Biochemical Engineering Fundamentals, Part 1 - Bailey, J., Ollis, D. 1989

Molecular Genetics and Regulatory Systems
Recombinant DNA Technology

In the late 1970s, a series of novel methodological approaches emerged in biochemistry, marking the dawn of the Introduction/32.html">Genetic Engineering era. Recombinant DNA technology revolutionized our understanding of biotechnology's potential to meet human needs, enabling the targeted design of genetic material, its introduction into living Cells, and the subsequent expression of this Genetic information. Furthermore, recombinant DNA technology has dramatically accelerated the expansion and deepening of our knowledge regarding DNA Functions, Gene Organization, regulation of expression, and protein Primary Structure. In turn, this lays the foundation for comprehending The Nature of various diseases and combating them. The ability to introduce arbitrary DNA segments into cells makes it possible to engineer industrial microorganisms capable of synthesizing highly valuable Proteins. In this section, we will focus primarily on the Industrial Applications of Genetic Engineering and The Use of the bacterium Escherichia coli as a host Organism.

Before delving into the core techniques and Methods of Genetic Engineering, it is useful to review the General Principles of the process and become familiar with specialized terminology. Fig. 6.21 depicts a simplified scheme for cloning a DNA fragment. Cloning is understood here as the generation of a colony of genetically identical cells containing the DNA segment of interest. This segment, designated in Fig. 6.21 as 'foreign DNA', is obtained by cleaving a larger DNA molecule into fragments using specific endonuclease Enzymes, or via chemical or enzymatic synthesis. The foreign DNA is joined (in vitro) with a vector—a specialized vehicle used to introduce foreign DNA into a bacterial Cell. In our example, the vector is a bacterial plasmid slightly modified to facilitate the cloning process. Subsequently, the recombinant DNA molecule (the complex of foreign DNA and plasmid) is introduced into a bacterial cell via transformation. The clone containing the plasmid with the foreign DNA is then identified using the methods described below.

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FIG. 6.21. MAIN STAGES OF foreign DNA fragment cloning.

Cloning a DNA fragment makes it possible to obtain it in quantities sufficient for detailed study and use as a reagent in subsequent biochemical and genetic workflows. A classic example of using cloned DNAs for analytical purposes is the research on fibroblast interferon (IFN-ß) in the late 1970s. This protein was extremely scarce; thus, even with the most sensitive amino acid sequencers, researchers could determine the nature of only a few N-terminal amino acid residues. However, shortly after cloning the fibroblast interferon gene, its nucleotide sequence was established, from which the exact Amino Acid Sequence of the corresponding protein could be directly deduced using METABOLISM/28.html">The Genetic Code! In Section 6.4.4, we will examine Examples of using cloned DNAs to develop microbial strains that produce large quantities of foreign proteins, while the following section will explore a range of enzymes essential to recombinant DNA technology.