Biotechnology

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Biotechnology

WHY YOU NEED TO KNOW

HISTORY

Traditional biotechnology has been used for thousands of years, most likely from the beginning of civilization. With the domestication of the dog during the Mesolithic Period of the Stone Age, to the time when the first human beings realized that they could breed plants to get their own crops, humans have used biotechnology to survive. The best animals and plants were bred, with each successive generation more likely to carry the traits desired. People discovered that fruit juices could be fermented into wine, milk could be converted into cheese or yogurt, and beer could be made by fermenting malt and hops together. At the end of the nineteenth century microorganisms were discovered, Mendel’s work on genetics was accomplished, and institutes for investigating microbial processes were established, including those of Koch and Pasteur.

The term “biotechnology” was created in 1919 by Karl Ereky, a Hungarian engineer. He introduced the term in his book entitled Biotechnologie der Fleisch-, Fett- und Milcherzeugung im landwirtschaftlichen Grossbetriebe (Biotechnology of Meat, Fat and Milk Production in an Agricultural Large-Scale Farm). Much later, in 1989, Robert Bud rediscovered this work and acknowledged Ereky as the father of the term biotechnology. During World War I, fermentation processes were developed, and microbes were used to produce chemicals such as acetone, ethanol, and citric acid. Since World War II, microorganisms have been grown on a large scale to produce penicillin.

Biotechnology is a subdiscipline of biology that uses microorganisms, cells, or cell components to produce a practical product. Although biotechnology has been a part of human lives for thousands of years to produce products such as bread, cheese, and alcohol, modern biotechnology involves recombinant DNA technology, also referred to as genetic engineering, for industrial, medical, and agricultural purposes.

Humans have used gene manipulations unknowingly for thousands of years by cross-breeding plants as well as animals. For example, a pluot is a cross between a plum and an apricot, and a mule is the result of breeding a female horse to a male donkey. However, genetic engineering should not be confused with this traditional breeding, by which the genes of organisms are manipulated indirectly. The primary goals of recombinant DNA technology include, but are not limited to:

Tools of Genetic Engineering

Genetic engineering is a sophisticated science performed largely in test tubes. Scientists are now able to take fragments of DNA from different species and splice them together, producing a novel form of DNA that changes the character of the organism. For example, gene-altered, insulin-producing bacteria are no longer identical to their native species, but successfully produce insulin that is harvested for human use.

Although there are several methods/processes to accomplish genetic engineering, all have several steps in common:

DNA technology has been made possible by extensive work with viruses, especially bacteriophages, and many bacterial species. The most popular bacterium for use in biotechnology has been Escherichia coli, the organism that is considered to be the “workhorse” for experiments in genetic engineering. The bacterial chromosome is ideal for DNA experiments because it consists only of DNA, whereas eukaryotic chromosomes consist of large amounts of proteins, called histones, associated with the DNA molecules. Furthermore, eukaryotic DNA exists in pairs and one chromosome can dominate the other, whereas bacterial DNA is a single entity. Also, bacterial DNA floats free in the cytoplasm, whereas eukaryotic DNA is enclosed by the nuclear envelope (membrane).

Because the genetic code is nearly universal, bacteria can express foreign DNA if it is attached to the bacterial DNA. This can be achieved through genetic engineering. Bacteria can also acquire fragments of DNA from the local environment and then express the proteins encoded by the genes in those fragments. This genetic recombination process is called transformation. Transformation reportedly takes place in less than 1% of the bacterial population, but it can bring about profound genetic changes. In this process several donor bacteria lyse, and their DNA bursts into fragments. If a recipient bacterium is present a segment of double-stranded DNA can pass through the wall and plasma membrane. An enzyme then dissolves one strand of the DNA, and the remaining strand incorporates itself into the recipient’s DNA (Figure 25.1). The transformation is complete when the foreign genes are expressed during protein synthesis. This process is one way by which bacteria can obtain drug resistance.

Another process used by bacteria to obtain DNA fragments from other bacteria is conjugation (Figure 25.2). This genetic recombination process occurs between two living bacteria: a donor and a recipient. The donor and recipient form a cytoplasmic bridge by which single-stranded DNA of the donor crosses to the recipient. On rare occasions the DNA may be integrated into the recipient’s chromosome, but most often the DNA remains in the cytoplasm as a free-floating loop, called a plasmid (see Chapter 6, Bacteria and Archaea).

A third form of recombination is transduction, which involves bacterial viruses called bacteriophages (see Chapter 7, Viruses). The virus enters a bacterial cell and integrates its DNA into the bacterial chromosome.

