCTACCTCGAGCTCTAGACGCAT GC-3' (SEQ ID NO:21). See Figure 2 for the polylinker sites.

EXAMPLE 6

Construction of TGV-Cat

For those experiments which can tolerate antibiotic selections, and furthermore so that efficiency of anti-λ selection and traditional antibiotic selection can be compared, versions of the TGV-Light with selectable antibiotic resistance cassettes have been constructed (see also Examples 7, 8, and 11).

TGV-Cat (Figure 3) was constructed by ligating the chloramphenicol-resistance cassette (chloramphenicol acetyl transferase, or cat gene) from pCAT19 (Fuqua, 1992) as a Sphl-Xbal fragment into Sphl-Xbal digested TGV-Light. The polylinker in an early version of this construct was also replaced with the same annealed oligonucleotides (SEQ ID NO:20 and SEQ ID NO:21) as for TGV-Light, but digested with^δαl and Agel into the Xbαl-Agel sites to give TGV-Cat.

EXAMPLE 7 Construction of TGV-Kan

TGV-Kan (Figure 4) was constructed by ligating the kanamycin-resistance gene from pUC4K (Vieira and Messing, 1982) as a Nspl digested PCR product into Sphl digested pTGV-Light using primers 5' TCAACATGTGTCTGCCTCGTGAAGAAG 3' (SEQ ID NO:22) and 5' TCAGCATGCAGCCAGGTTGTGTCTCAA 3' (SEQ ID NO:23) to create the Nspl and Sphl sites. The desired orientation, in which the Sphl site in the multiple cloning cassette was recreated, was determined by Hz^'«dIII digestion. The presence of H dIII, CM, and ^T oI restriction sites in the kanamycin resistance gene excludes their use for cloning.

EXAMPLE 8 Construction of TGV-Tet

pTGV-Tet (Figure 5) was constructed by ligating the tetracycline resistance (tet) gene from pACYC184 (Chang and Cohen, 1978) as a Styl-Xbal digested PCR product i n t o Xb a l d i g e s t e d p T G V - L i g h t u s i n g p r i m e r s 5 ' TCATCTAGATTAATGCGGTAGTTTATC 3' (SEQ ID NO:24) and 5' TCACCTAGGTGCAGCAGCAGTCGCTTC 3' (SEQ ID NO:25) to create the Styl and Xbal sites. Orientation was determined by digestion with Sphl. The presence of a Sphl site in the tet gene excludes its use for cloning.

EXAMPLE 9 Construction of TGV-ProkExpress

The TGV-Light vectors are best used when expressing a gene from its natural promoter. However, for overexpression work regulatable promoters would be advantageous (see also Example 10 and 11). Therefore, the P_BAD inducible promoter has been included in the described vectors.

TGV-ProkExpress (Figure 6) contains the P_BAD promoter and the araC gene (encoding the AraC regulatory protein) amplified from the bacterial chromosome using primers 5'- CAGTCAGCTAGCTCCCGCCATTC-3' (SEQ ID NO:26) and

5'- CATCGCTCTAGAAAAACGGGTATGGAG-3' (SEQ ID NO:27). The P_BAD promoter allows tight regulation of gene expression by the AraC protein via arabinose inducibility and catabolite repression (Guzman et al, 1995). Nhel and Xbal sites placed in the primers of SEQ ID NO:26 and SEQ ID NO:27 were used to create ends for ligation into _^Yδαl-digested TGV-Light. The insert orientation that placed the polylinker downstream of the promoter sequences was chosen. The polylinker in this plasmid was also replaced using the same annealed oligonucleotide polylinker as pTGV-Light but cut with &αl and SnaSl. The presence of an Agel site in the promoter excludes its use for cloning.

EXAMPLE 10 TGV-Express

For expression of eukaryotic genes, and in addition to the tightly regulated inducible P_BAD promoter, the vectors described in Examples 10 and 11 also provide a prokaryotic translation-start signal.

