History, scope and development of biotechnology

The utilization of biological processes, organisms or systems to produce products that are anticipated to improve human lives is termed biotechnology. Broadly, this can be defined as the engineering of organisms for the purpose of human usage. It can also be defined as the skill set required for the utilization of living systems or the influencing of natural processes so as to produce products, systems or environments to help human development. Currently biotechnology places more emphasis on the establishment of hybrid genes followed by their transfer into organisms in which some, or all, of the gene is not usually present. In prehistoric times, a primitive form of biotechnology was practised by agriculturalists who established better-quality species of plants and animals by methods of cross-pollination or cross-breeding. Previous forms of biotechnology include the training and selective breeding of animals, the cultivation of crops and the utilization of micro-organisms to produce products such as cheese, yogurt, bread, beer and wine. Early agriculture concentrated on producing food.

The most primitive type of biotechnology is the cultivation of plants and the training (in particular the domestication) of animals. The domestication of animals stretches back over 10 000 years, when our ancestors also started maintaining plants as a reliable source of food. The earliest examples of such domesticated plants are rice, barley and wheat. Wild animals were also controlled to produce milk or meat. The ancient production of cheese, yogurt and bread from micro-organisms is also reported. Various alcoholic drinks such as beer and wine were developed during this period, when the process of fermentation was first discovered.

Later, it was discovered that micro-organisms, e.g. bacteria, yeast or molds, hydrolyze sugars when they lack oxygen and are ultimately responsible for fermentation. This process results in the formation of products (food and drink). Consequently, fermentation was perhaps first explored by chance, since in earlier times nobody knew how it worked. During the prehistoric era some civilizations considered fermentation to be a gift from their gods. Scientific evidence for fermentation was first described by Louis Pasteur in the late 1800s. He demonstrated a theory known as germ theory, presenting the survival of micro-organisms and their further effects on the process of fermentation. Pasteur's efforts contributed towards several branches of science. In earlier times several traditional medicines were used as biotechnology products, such as honey, which could be used to treat several respiratory ailments and as an ointment for wounds. Since honey contains several antimicrobial compounds it is considered to be a natural antibiotic and is effectively used in wound healing. Similarly, in China as far back as 600 BC, soybean curds were used to treat boils. Ukrainian farmers once used utilized moldy cheese to treat infected wounds. It was later observed that antibiotics present in such molds killed bacteria and averted the spread of infection. In 1928 Alexander Fleming extracted penicillin, the first antibiotic, from mold [1]. This discovery revolutionized the available treatments, with antibiotics having more potential and being more effective than earlier medicines. The development of biotechnology in terms of crop rotation (including leguminous crops), vaccinations and animal-drawn technology, was realized between the late eighteenth century and the commencement of the nineteenth century [1]. The late nineteenth century was known to be a milestone in biology. Some of the key developments during this period are highlighted below:

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  Structures for examining fermentation and other microbial developments were identified by Robert Koch, Pasteur and Joseph Lister.
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  Gregor Mendel's work on genetics was carried out.
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  Micro-organisms were discovered.

In the 1920s a start was made on the production of useful chemicals through biological processes, when Chaim Weizmann used Clostridium acetobutylicum for the conversion of starch into butanol and acetone (the acetone thus produced was used as an essential component of explosives during World War I) [2]. At the beginning of the eighteenth century, developments in biotechnology tended to bring industry and agriculture together. Later, one some basic processes of biotechnology such as fermentation were refined to develop paint solvents for the emerging automobile industry and acetone from starch. These processes were promoted during World War I. In the 1930s the processes of biotechnology moved more into utilizing surplus agricultural goods to supply industry as a replacement for imports or petrochemicals.

The advent of World War II brought the manufacture of penicillin. The production of antibiotics from micro-organisms became possible when Fleming discovered penicillin, which was later produced at a large scale from cultures of Penicillium notatum (this proved useful for the treatment of wounded soldiers during World War II) [1]. The focus of biotechnology shifted to pharmaceuticals. The Cold War years were ruled by work on micro-organisms for the preparation for biological products along with antibiotics and fermentation processes [3].