Viruses other than bacteriophages are also useful in genetic engineering experiments. Viruses (see Chapter 7) consist of nucleic acid fragments, either DNA or RNA, surrounded by a protein coat. They can replicate only in living cells, taking over the host cell machinery. Some viruses integrate their own DNA into the host cell’s DNA and become part of the cell’s genome. An example is herpesvirus, which integrates into a nerve cell genome and remains within that cell for years, causing recurrent herpes infections. DNA technologists realized that this was a way to bring genes into a cell of choice. The tools of genetic engineering include the following:

Restriction Enzymes

In the 1950s Salvador Luria and colleagues discovered that E. coli could resist destruction by bacteriophages; the organism could “restrict” the replication of the virus. Later, in 1962 Werner Arber and his research group identified the mechanism by which E. coli could achieve this restriction. They showed that an enzyme system was able to cleave the phage DNA before it could get to the bacterial cytoplasm. Subsequently they isolated the DNA-cleaving enzyme, which they called endonuclease, now known as a restriction enzyme. The enzyme is capable of cutting viral DNA but does not cut the host DNA because the organism modifies the bacterial DNA by adding a methyl group to it. Since the work of these two research groups other restriction enzymes have been discovered. More than 800 restriction enzymes have been identified so far, recognizing more than 100 different nucleotide sequences. Restriction enzymes are named after the bacterium from which they have been isolated. The name includes three letters from the bacterium, one from the strain, and roman numerals indicating the order in which enzymes from the same type of bacterium were identified (Box 25.1).

A restriction enzyme scans a DNA molecule, recognizing and cutting DNA only at a particular sequence of nucleotides. Some enzymes cut the DNA straight across both of the strands of the double helix and therefore produce “blunt” ends (Table 25.1). On the other hand, many restriction enzymes cut in an offset fashion and the ends of the cut have an overhanging piece of single-stranded DNA. These ends are called “sticky” ends (Table 25.2) because they are able to form base pairs with any DNA molecule that contains the complementary sticky end. Every cleavage performed with the same enzyme will occur at the same restriction site, regardless of the source of the DNA. When such molecules from different DNA sources are mixed together they will join with each other by base pairing between their sticky ends (Figure 25.3, A). This combination can be made permanent by DNA ligase, an enzyme that forms covalent bonds along the backbone of each strand. The result of this reaction is a molecule of recombinant DNA (rDNA) (Figure 25.3, B). The ability to produce rDNA molecules has revolutionized the study of genetics and formed the foundation for the biotechnology industry.

TABLE 25.1

Examples of Restriction Enzymes That Produce “blunt” Ends

Enzyme Source Recognition Site
AluI Arthrobacter luteus

EcoRII Escherichia coli R245

HaeIII Haemophilus aegyptius HpaI Haemophilus parainfluenzae SmaI Serratia marcescens

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TABLE 25.2

Examples of Restriction Enzymes That Produce “Sticky” Ends

Enzyme Source Recognition Site
BamHI Bacillus amyloliquefaciens H
EcoRI Escherichia coli RY13
HindIII Haemophilus influenzae Rd
PstI Providencia stuartii
SalI Streptomyces albus

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Gene Libraries

A gene library is composed of a series of cells or unicellular organisms that are used to store genes that have been obtained from foreign cells. To form a gene library a mass of cells that contain the desired gene must be acquired. The desired gene must then be isolated from the total DNA of the cells. For example, human cells have a total of 100,000 genes and only 1 gene may be the target of interest, such as the gene for insulin (pancreatic cells), or the gene for human growth hormone (anterior pituitary).

Creating a Gene Library

To establish a cell line containing the gene of interest, the particular DNA containing the gene must be isolated. A restriction enzyme is used to cut the cell’s DNA into fragments. After this process a host organism for the fragments needs to be chosen. An example of such an organism is Escherichia coli. The plasmids of E. coli are then isolated and the DNA fragments that were previously obtained are inserted into the plasmids (Figure 25.4). The recombined plasmids contain all the DNA fragments and are then inserted into E. coli cells, and together these E. coli cells now are a warehouse of all the DNA of the original cell of interest. The cell collection is the library, the individual cells represent the books of the library, and the individual DNA fragments contain the information in each of the books. This type of library is referred to as a genomic library. The next step is to find the particular gene of interest and therefore the library must be screened.

Screening the Gene Library

The screening process begins with growing the transformed E. coli cells on nutrient agar. Colonies derived from each individual cell will be visible within 24 to 48 hours, and each colony contains cells with the DNA insert. Now each colony needs to be examined for the presence of the insert that contains the gene of interest. To identify the specific colony a copy of the plate must be made for further work. This is done by a process called replica plating (Figure 25.5). This technique is performed by pressing a piece of sterile velvet onto the gel containing the colonies, followed by pressing the velvet to a new petri dish of nutrient agar to establish another plate, which then can be used for analysis of the target gene.

The analysis can be done a number of ways, one of which is by using a radioactive mRNA probe to identify the gene of interest. Bacteria from a particular colony are taken; their DNA is fragmented and then separated in a process called electrophoresis.

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