TGV-Express (Figure 7) contains the P_BAD promoter and the araC gene, but also contains an initiating methionine and a Shine-Dalgarno sequence (prokaryotic translation initiation sequence) upstream of the Met codon to allow efficient translation. The P_BAD promoter and araC were amplified from the bacterial chromosome by PCR with primers 5'- TCACATGTCTGAGCTCTCCCGCCATTCAGAGAAGAAAC-3' (SEQ IDNO:28; creating an Nspl site and a Sad site) and

5'- TCAGCATGCTCAGT A.CCTCCTAAAACGGGTATGGAGAAACAGTAG-3' (SEQ ID ΝO:29; creating an Sphl site and providing the initiating methionine codon in the Sphl recognition site and the Shine-Dalgarno sequence). The Nspl-Sphl digested PCR product was ligated into Sphl digested TGV-Light to make TGV-Express. The correct orientation, which recreated the Sphl site, was determined by digestion with S cl. As with pTGV-ProkExpress, the Agel site is not available for cloning.

EXAMPLE 11 Construction of TGV-Express plus Antibiotic Resistance

pTGV-Express-CAT (Fig. 8), -Express-Kan (Fig. 9), and -Express-Tet (Fig. 10) were constructed using the cat, kan, or tet genes as Sphl fragments from the appropriate pKRP plasmid (Reece and Phillips, 1995). Annealed oligonucleotides, which contained a Sphl compatible end and a Sad end to give products with Sad overhangs (5' ACAGGAGCT 3' (SEQ ID NO:30) and 5' CCTGTCATG 3' (SEQ ID NO:31)), were added to each Sphl digest. These were ligated into pTGV-Express, using partial Sad digestion because of the presence of a Sad site in the multiple cloning site. In the pKRP vectors, the H dIII site in the kanamycin gene and the Sphl site in the tetracycline gene have been eliminated making them available for use in cloning with the Kan and Tet versions of pTGV-Express, respectively. EXAMPLE 12 Generating Linear Transformants with the TGV System

Escherichia coli cells were elecfroporated with either no DNA or with 1.5μg

TGV-Light digested with Ndel to yield lμg of the 4.2 kilobase pair linear fragment containing the polylinker and flanking chromosomal DNA. Transformants were selected at 42°C. Temperature-resistant colonies were obtained at frequencies expected for homologous replacement reactions with linear DNA in Escherichia coli. In Table 2, the efficiencies of linear replacement are compared with linear replacements reported by others previously. The efficiencies of transformation using a TGV vector are comparable with what others obtain using selections for antibiotic resistance cassettes in their integrating DNA. In comparisons of antibiotic selection with TGV-Cat versus anti-λ- prophage selection with TGV-Light for integration into the SMR5078 cells in parallel, drug selection is found to be somewhat more efficient.

Table 2. Efficient recovery of homologous linear replacements of a λ prophage with the TGV- transformation of SMR5078 cells

No. of No. colonies per

Exp. DNA molecule

Bacterial strain used (n) transformed Reference

SMR5078 = recBCsbcBC 4 7.7 x l0^"9 ± l x l0-⁹

(λ xis λ cI857) monolysogen This work

recD 1 5.5 x 10-¹² Shevell, et al, 1988 recD 1 1.9 x lO^"8 Russell, et al, 1989 recD 1 4.6 x lO^"9 K.C. Murphy, 1998 recBC sbcBC 1 2.3 x lO^"8 K.C. Murphy, 1998 recBCD::red⁺ 4 3.3 x l0-⁶ ± l x l0^"6 K.C. Murphy, 1998 EXAMPLE 13 Verification of Gene Insertion

The basic vector, pTGV-Light, was used without an insertion to test for replacement of the prophage using the anti-λ prophage selection. Linearized pTGV-Light was elecfroporated into a recombination-proficient strain carrying the temperature-sensitive prophage, and transformants were selected at 42°C. Although some colonies appeared in control platings of cells that were mock-transformed (without DNA), ten- to thirty-fold more appeared in platings of cells with DNA (Fig. 11a and l ib). The colonies on the control plates may result from reversion of the cits and subsequent survival of lysogens at high temperature. Replacement of the prophage was confirmed by polymerase chain reaction (PCR) across the multiple cloning site (Fig. 12a and 12b) as follows.