Biotechnology is now being used in numerous disciplines including bioremediation, energy production and food processing agriculture. DNA fingerprinting is often practiced in forensics. Insulin production and other biotech-based medicines (biopharmaceuticals) are produced through cloning of vectors with genes of interest (GOIs). Immunoassays are frequently utilized in medicine for drug efficiency and pregnancy testing. In addition, immunoassays are also utilized by farmers to find hazardous levels of pesticides, herbicides and toxins in crops and animal-based products. These tests also offer rapid field tests for the determination of industrial chemicals, in particular, in ground water, sediment and soil. Biotechnology also has vast scope in agriculture for the production of plants that are resistant to insects, weeds and plant diseases. This can be achieved by the introduction of GOIs using genetic engineering.

Selective breeding of plants and animals was practiced in the past without awareness of the basic concepts of biotechnology. In this procedure organisms with desirable traits were allowed to mate to further enhance these traits in their offspring. Consequently, it was revealed that selective breeding could improve yields as well as productivity. During this time farmers were not aware that selective breeding innovators were modifying the genetic make-up of organisms. An outstanding example is the corn plant, which has been enriched by selective breeding to develop an improved source of food and has given a platform for plant breeders to develop more hybrid varieties. Regarding animals, dogs are another example of selective breeding. Breeding between different dogs was promoted to improve traits e.g. size, agility, shape and color, resulting in breeds from the tiny Chihuahua to the Great Dane. Another revolutionary development in biotechnology that initiated the era of genetics was started in 1865 by a monk, Gregor Mendel, who recognized genes as the unit of inheritance. It took almost another 90 years of research to determine that genes are made up of DNA. This breakthrough was the beginning of modern biotechnology. Recent developments in biotechnology have led to an expansion in its sophistication, scope and applicability. As mentioned above, the simplest way to define biotechnology is to split this word into its two constituent parts (biotechnology = biology + technology). By considering these two key words we can define biotechnology as a set of techniques that are employed to manipulate living organisms, or utilize biological agents or their components, to produce useful products/services. The vast nature of biotechnology has frequently made a detailed definition of the subject rather difficult. Some definitions of biotechnology are as follows:

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  'Biotechnology means any scientific application that uses biological systems, living organisms or derivatives thereof, to produce or alter products or processes for particular use' [4].
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  'The utilization of living organisms, systems or processes constitutes biotechnology' [5].
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  Based on the Collins English Dictionary definition [6], biotechnology is the employment of living organisms, their parts or processes, to develop active and useful products and to provide services e.g. waste treatment. The term signifies a broad range of processes, from the use of earthworms as a source of protein to the genetic modification of bacteria to offer human gene products, e.g. growth hormones.
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  According to the Golden Treasury of Science and Technology [7], biotechnology is a discipline based on the harnessing of life processes which are controlled for the bulk production of valuable substances.

It is obvious from the above definitions that biotechnology includes different technologies that rely on information gained by modern discoveries in biochemistry, cell biology and molecular biology. These technologies are already having a huge impact on diverse areas of life, including agriculture, food processing, medical technology and waste treatment.

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  Biotechnology consists of 'the controlled employment of biological agents, e.g. micro-organisms or cellular components, for favorable use' [8].
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  Biotechnology has been defined as 'Janus-faced' [9]. This means that there are two sides to it. On one side, we know that the technology allows DNA to be modified so that genes can be moved from one organism to another. On the other, it also entails comparatively new techniques whose results are untested and should be met with care.
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  Biotechnology is 'the integrated use of microbiology, biochemistry and engineering sciences in production or as service operation' [10].
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  Biotechnology is the commercial employment of micro-organisms and living plant and animal cells to create substances or effects beneficial to people. It includes the production of antibiotics, vitamins, vaccines, plastics, etc [11].
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  'Bio' refers to life and 'technology' refers to the application of information for practical use, i.e. the application of living organisms to create or improve a product [11].
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  It involves the industrial application of living organisms or their products, which entails the intentional manipulation of their DNA molecules. It may mean making a living cell execute a particular task in a predictable and controllable way [11].
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  The term biotechnology is occasionally also applied to processes in which micro-organisms such as yeasts and bacteria are cultured under strictly controlled environmental conditions. For this reason, fermentation is occasionally called the oldest form of biotechnology. Genetic engineering techniques are frequently, but not always, used in biotechnology [11].
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  The Universities Press Dictionary of Biology defines biotechnology as 'the application of technology to biological processes for industrial, agricultural and medical purposes' [12].
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  The Oxford Dictionary of Biology [13] defines biotechnology as 'the development of techniques for the application of biological processes to the production of materials of use in medicine and industry.'
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  The employment of cells and biological molecules to explain problems or make valuable products. These biological molecules include DNA, RNA and proteins.
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  Biotechnology may be defined as 'the utilization of living organisms in systems or processes for the production of valuable products; it may involve algae, bacteria, fungi, yeast, cells of higher plants and animals or subsystems of any of these or isolated components from living matter' [14].

