Biotechnology - Y.O. Sazykin 2006

General Biotechnology
Biological Objects: Methods of Creation and Improvement
Creation of Biological Objects via Genetic Engineering - General Overview

Introduction/32.html">Genetic Engineering is much more specific and precise than cellular engineering in terms of the objects it utilizes, dealing primarily with Cell fragments that vary in shape and size. Note that the terms "genetic engineering", "Gene engineering", and "recombinant DNA" are synonymous.

METABOLISM/2.html">THE CONCEPT OF genetic engineering is extremely broad and therefore cannot be concisely defined. One way to describe genetic engineering is as the joining of DNA fragments of natural and synthetic origin or their combination in vitro, followed by the introduction of the resulting recombinant structures into a living cell so that the inserted DNA fragment, once integrated into the chromosome, either replicates or is autonomously expressed. Consequently, the introduced genetic material becomes part of The Cell's genome.

Before detailing the stages of a genetic engineer's work, let us outline what must be at their disposal:

a) genetic material (host cell);

b) a transport device—a vector that transfers the genetic material into the cell;

c) a set of specific Enzymes—the "tools" of genetic engineering.

The principles and Methods of Genetic Engineering were primarily developed using microorganisms: prokaryotic Bacteria and eukaryotic Yeasts.

In the context of pharmaceutical biotechnology, the greatest practical successes in genetic engineering have currently been achieved in creating microbial strains that act as producers of human species-specific Proteins. Such proteins are foreign to the microbial cell, whereas in The Human Body, some act as BIOREGULATORS (protein Hormones), while others function as components of innate Immunity (interferons, etc.).

The strategic goal of a genetic engineer is to create a fundamentally new biological object for biotechnological production—a microorganism carrying a human gene.

When selecting a microorganism as a potential producer, several factors are taken into consideration.

1. Since the microorganism will be cultivated under industrial conditions in large quantities and handled by numerous facility personnel (biologists, chemists, etc.), it is desirable that it be non-pathogenic. Furthermore, the targeted genetic engineering product extracted from the microorganism must be guaranteed to be entirely free of microbial toxin traces.

2. Once the vector carrying the foreign gene (or gene cluster) enters the microbial cell, it must not be cleaved by the cell's endonucleases; in other words, the genetic material must be preserved. At the same time, the potential producer's Ribosomes must be able to translate the Messenger RNA corresponding to the foreign material.

3. The resulting foreign protein (the target product for the biotechnologist) must not be degraded by the cell's proteases—meaning it should be protected from the cell's repair systems that hydrolyze foreign proteins. Mitigating The activity of such systems is also one of the preliminary steps a genetic engineer takes when working with a producer strain.

4. Ideally, the potential producer of the foreign protein (target product) should secrete it out of the cell and into the nutrient medium, which significantly facilitates subsequent extraction and purification.

The preliminary work of a genetic engineer begins with the gene encoding the target protein itself. A nucleotide sequence is attached to this gene, which in turn encodes a so-called leader sequence of Amino Acids (predominantly hydrophobic). Once synthesized inside the cell, the target product, equipped with this amino acid leader sequence, is guided through the lipid layers of the cytoplasmic membrane out of the cell. However, this requires the producer cell to be modified by the genetic engineer. Specifically, the membrane must contain a "signal protease" that cleaves the leader sequence off the gene product before it is released into the medium.

To enable the vector carrying the foreign gene to penetrate the cell, the cell is subjected to a special Treatment with lithium or calcium salts, depending on the species of microorganism. As a result, small-diameter pores form in The Cell wall, allowing vector molecules to enter. Cells treated in this manner are called competent cells: they are capable of taking up the information carried by the vector.

An important preliminary step in a genetic engineer's work is selecting the vector. Vectors used in work with microorganisms are most often constructed on The basis of temperate phages or Plasmids. The advantage of plasmids over phages lies in the absence of cell lysis, which can occur when working with temperate phages.

When creating a new recombinant producer, the key moment in the genetic engineer's work is the insertion of the gene (or gene cluster) into the vector—more precisely, into the DNA of the vector molecule, such as a plasmid. This is made possible by the fact that genetic engineers have access to a large assortment of endonucleases with varying substrate specificities, commonly referred to as restriction enzymes or Restriction Endonucleases (from the English *restriction*—cutting). Currently, many dozens of different restriction enzymes are known, categorized into those that cleave only one of the two complementary DNA strands or both strands simultaneously.

For the biotechnologist, restriction enzymes that catalyze the Cleavage of only a single strand in the sugar-phosphate DNA backbone are of primary interest. In addition, it is essential that the restriction enzyme performing this cut has a sufficiently high Specificity. This means that The nucleotide sequence required for the enzyme to select the cleavage site on the sugar-phosphate backbone must be relatively long. For instance, the restriction enzyme EcoRI, frequently used in genetic engineering research and isolated from *E. coli* (*Escherichia coli*), recognizes a nucleotide sequence where the nitrogenous bases are arranged in the following order: —GAATTC—; the cut (break) in the sugar-phosphate backbone of one of the two complementary DNA strands occurs between G and A. However, the second complementary strand has virtually the same sequence: —CTTAAG—.