PCR was performed using primers flanking attB to determine whether the prophage was absent (short PCR product) or not (λ prophage of 48 kilobases is too large to PCR across). Ten transformants from the transformation with TGV-Light DNA and ten colonies from a control transformation with no DNA were plated at 42°C and subsequently were transferred into 50μl water. These cell suspensions were used to provide template DNA for PCR with attB primers attBL: 5'-GGATTCGGTGTTATCG-3' (SEQ ID NO: 32) and attBR: 5'-GGATCCGGCCTTTTG-3'(SEQ ID NO:33). If the λ prophage is still present, no product is observed due to the large distance between the primers, approximately 54 kilobase pairs. If the polylinker has replaced the prophage in a homologous recombination event, a 1.75 kilobase pair product is observed.

In Figure 12 A PCR products were run on a 1 % agarose gel. Lanes 1-10 represent colonies picked from mock transformed plates. Lanes 11-20 represent colonies picked from plates transformed with linear pTGV-Light. The PCR product is approximately

1.73kb. In Figure 12a, no product is seen in the cells that received no DNA, and the expected product is seen in the cells that received DNA.

To verify that TGV-Light DNA was present and had replaced the prophage, the PCR products generated above were digested with restriction enzymes that cut the polylinker which should be present in the replaced DNA, but would not cut wild-type bacterial attB region DNA. This distinguishes the unlikely possibility that the temperature-resistant colonies obtained after transformation with TGV-Light DNA was simply cured of their prophage, which would return them to the natural attB region sequence. Ten μl of each transformant PCR reaction was digested with BgM, a restriction enzyme whose site is present only in the polylinker of the 4.2 kilobase pair TGV linear replacement fragment. Digestion of the 1.75 kilobase pair PCR product results in 966 base pair and 783 base pair fragments. Colonies from control plates gave no PCR product (because lambda is too large to be amplified (~48kb)), whereas transformant colonies allowed amplification of the 1.73 kb region between the primers (Fig. 12a). In Figure 12b, PCR products from lanes 11-20 in IB were digested with Bglϊl (+) and run with uncut samples (-) on a 1.5% agarose gel. Digest products migrate as expected for 955bp and 775bp. Digestion of this PCR product with a restriction enzyme that cuts in the multiple cloning site yielded products of the correct size (Fig. 12b).

EXAMPLE 14 Determination of an Efficient rec Genotype for Linear Replacement.

Four different recombination-proficient E. coli strains were compared to determine which genotype allows the most efficient linear replacement of the prophage.

Only non- wild-type E. coli genotypes have been shown to incorporate transformed linear

DNA efficiently (El Karoui et al, 1999; Murphy, 1998; Russell et al, 1989; Shevell et al., 1988). Each strain was tested first in both the mono- and multi-lysogenic state because previous data suggested that linear replacement is decreased in multi- versus mono-lysogens (Powell et al., 1994; Shimada et al, 1972). Each strain was transformed with linear pTGV-Light, or with no DNA (Fig. 11 A). As shown, strains containing one lambda prophage (mono) are more efficient at linear replacement than strains with multiple copies. Among mono-lysogens, recBC sbcBC had the highest efficiency with the lowest background of colonies in mock transformations. recD was the least efficient strain. Separate experiments showed that a strain containing the re d recombination genes from phage lambda in place of recBCD (Murphy, 1998) were equally as efficient at linear replacement whether a mono- or multi-lysogen (Fig. 11 A). For No DNA controls, units are transformants/equal reaction volume without DNA. Each bar represents an average of three experiments performed on different days with different batches of competent cells. The ArecBCD::rec strain was grown with IPTG (lmM) for red induction and tested separately from the other strains. Strains used (from left to right) are SMR5073, SMR5074, SMR5078, SMR5079, SMR5080, SMR5081, SMR5076, and SMR5077.