It may be seen that the diverse definitions of biotechnology above differ in their approach, content and emphasis. But there are two main characteristics common to them all are. First, biotechnology involves the exploitation of biological entities (i.e. micro-organisms, cells of higher organisms—either living or dead), their components or constituents (e.g. enzymes), in such a way that some functional product or service is generated. Second, this product or service should aim to improve human welfare.

In summary, biotechnology is the '[a]pplication of the theory of engineering and biological science to generate new products from raw materials of biological origin, e.g. vaccines or food', or, in other words, it can also be defined as 'the exploitation of living organism/s or their product/s to change or improve human health and human surroundings' [15].

Hungarian engineer Karl Ereky first coined the term 'biotechnology' in 1919, meaning the production of products from raw materials with the aid of living organisms [16, 17]. As mentioned above, biotechnology is not new, since human civilization has been exploiting living organisms to solve problems and improve our way of life for millennia. The production technologies and processes involved in animal husbandry, agriculture, horticulture, etc, utilize plants and animals to produce useful products. However, such technologies are not regarded as biotechnology since they are long recognized and well-established disciplines in their own right. Today, the exploitation of animal and plant cells cultured in vitro as well as their constituents for generating products/services is an integral part of biotechnology.

The definition of biotechnology can be further divided into different areas known as red, green blue and white.

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  Red biotechnology: This area includes medical procedures such as utilizing organisms for the production of novel drugs or employing stem cells to replace/regenerate injured tissues and possibly regenerate whole organs. It could simply be called medical biotechnology.
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  Green biotechnology: Green biotechnology applies to agriculture and involves such processes as the development of pest-resistant grains and the accelerated evolution of disease-resistant animals.
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  Blue biotechnology: Blue biotechnology, rarely mentioned, encompasses processes in the marine and aquatic environments, such as controlling the proliferation of noxious water-borne organisms.
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  White biotechnology: White (also called gray) biotechnology involves industrial processes such as the production of new chemicals or the development of new fuels for vehicles.

A distinction is made between 'non-gene biotechnology' and 'gene biotechnology':

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  Non-gene biotechnology: Non-gene biotechnology works with whole cells, tissues or even individual organisms. Non-gene biotechnology is the more popular practice, involving plant tissue culture, hybrid seed production, microbial fermentation, production of hybridoma antibodies and immunochemistry.
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  Gene biotechnology: Gene biotechnology deals with genes, the transfer of genes from one organism to another and genetic engineering.

Biotechnology, like other advanced technologies, has the potential for misuse. Concern about this has led to efforts by some groups to enact legislation restricting or banning certain processes or programs, such as human cloning and embryonic stem cell research. There is also concern that if biotechnological processes are used by groups with nefarious intent, the end result could be biological warfare. Apart from their beneficial applications, biotechnological principles also have the potential for destruction, the best example of which is bioterrorism. A representation of the potential pitfalls of biotechnology in fiction can be found in the novel Frankenstein [18, 19]. In this science fiction story, the character of Frankenstein has created a human life which becomes a monster; this monster becomes the cause of the destruction of Frankenstein, the creator of human life. The real-life changes that can be brought about by biotechnology can easily be seen in figure 1.1, illustrating a 14-month-old genetically engineered ('biotech') salmon (left) and a standard salmon (right).

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It is well known that the technical application of biological material is considered to be biotechnology. To understand how biotechnology works it is important to think about the starting point or material for biotechnology processes. Generally, biotechnology utilizes living material or biological products to generate new products for use in various medical, agricultural, pharmaceutical and environmental applications. The ultimate goal of biotechnology is to benefit humanity by, for example, the production of resistant crops, vegetables, recombinant proteins, higher milk-producing animals, etc.

