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f inate cells that have not taken up vector sequences. If the selectable marker is antibiotic resistance, then only those cells that have taken up the vector or a recombinant molecule can grow on antibiotic-containing medium. ■ bacteriophage, p. 323 ■ antibiotic, p. 508 ■ ampicillin, p. 513

Vectors also must have at least one restriction enzyme recognition site so that the circular vector can be cut to form a linear molecule to which the insert can be joined. Many vectors have been engineered to contain a short sequence called a multiple-cloning site that has the recognition sites of several different restriction enzymes. The value of a multiple-cloning site is its versatility; a fragment obtained by digesting with any of a number of different restriction enzymes can be inserted into the site.

Most vectors have a second genetic marker, in addition to the selectable marker, that is used to differentiate cells contain-

■ Expression Vectors These have been developed to facilitate the expression of eukaryotic genes cloned into bacteria such as E. coli. With the human insulin gene or any other gene cloned for the purpose of protein production, cloning is only successful if the gene is expressed in the new host. For transcription to occur, the promoter of the cloned gene must be recognized by the host's RNA polymerase. At the same time, sequences recognized by the host's ribosomes are required for translation to occur. Expression vectors have been engineered to contain an appropriate promoter and ribosome-binding site adjacent to the multiple-cloning site. The vector may also contain regulatory regions such as an operator, which enables the researcher to control the level of gene expression. ■ promoter, p. 174 ■ transcription, p. 173 ■ translation, p. 175 ■ ribosome-binding site, p. 178 ■ operator, p. 183

234 Chapter 9 Biotechnology and Recombinant DNA

■ Bacterial Artificial Chromosomes

(BACs) These have been developed to clone extremely large inserts, such as those used to create a DNA library of a eukaryotic organism. BACs are derivatives of the F plasmid and can contain inserts as large as 300,000 nucleotides in length. The cloned DNA can then be used for a variety of purposes, including sequencing reactions. ■ F plasmid, p. 206

Introducing the Recombinant DNA into a New Host

Once a group of recombinant plasmids is generated, they must be transferred into a suitable host where the molecules can replicate.

Selecting a Suitable Host For routine cloning experiments, one of the many well-characterized laboratory strains of E. coli is generally used as a host. E. coli is a desirable host because it is easy to grow and much is known about its genetics and biochemistry. These strains also have known pheno-typic characteristics such as sensitivity to specific antibiotics. ■ phenotype, p. 192 ■ antibiotics, p. 508

If protein production is the goal of cloning, a host must be selected that can express the cloned DNA. As a general rule, the more closely two organisms are related, the more likely the genes of one will be expressed in the cells of the other. E. coli seems to be especially able to express foreign genes from other bacteria. Like all Gram-negative organisms, however, E. coli has an outer membrane that contains endotoxin, which is toxic when injected into humans and other animals. Therefore, great pains must be taken to purify any cloned gene product used intravenously, such as insulin, lest any outer membrane material contaminate the preparation. ■ endotoxin, p. 59

Introducing DNA Into Cells A common method of introducing DNA into a bacterial host is DNA-mediated transformation. In this process, cells in a physiological state called competence take up naked DNA from their surrounding environment. While some bacteria are naturally competent, E. coli must be specially treated in a dilute calcium chloride solution to induce them to take up DNA. Even though this method is used routinely by a multitude of laboratories, the exact mechanism by which it enables cells to become competent is not clear. An alternative technique is to introduce the DNA by electroporation, a procedure that involves treating the cells with an electric current. This procedure works with E. coli as well as most other bacteria. ■ DNAmediated transformation, p. 203 ■ electroporation, p. 204

Selecting for Transformants After the DNA is introduced into the new host, the transformed bacteria are cultivated on a medium that both selects for cells containing vector sequences and dif

Origin of replication

Selectable Marker

A gene encoding resistance to an antibiotic such as ampicillin

Origin of replication

Selectable Marker

A gene encoding resistance to an antibiotic such as ampicillin

Second Genetic Marker

A gene such as lacZ'that encodes an observable phenotype

Multiple-Cloning Site

Contains the recognition sequence of several different restriction enzymes

Figure 9.15 Typical Properties of an Ideal Vector Most vectors have an origin of replication, a selectable marker, and a multiple-cloning site. A second genetic marker, used to differentiate cells containing recombinant plasmids from those that contain intact vector, spans the multiple-cloning site.

Second Genetic Marker

A gene such as lacZ'that encodes an observable phenotype

Multiple-Cloning Site

Contains the recognition sequence of several different restriction enzymes

Figure 9.15 Typical Properties of an Ideal Vector Most vectors have an origin of replication, a selectable marker, and a multiple-cloning site. A second genetic marker, used to differentiate cells containing recombinant plasmids from those that contain intact vector, spans the multiple-cloning site.

ferentiates those that carry recombinant plasmids. The medium exploits the selective marker encoded on the vector to permit the growth of only those cells that have taken up plasmids containing vector sequences. If, for example, the vector encodes resistance to the antibiotic ampicillin, then the transformed cells are grown on ampicillin-containing medium. Cells that have not been transformed with a vector sequence are killed by the ampi-cillin. To differentiate cells that carry a recombinant plasmid, the medium exploits the second marker. In the case of vectors that use the lacZ' gene as a second marker, the chemical x-gal is added to the medium. Colonies of cells containing intact vector turn blue on the medium, whereas those containing recombinant plasmid are white. The white colonies are then further characterized to determine if they carry the gene of interest. ■ ampicillin, p. 513

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