To compare the different rec genotypes directly, isogenic strains were compared in the mono-lysogenic form (Fig.1 IB). Each bar represents an average of four experiments performed on different days with different batches of competent cells. rec⁺ was done separately from the others in three experiments, but in parallel with recBC sbcBC. A subset of these transformants was confirmed by PCR. Strains used (from left to right) are SMR5078, SMR5080, SMR5076, SMR5220, SMR5221, SMR5078, and SMR5449. Error bars represent one standard error of the mean. All strains showed a similar transformation efficiency, except for recD and rec⁺ which were lower.

EXAMPLE 15 Efficiency of Anti-lambda Versus Drug Selection

The recBC sbcBC strain was used as a mono-lysogen and a non-lysogen to compare the efficiencies of anti-λ prophage versus drug selection. The same number of molecules of linearized pTGV-CAT was elecfroporated into each strain, and selection was performed either against the prophage (at 42°C) or for the presence of the chloramphenicol resistance gene (Table 3). Recovery of antibiotic resistant transformants was four- fold higher than recovery of transformants after selection against the prophage. These data indicate that the anti-λ prophage selection is nearly as efficient as drug selection for obtaining linear transformants, and show that cat is expressed from the attB site.

Experiments were performed by transforming (by electroporation) 2.18 x 10^u molecules (1.5μg) of pTGV-CAT linear fragment for each strain (see methods in Example 19). After recovery, dilutions were plated, and 28 to 1000 colonies were scored Table 3. Comparison of drug versus anti-λ selection.

rec Transformants / Mean ± SE genotype Selection Expt. # molecule (x10-8) recBC sbcBC CAM @ 32° 1 >4.7x10^"9

2 2.6x10-⁸ 1.1 ±0.74

3 2.8x10-⁹ recBC sbcBC Cit @ 42° 1 1.4x10-⁹

(λ xisl c\ts857) 2 4.9x10-9 0.27 ±0.11

3 1.8x10-9

for each transformation. Strains compared are CES202 (Table 5) and SMR5078, a λ xisl clts857 mono-lysogen of CES202 (see methods herein).

EXAMPLE 16 The P_BAD Promoter is Functional

To demonstrate repression and induction of transgene expression by the P_BAD promoter, we used a strain in which the l-Scel restriction endonuclease (Colleaux et al, 1986) is under P_BAD control (Δαtt5::P_BAD-I-5ceI, placed in the chromosome using pTGV-Express). l-Scel expression was measured using an in vivo assay for its endonuclease activity. In this assay, l-Scel endonucleolytic cleavage of introduced plasmids carrying an l-Scel site should decrease the transformation efficiency of that plasmid, but not that of a control plasmid. The ratio of transformants recovered from a plasmid carrying the l-Scel site (pHW249) versus a control plasmid lacking an l-Scel site (pACYCl 84; Chang and Cohen, 1978) present in the same DNA mixture was compared for Δ«tti?::P_BAD-I-SceI competent cells and Δαttβ::P_BAD competent cells prepared in rich medium supplemented with either glucose or arabinose (Table 4). The data show that recovery of the plasmid carrying the l-Scel site is reduced by 1000-fold or more by arabinose induction of l-Scel expression. In addition, glucose seems to repress l-Scel expression efficiently, as the ratio for that strain is similar to the P_BAD control strain grown in either glucose or arabinose. Thus, the P_BAD promoter and the prokaryotic translation-initiation signal are functional in a chromosomal transgene made with pTGV-Express.

The strains in Table 4 also carry the AaraBAD_Aim (Haldimann et al, 1998) deletion. The AraC regulatory protein is expressed from its normal chromosomal location. Electro-competent cells were prepared (Sambrook et al, 1989) and were diluted 1:200 from LBH cultures into LBH plus 0.2% arabinose or 0.2% glucose followed by 5-6 hours of growth before harvesting. Following electro-transformation of a mix of 40-80ng of each plasmid, transformants were counted by dilution and plating Table 4. Inducible expression of a transgene inserted with TGV-Express.

