Definition Biotechnology is the use of living organisms for industrial or commercial application. It depends heavily on techniques of genetic engineering. Much of genetic engineering is based on molecular cloning, in which a double- stranded DNA fragment from any source is recombined with a vector and introduced into a suitable host. Commonly employed cloning vectors include plasmids and bacteriophages.
Essentials of Molecular Cloning A plasmid or virus is used as the cloning vector to isolate a specific gene or region of a chromosome by molecular cloning (Figure 10.35). An in vitro recombination procedure uses restriction enzymes and DNA ligase to produce the hybrid DNA molecule. Once introduced into a suitable host, the cloning vector can control production of large amounts of the target DNA.
Essentials of Molecular Cloning Making a gene library by cloning random fragments of a genome is called shotgun cloning, and it is a widely practiced technique in gene cloning and genomic analyses. Gene library is defined as a large collection of DNA fragments cloned (MOLECULAR CLONING) from a given organism, tissue, organ, or cell type. It may contain complete genomic sequences (GENOMIC LIBRARY) or complementary DNA sequences, the latter being formed from messenger RNA and lacking intron sequences.
Plasmids as Cloning Vectors Plasmids are useful cloning vectors (Figure 10.36) because they are easy to isolate and purify and can multiply to high copy numbers in bacterial cells. Antibiotic resistance genes of the plasmid are used to identify bacterial cells containing the plasmid (Figure 10.37).
Bacteriophage Lambda as a Cloning Vector Bacteriophages such as lambda have been modified to make useful cloning vectors (Figures 10.38, 10.39). Larger amounts (longer) of foreign DNA can be cloned with lambda than with many other plasmids. In addition, the recombinant DNA can be packaged in vitro for efficient transfer to a host cell. Plasmid vectors containing the lambda cos sites are called cosmids, and they can carry a large fragment of foreign DNA.
Genetic Engineering The techniques of genetic engineering are based on fundamental concepts in molecular genetics and biochemistry (Figure 31.1). Successful genetic engineering depends not only on being able to carry out molecular cloning but also on knowledge of replication, transcription, translation, and the regulatory aspects that control all of these processes.
Hosts for Cloning Vectors The choice of a cloning host depends on the final application. In many cases, the host can be a prokaryote, but in others it is essential that the host be a eukaryote (Figure 31.2). Any host must be able to take up DNA, and there are a variety of techniques by which this can be accomplished, both natural and artificial. Figure 31.3 shows a nucleic acid gun for transfection of certain eukaryotic cells.
Finding the Right Clone Special procedures are needed to detect the foreign gene in the cloning host (Figure 31.4). If the gene is expressed, the presence of the foreign protein itself, as detected either by its activity or by reaction with specific antibodies, is evidence that the gene is present. However, if the gene is not expressed, its presence can be detected with a nucleic acid probe.
Specialized Vectors Shuttle vectors allow cloned DNA to be moved between unrelated organisms (e.g., from one bacterium to another). A shuttle vector is a cloning vector that can stably replicate in two different organisms. Many cloned genes are not expressed efficiently in a new host. Expression vectors have been developed for both prokaryotic and eukaryotic hosts. These vectors contain genes that will increase the level of transcription of the cloned gene and make its transcription subject to specific regulation (Figure 31.5). Signals to improve the efficiency of translation may also be present in the expression vector.
Reporter Gene Reporter genes are incorporated into vectors because they encode proteins that are readily detected. These genes can be used to signal the presence or absence of a particular genetic element or its location. They can also be fused to other genes or to the promoter of other genes so that expression can be studied.
Expression of Mammalian Genes in Bacteria It is possible to achieve very high levels of expression of mammalian genes in prokaryotes. However, the expressed gene must be free of introns. This can be accomplished by using reverse transcriptase to synthesize cDNA from the mature mRNA encoding the protein of interest (Figure 31.8).
Expression of Mammalian Genes in Bacteria One can also use the amino acid sequence of a protein to design and synthesize an oligonucleotide probe that encodes it. This process is in effect reverse translation and is illustrated in Figure 31.9. Fusion proteins are often used to stabilize or solubilize the cloned protein (Figure 31.10).
Practical Applications: Production of Insulin Production of insulin began the era of commercial biotechnology. This was the first human protein made commercially using engineered bacteria (Figure 31.11), but many other hormones and human proteins are now being produced in this way. In addition, many recombinant vaccines have been produced.