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Fig. 3. Diagram of double-stranded DNA cleavage by a restriction enzyme:

A — adenine; C — cytosine; G — guanine; T — thymine

To prepare for the incorporation of a gene into a vector, a restriction enzyme of the exact same specificity must be used. If the restriction enzyme EcoRI was used to generate sticky ends in the vector, it must also be used to generate sticky ends in the fragment being inserted. Naturally, sticky ends must not form within the gene itself. This highlights the advantage of restriction enzymes that recognize long, specific sequences of nucleotide Base Pairs.

The number of such sequences recognized by a restriction enzyme in a DNA molecule decreases sharply as the number of nucleotide pairs in the sequence increases. Consequently, the risk of the restriction enzyme damaging the gene itself is reduced; the gene, remaining nestled between the binding sites of the restriction enzyme on its DNA substrate, is incorporated into the vector intact.

Another technique available to the genetic engineer is flanking the gene with synthetic nucleotide sequences—that is, using bioorganic chemistry methods to generate sticky ends with a predetermined order of NUCLEOTIDES.

A gene (or gene cluster) integrated into a vector is initially held in place solely by Hydrogen Bonds between complementary sticky ends. This stage is known as “annealing.” For the gene to be permanently embedded within the vector, it must be secured by covalent bonds. This is achieved using ligase enzymes (derived from “ligare,” meaning to tie or bind), which seal the gaps in the sugar-phosphate backbone of the DNA. Following this step in genetic engineering, the vector with its securely anchored gene can be introduced into a microbial cell. However, the success rate of vector uptake by the cell is typically extremely low.

After incubation, the suspension of microorganism cells containing the vector is plated onto a solid nutrient medium, and the resulting colonies are then transferred to Agar slants. The resulting cultures (clones) must be screened to verify whether their cells contain the vector with the gene (or gene cluster) encoding the target product, such as a human species-specific protein hormone or a species-specific innate immunity factor, etc.

If a low vector uptake frequency means that only 0.01–0.1% of the cells take up the vector, it is easy to imagine the huge number of cultures that must be screened to find one that synthesizes the target product. To detect this product based on its function, it must first be identified, isolated, purified, and tested in vitro or in animal experiments. However, analyzing thousands of cultures in this manner is virtually impossible. Therefore, a method has been developed for the preliminary Selection of clones containing the vector.

The concept of a marker gene is introduced. Such a gene easily “announces its presence,” meaning it labels the cell and, consequently, the clone. The marker gene is also inserted into the vector, but of course using a different restriction enzyme that targets a different nucleotide sequence, thereby precluding its insertion into the “working” gene. The marker gene occupies its designated place within the vector. While this gene plays no role in the future biotechnological process, it is essential for selecting the producer of the target product encoded by the “working” gene. An example of a marker is the gene encoding the enzyme beta-lactamase. This enzyme inactivates beta-lactam Antibiotics by catalyzing the Hydrolysis of their beta-lactam ring.

E. coli cells of a microorganism frequently used in the engineered Production of Human proteins are checked for the uptake of the vector carrying two genes—the “working” gene and the “marker” gene; they are then plated on a solid nutrient medium containing ampicillin (a broad-spectrum antibiotic). Since the initial E. coli culture is sensitive to ampicillin, its growth as a colony on the medium indicates that ampicillin has been degraded by beta-lactamase; in turn, this beta-lactamase can only be encoded by a gene residing on the vector, as the host cells lack such a gene. This confirms that the vector incorporated not only the marker gene but also the gene encoding the target product.

Next, the culture is tested for its ability to produce the human species-specific protein. The number of cultures requiring direct, prolonged, and labor-intensive screening is reduced hundreds of times over thanks to the marker gene. As a result, the entire workflow of selecting recombinant producers is greatly simplified. Sometimes, two different marker genes are introduced into the vector, which further enhances the efficiency and precision of screening for clones carrying the gene encoding the target product.

Microbial genes correspond to the “original” or classical concept of a gene; that is, a structural gene is a segment of DNA that is transcribed into messenger RNA. The sequence of codons in the latter directly dictates the Amino Acid Sequence of the protein.

In eukaryotes, the information-transfer function of most structural genes operates differently. Relatively recently, the interrupted or discontinuous nature of genes in mammals and, consequently, in humans, was discovered. These genes contain alternating exons and introns. Both are transcribed, meaning that the primary transcript messenger RNA reflects both exons and introns. The introns are spliced out of the primary transcript, while the exons are joined together. This produces mature messenger RNA, which becomes a component of the ribosomal template system. This phenomenon, unique to eukaryotes, is known as splicing.

Thus, The nucleotide sequences of introns do not convey Genetic information for proteins. Because prokaryotic microbial cells lack splicing, genetic engineers must use the enzyme Reverse Transcriptase to transcribe the mature messenger RNA of the human gene back into DNA in order to achieve the synthesis of human proteins in Prokaryotic Cells. This shortened DNA (lacking introns) can then be used for vector insertion.

Even individual areas of genetic engineering currently comprise the Subject Matter of foundational monographs. Knowledge in this field is growing at a staggering pace, and its capabilities are rapidly expanding.