Strains PBAD promoter ratio of pHW249/pACYC184 activity transformants

Exp. 1 Exp. 2 attB: :PBAD-'-^Scel induced 0.00019 0.00054 repressed 0.24 0.22 attB::P_BAD induced 0.86 1.2 repressed 2.5 0.32

on either LBH + 50 μg/ml kanamycin to select for pHW249 transformants or LBH + 25 μg/ml chloramphemcol to select for pAYCY184 transformants. Control platings revealed no chloramphemcol resistant tran 9 and vice versa. The ratio is kanamycin-resistant transformants tant transformants.

EXA Movement of Trans

Once incorporated, a transgene is background and into

other strains simply by phage PI -mediate 1 xisl clts857 lysogen by selecting for temperature-resistance. ces are added in the process.

EXA

Bacterial Stra

All combinations of E. coli allel dard PI transduction methods (Miller, 1992) (Table 5). Allel and A(recC ptr recB recD): :P_lac bet exo kan are from DPB271 ( and KM22 (Murphy,

1998), respectively. Lambda lysogen deri

tested using standard

lambda methods (Murray, 1983). Mono- versus multi-lysogeny was determined using lambda cl90cl 7 as described (Shimada et al, 1972). Lambda xisl clts857 is λSR446.

Table 5. Bacterial strain backgrounds.

Straina Relevant genotype Source or reference

CES202 recB21 recC22 sbcB15 sbcC201 hsdr_r^mκ⁺ 30

JC11450 rec⁺ A. J. Clark (Berkley)

5K hsdr^m^ M. Meselson via F. W. Stahl

SMR12 recB21 sbcA20 hsdrfc-mκ⁺ Lab collection

SMR423 recD 7903:: mi ni-tef hsdr^m^ Lab collection

<7l

SMR5075 A(recC ptr recB recD)::P/_ac red⁺ kan hsdrχ-mκ⁺ This work

SMR5220

sbcB15 sbcC201 hsdr_rζτnκ⁺ (λ xisl c\ts857) mono This work

SMR5221 A(recC ptr recB recD)::Pι_acred⁺kan sbcB15 sbcC201 hsdr^ ^ (λ xisl c\ts857) j js work

mono

SMR5075 are derived from C600 (Bachmann, 1996) and also contain thi, thr, leu, and supE. SMR423 also contains trp::Tn5 and supF.

EXAMPLE 19

Exemplary Methods

TGV-Light and TGV-CAT (or any vector of the present invention) were linearized by digestion with Ndel (or an appropriate enzyme) overnight at 37°C. Shrimp alkaline phosphatase was included in the reaction mixture and was inactivated following digestion by incubation at 65°C for 20 minutes. 1 μg of the linear fragment was delivered to electrocompetent cells by electroporation (according to manufacturer's instructions for Bio-Rad E. coli Pulser) and allowed to recover for one hour in SOC medium (Sambrook et al, 1989) at 32°C. Transformed cells were then concentrated, and either one-half or a dilution of the entire transformation mixture was plated on LBH27 with 20mM sodium citrate to inhibit infection of recombinants by free lambda phage. The plates were incubated at 42°C overnight. TGV-CAT transformations were treated similarly but plated on LBH with 25 μg/ml chloramphemcol, and were incubated overnight at 37°C. Verification of linear replacement was done by colony PCR on transformants using primers attBL (5' GGATTCGGTGTTATCG 3'; SEQ ID NO:32) and attBR (5' GGATCCGGCCTTTTG 3'; SEQ ID NO:33) which anneal at sequences -900 and -700 bp from the core att site respectively. When linearizing with rare-cutter enzymes other than Ndel, it was determined that gel purification of the linear fragment to be transformed is required for high linear replacement efficiency.

