The advantage of this procedure is the ability to easily transfer the target gene to a variety of vectors that have been developed for different purposes. For example, to produce high levels of the protein encoded on the cloned gene, the target DNA can be transferred to a destination vector that carries a promoter and other expression signals. An entry clone is mixed with a destination vector that has attR1 and attR2 sites flanking a toxin gene (Fig. 2.13B). In the presence of integration host factor, integrase, and bacteriophage λ excisionase, the attL1 and attL2 sites on the entry clone recombine with the attR1 and attR2 sites, respectively, on the destination vector. This results in the replacement of the toxin gene on the destination vector with the target gene from the entry clone, and the resultant plasmid is designated an expression clone. The reaction mixture is transformed into E. coli, and a selectable marker is used to isolate transformed cells that carry an expression clone. Cells that carry an intact destination vector or the exchanged entry plasmid (known as a by-product plasmid) will not survive, because these carry the toxin gene. Destination vectors are available for maintenance and expression of the target gene in various host cells such as E. coli and yeast, insect, and mammalian cells.
Genomic Libraries
A genomic library is a collection of DNA fragments, each cloned into a vector, that represents the entire genomic DNA, or cDNA derived from the total mRNA, in a sample. For example, the genomic library may contain fragments of the entire genome extracted from cells in a pure culture of bacteria or from tissue from a plant or animal. A genomic library can also contain the genomes of all of the organisms present in a complex sample such as from the microbial community on human tissue. Such libraries are known as metagenomic libraries. Whole-genome libraries may be used to identify genes that contain specific sequences, encode particular functions, or interact with other molecules.
To create a genomic library, the DNA extracted from the cells (cell cultures or tissues) of a source organism (or a community of organisms for a metagenomic library) is first digested with a restriction endonuclease. Often a restriction endonuclease that recognizes a sequence of four nucleotides, such as Sau3AI, is used. Although four-cutters will theoretically cleave the DNA approximately once in every 256 bp, the reaction conditions are set to give a partial, not a complete, digestion to generate fragments of all possible sizes (Fig. 2.14). This is achieved by using either a low concentration of restriction endonuclease or shortened incubation times, and usually some optimization of these parameters is required to determine the conditions that yield fragments of suitable size. The range of fragment sizes depends on the goal of the experiment. For example, for genome sequencing, large (100- to 200-kb) fragments are often desirable. To identify genes that encode a particular enzymatic function, that is, genes that are expressed to produce proteins in the size range of an average protein, smaller (∼3- to 40-kb) fragments are cloned.
Figure 2.14 Construction of a genomic DNA library. Genomic DNA extracted from cells or tissues is partially digested with a restriction endonuclease. Conditions are set so that the enzyme does not cleave at all possible sites. This generates overlapping DNA fragments of various lengths that are cloned into a vector.
The number of clones in a genomic library depends on the size of the genome of the organism, the average size of the insert in the vector, and the average number of times each sequence is represented in the library (sequence coverage). To ensure that the entire genome, or most of it, is contained within the clones of a library, the sum of the inserted DNA in all of the clones of the library should be at least three times the amount of DNA in the genome. For example, the size of the E. coli genome is approximately 4.6 × 106 bp; if inserts of an average size of 1,000 bp are desired, then 13,800 clones are required for threefold coverage (i.e., 3[(4.6 × 106)/103]). For the human genome, which contains 3.3 × 109 bp, about 80,000 clones with an average insert size of 150,000 bp are required for fourfold coverage (i.e., 4[(3.3 × 109)/(15 × 104)]). Statistically, the number of clones required for a comprehensive genomic library can be estimated from the relationship N = ln(1 − P)/ln(1 − f), where N is the number of clones, P is the probability of finding a specific gene, and f is the ratio of the length of the average insert to the size of the entire genome. On this basis, about 700,000 clones are required for a 99% chance of discovering a particular sequence in a human genomic library with an average insert size of 20 kb.
Several strategies can be used to identify target DNA in a genomic library. Genomic or metagenomic libraries can be screened to identify members of the library that carry a gene encoding a particular protein function. In functional complementation, the host cell does not have the protein activity of interest, in some cases because the host gene encoding the protein carries a mutation that abolishes the activity of the protein. A DNA library is constructed that carries fragments of genomic DNA from an organism that has the desired protein activity. Host cells with the genetic deficiency are transformed with plasmids of the DNA library, and transformed cells that have restored normal protein function are selected. The genomic DNA that is used to prepare the library can be from a variety of donor organisms, such as the wild-type strain of the host bacterium that carries a functional copy of the gene encoding the protein, a different organism that can be either another prokaryote or perhaps a eukaryote, or uncultured organisms that are present in an environmental sample.
Many genes encoding enzymes that catalyze specific reactions have been isolated from a variety of organisms by plating the cells of a genomic library on medium supplemented with a specific substrate. For efficient screening of thousands of clones, colonies that carry a cloned gene encoding a functional catabolic enzyme must be readily identifiable, often by production of a colored product or a zone of substrate clearance around the colony. Alternatively, genes may be identified that confer desired properties on host cells. In one example, genes encoding proteins that confer resistance to salt were isolated from a metagenomic library of DNA from microbes present in a hypersaline environment (Fig. 2.15). A library of 192,000 clones with an average insert size of 3 kb representing 1.2 × 109 bp of metagenomic DNA was constructed in an osmosensitive strain of E. coli from DNA extracted from bacteria collected from the saline soil around the roots of the halophyte Arthrocnemum macrostachyum. The library was screened by plating the transformed E. coli cells on media containing 3% sodium chloride, which is normally lethal for the host E. coli strain. Eight different genes that conferred salt resistance were identified. Based on sequence similarity to known genes, these are predicted to encode enzymes involved in nucleic acid structure and repair, an outer membrane protein, a glycerol permease, a proton pump, and two hypothetical proteins.
Figure 2.15 Isolation of genes involved in salt resistance from a metagenomic DNA library of halophilic (thrive in high salt concentrations) bacteria. Genomic DNA extracted from bacteria found in the soil around the roots of a plant (rhizosphere) growing in a hypersaline soil was fragmented and cloned into a plasmid to generate a metagenomic library. E. coli host cells carrying the cloned DNA were plated on a solid medium containing 3% sodium chloride. The E. coli strain normally does not grow on 3% sodium chloride. The cloned DNA fragments from colonies that developed on the plates were sequenced to identify genes that confer resistance to salt. Mirete et al., Front. Microbiol. 6:1121, 2015.
The presence of particular proteins produced by a genomic library can also be detected using an immunological assay. Rather than screening for the function of a protein, the library is screened using an antibody that specifically binds to the protein encoded by a target gene. The colonies are arrayed on a solid medium, transferred to a matrix, and then lysed to release the cellular proteins (Fig. 2.16). An antibody (primary antibody) is applied that specifically