EXAMPLE 20 Significance of TGV Vector System

Cloning and expression of genes in single copy in the bacterial chromosome is sometimes required to avoid the complications encountered with plasmids. In addition to plasmid loss, cell inviability, and selection for mutants in the presence of toxic gene products, gene overexpression from plasmids can produce aberrant phenotypes not representative of the normal function of the gene product. Several methods exist for cloning and expressing genes in the bacterial chromosome in single copy, but all use antibiotic resistance markers to select transgene insertion (Boyd et al, 2000; El Karoui et al, 1999; Murphy, 1998; Russell et al, 1989; Shevell et al, 1988; Hamilton et al, 1989; Atlung etal, 1991; Le Borgne etal, 1998; Martinez-Morales etal, 1999; Yu and Court, 1998). Bacterial artificial chromosomes (BACs) have also been used for single copy expression, but much larger pieces of DNA are used and instability and loss of DNA can still be a problem (Rondon et al, 1999).

Demonstrated herein is a simple new method for single-copy gene cloning and expression in the E. coli chromosome without the need for an antibiotic selection. The counter-selection of a lambda prophage containing a temperature-sensitive repressor is useful when antibiotic resistance markers are not available for selection, and was shown herein to be efficient. Additionally, comparisons are provided herein regarding efficiencies of linear replacement recombination in various recombination-proficient strains using the same linear DNA molecules in an isogenic strain set. All of the genetic backgrounds were roughly equivalent, except for recD and rec⁺, which were lower. As presented herein, cells expressing phage λ red recombination genes were not as efficient as had been reported (Murphy, 1998). However, recent reports of new constructs expressing these genes indicate much more efficient recombination, and appear promising for use with this system (Datsenko and Wanner, 2000; Yu et al, 2000). Finally, inducible expression of a transgene using pTGV-Express which carries the P_BAD promoter and the Shine-Dalgarno sequence is presented herein. Once a transgene is inserted into the chromosome with the TGV system, it can be moved easily into any genetic background using standard PI transduction (Miller, 1992). The desired genetic background is first lysogenized with the xisl clts857 lambda phage using a simple procedure (Murray, 1983). A PI lysate made from the strain containing the transgene is then used to transduce the gene into the desired lysogenized background, selecting again at the non-permissive temperature. This method allows expression of transgenes in a multitude of genetically relevant backgrounds. REFERENCES

All patents and publications mentioned in the specification are indicative of the level of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

U.S. PATENT DOCUMENTS

U.S. Pat. No. 4,506,013 issued on March 19, 1985 with Hershberger et al. listed as inventors. U.S. Pat. No. 4,650,761 issued on March 17, 1987 with Hershberger et al. listed as inventors. U.S. Pat. No. 4,673,640 issued on June 16, 1987 with Backman listed as the inventor. U.S. Pat. No. 5,364,779 issued on November 15, 1994 with De Lafonteyne listed as the inventor. U.S. Pat. No. 5,733,753 issued on March 31 , 1998 with J.o slashed.rgensen listed as the inventor.

U.S. Pat. No. 5,736,367 issued on April 7, 1998 with Haun et al listed as inventors.

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Jones, D and J Errington 1987. J. Gen. Microbiol. 133: 483-492.

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Martinez-Morales, F. Borges, AC, Martinez, A, Shanmugam, KT and Ingram, LO 1999. J Bacteriol 181: 7143-7148.

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Laboratory Press: Cold Spring Harbor, NY. p. 395-432.

Murphy, KC 1998. J. Bact. 180:2063-2071.

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Perez-Casal, J, JA Price, E Maguin, and JR Scott 1993. Molec. Microbiol. 8: 809-819. Powell, BX, Rivas, MP, Court, DL, Nakamura, Y and Turnbough, CL 1994. Nucl Acids

Res 22: 5765-5766. Prozorov, AA, VI Bashkirov, FK Khasanov, EF Glumova and VY Irich 1985. Gene 34: 39-46. Razavy, H, SK Szigety, and SM Rosenberg 1996. Genetics 142: 333-339. Reece, KS and Phillips, GJ 1995. Gene 165: 141-142. Rondon, MR, Raffel, SJ, Goodman, RM and Handelsman, J 1999. Proc Natl Acad Sci

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Rosenberg, SM, MM Stahl, I Kobaayashi, and FW Stahl 1985. Gene 38: 165-175. Rosenberg, SM 1987. Metnods In Enzymology: Recombinant DNA Techniques, R. Wu and L. Grossman, editors, p. 95-103. Russell, CB, DS Thaler, and FW Dahlquist 1989. J. Bacteriol. 171 : 2609-2613. Sambrook, J., Fritsch, E.F. and T. Maniatis 1989 Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York

Shevell, D.E., Abou-Zamzam, A.M., Demple, B. and G.C.Walker. 1988. J. Bact. 170:3294-3296. Shimada, K, Weisberg, RA and Gottesman, ME 1972. J Mol Biol 63: 483-503.

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1628. Vieira, J. and J. Messing 1982. Gene 19:259.

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250 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1983). Yu, D and Court, DL 1998. Gene 223: 77-81. Yu, D et al. 2000. Proc Natl Acad Sci USA 97: 5978-5983. One skilled in the art readily appreciates that the patent invention is well adapted to carry out the objectives and obtain the ends and advantages mentioned as well as those inherent therein. Vectors, plasmids, cells, phage, prophage, lysogens, methods, procedures and techniques described herein are presently representative of the preferred embodiments and are intended to be exemplary and are not intended as limitations of the scope. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention or defined by the scope of the pending claims.

Claims

We claim:

1. A method for making a transgenic cell comprising the steps of: introducing a linear vector into the cell wherein said vector contains a cassette comprising sequentially from 5' to 3': a 5' flanking sequence, a polylinker site containing a nucleic acid sequence of interest, and a 3' flanking sequence wherein the 5' and 3' flanking sequences are homologous to a sequence in a chromosome in the cell at a location where the nucleic acid sequence of interest is to be inserted; integration of said cassette into the chromosome through recombination between the flanking sequences and the homologous DNA sequences, wherein said homologous DNA sequences flank a conditional killing module in the chromosome; and negatively selecting against cells retaining said conditional killing module and deficient for integration of said cassette.

2. The method of Claim 1 wherein said cassette further comprises a 5' rare- cutter restriction site which is 5' to the 5' flanking sequence and a 3' rare-cutter restriction site which is 3' to the 3' flanking sequence, and wherein said 5' and 3' rare-cutter restriction sites have one or more sequences which are cut by rare-cutter restriction enzymes.

3. The method of Claim 2 wherein said 5' and 3' rare-cutter restriction sites are selected from the group consisting of Srfi, Ndel, Sfil, Avrll, and Ascl.

4. The vector of Claim 1 or 2 wherein the 5' and 3' flanking sequences comprise of nucleic acid sequences which flank attB in the genome of Escherichia coli.

5. A method for making a transgenic cell comprising the steps of: introducing a linear vector into the cell wherein said vector contains a cassette comprising sequentially from 5' to 3': a 5' flanking sequence, a nucleic acid sequence of interest, and a 3' flanking sequence wherein the 5' and 3' flanking sequences are homologous to a sequence in a chromosome in the cell at a location where the nucleic acid sequence of interest is to be inserted; integration of said cassette into the chromosome through recombination between the flanking sequences and the homologous DNA sequences wherein said homologous DNA sequences flank a conditional killing module in the chromosome; and negatively selecting against cells retaining said conditional killing module and deficient for integration of said linear vector.

6. The method of Claim 5 , wherein said cassette is generated by polymerase chain reaction.

7. The conditional killing module of Claim 1, 2 or 5, wherein said module includes a conditional repressor.

8. The conditional killing module of Claim 1, 2 or 5, wherein said module contains temperate prophage sequences including a temperature sensitive conditional repressor.

9. The method of Claim 1 , 2 or 5, wherein said cell is Escherichia coli and said conditional killing module is the lambda xisl clts857 prophage.

10. The method of Claim 1, 2 or 5, wherein said cell is Escherichia coli and said conditional killing module is the defective lambda prophage lambda clts857 A(cro- bioA).

11. The method of Claim 1 , 2 or 5 , wherein said cell is Escherichia coli and said conditional killing module is the defective lambda prophage lambda clts857 clind A(cro-bioA).

12. The method of Claim 1, 2 or 5, wherein said cell is Escherichia coli and said conditional killing module is the defective lambda prophage lambda clts857 clind A(cro-attR).

13. The method of Claim 1 , 2 or 5 , wherein said cell is Escherichia coli and said conditional killing module is the defective lambda prophage lambda clts857 clind P_BAD A(cro-attR).

14. The method of Claim 1, 2 or 5, wherein said cell is Escherichia coli and said conditional killing module is the defective lambda prophage lambda xisl clts857

A(cro-bioA).

15. The method of Claim 1, 2 or 5, wherein said cell is Escherichia coli and said conditional killing module is the defective lambda prophage lambda xisl clts857 clind^' A(cro-bioA).

16. The method of Claim 1, 2 or 5 wherein said integrated cassette is transduced by recombination between cells.

17. The method of Claim 1, 2 or 5 wherein said integrated cassette is transduced between cells with the assistance of a helper phage.

18. The method of Claim 1, 2 or 5 wherein said cell is Escherichia coli and said integrated cassette is transduced between Escherichia coli cells which contain the prophage lambda xisl clts857 with the assistance of a P 1 helper phage.

19. The method of Claim 1, 2 or 5 wherein said cell is a proficient host for linear replacement using homologous recombination and has a genotype selected from the group consisting of recD, recBC sbcBC, recBC sbcA, recB carrying λ red" genes and rec⁺ carrying λ red" gam⁺ genes.

20. A vector selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

21. A vector for introduction of a nucleic acid sequence of interest into a cell wherein said vector includes a cassette, said cassette comprising sequentially from 5' to 3': a 5' rare-cutter restriction site, a 5' flanking sequence, a polylinker site containing the nucleic acid sequence of interest, 3' flanking sequence and a 3' rare-cutter restriction site, wherein said 5' and 3' rare-cutter restriction sites have one or more sequences which are cut by rare-cutter restriction enzymes and wherein said 5' and 3' flanking sequences are homologous to a sequence in a chromosome in the cell at a location where the nucleic acid sequence of interest is to be inserted.

22. The cassette of Claim 21 wherein the 5' and 3' flanking sequences consist of nucleic acid sequences which flank attB in the genome of Escherichia coli.

23. The cassette of Claim 21 wherein the 5' and 3' rare-cutter restriction sites are selected from the group consisting of Srfi, Ndel, Sfil, Avrll, and Ascl.

24. A vector for introduction of a sequence of interest into a cell wherein said vector includes a cassette comprising sequentially from 5' to 3': a 5' rare-cutter restriction site selected from the group consisting of Srfi, Ndel, Sfil, Avrll, and Ascl, a 5' nucleic acid flanking sequence, a polylinker site containing the nucleic acid sequence of interest, a 3' flanking sequence and a 3' rare-cutter restriction site selected from the group consisting of Srfi, Ndel, Sfil, Avrll, and^cl.

25. The cassette of Claim 24, wherein the 5' and 3' flanking sequences consist of nucleic acid sequences which flank attB in the genome of Escherichia coli.

26. The vector of Claim 24, wherein the expression of said nucleic acid sequence of interest is regulated by nucleic acid sequence selected from the group consisting of promoter sequence and bacterial translation-initiation sequence.

27. The vector of Claim 26, wherein said promoter is an inducible promoter.

28. The vector of Claim 26, wherein said promoter is the P_BAD promoter.

29. The vector of Claim 26, wherein said bacterial translation-initiation sequence is a Shine-Dalgarno sequence.