Different developmental stages have taken place in biotechnology to meet the various needs of humans at the time. Its development was principally based on observations, and the application of these observations to practical scenarios. Owing to the evolution of new technologies and a better understanding of various principles of life science, the complexity of biotechnology has increased. Table 1.1 gives a breakdown of historical events in biotechnology. The development of biotechnology can be divided into broad stages or categories, including:

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  Ancient biotechnology (8000–4000 BC): Early history as related to food and shelter; includes domestication of animals.
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  Classical biotechnology (2000 BC; 1800–1900 AD): Built on ancient biotechnology; fermentation promotes food production and medicine.
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  1900–1953: Genetics.
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  1953–1976: DNA research, science explodes.
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  Modern biotechnology (1977): Manipulates genetic information in organisms; genetic engineering; various technologies enable us to improve crop yield and food quality in agriculture and to produce a broader array of products in industries.

1.3.1. Old and new biotechnology

1.3.2. Ancient biotechnology (pre-1800)

1.3.3. Classical biotechnology

1.3.4. Modern biotechnology

Biotechnology is the science of the controlled application of biological agents for beneficial use. Since biotechnology is not an independent discipline, its well-known integration with allied fields such as biochemistry, molecular biology and microbiology facilitates the technological application of biological agents. Therefore, modern biotechnology has developed as a science with enormous potential for human welfare in areas ranging from food processing to human health and environmental protection. The major significance of this field of science in different fields will be evident from the following examples.

1.4.1. Biotechnology in medicine

1.4.2. Industrial biotechnology

1.4.3. Biotechnology and the environment

1.4.4. Biotechnology and agriculture

Some of the basic tools that are frequently experimented with in biotechnology to explore surrounding applications are listed in table 1.4.

1.5.1. Bioreactors

1.5.2. Cell fusion

1.5.3. Liposome-based delivery

1.5.4. Cell or tissue culture

1.5.5. Genetic engineering

1.5.6. DNA fingerprinting

1.5.7. Cloning

1.5.8. Artificial insemination and ET technology

1.5.9. Stem cell technology

The enormous potential of biotechnology is exploited to yield high therapeutic compounds (table 1.5).

1.6.1. Basic applications of biotechnology

1.6.2. Most common applications

Some of the most common applications of biotechnology are highlighted below.

1.6.2.1. Cloning

The term cloning describes a number of different processes that can be used to produce genetically identical copies of a biological entity. The copied material, which has the same genetic make-up as the original, is referred to as a clone. Various historical events that led to the development of cloning are listed in table 1.7.

Table 1.7.  The timeline of cloning.

Year	Cloning event
1885	First ever display of artificial embryo twinning.
1901	Cloning hypothesized: Hans Spemann splits a two-celled newt embryo into two parts, effectively producing two larvae. (Later, in 1938, Spemann hypothesized that animals could be cloned by fusing an embryo with an egg cell.)
1902	Artificial embryo twinning in a vertebrate achieved.
1928	It was found that the cell nucleus regulates embryonic development.
1952	First thriving nuclear transfer (frog).
1958	Nuclear transfer from a differentiated cell (frog).
1963	Term 'clone' coined: John Burdon Sanderson Haldane used 'clone' in his speech on the biological potential for the human species over the next ten thousand years.
1963	First cloned fish: Tong Dizhou, an embryologist from China, developed the world's first cloned fish by incorporating the DNA from a cell of a male carp into an egg from a female carp.
1973	Stanley Norman Cohen and Herbert Boyer discovered the tool of DNA cloning, which copies genes to facilitate their transplantation between various biological species.
1975	First mammalian embryo produced by nuclear transfer (rabbit).
1980	First transgenic (genetically modified) mouse.
1982	Giant mouse created by transferring GH genes from a rat.
1984	First mammal produced by nuclear transfer (sheep).
1985	First transgenic domestic animal, a pig.
1987	A sequence of transgenic mice developed carrying human genes.
1987	Nuclear transfer from an embryonic cell (cow).
1995	Transgenic pig hearts made: researchers discover transgenic pig hearts that endure up to 30 h when transplanted to baboons. The FDA sanctions the use of transgenic pig livers as bridge organs for transplant candidates awaiting organs.
1996	Nuclear transfer from laboratory cells (sheep).
1996	The birth of the first cloned animal, Dolly the sheep, was proclaimed.
1997	First primate engineered by embryonic cell nuclear transfer (rhesus monkey).
1997	Cloning of a transgenic lamb (Polly) from cells engineered with a marker gene and a human gene was announced. In this fashion, the genetic alteration of a lamb was combined with the techniques of cloning, thereby generating animals that produce a new protein.
1997	Nuclear transfer from genetically engineered laboratory cells (sheep).
1998–1999	Additional mammals cloned by somatic cell nuclear transfer (mice, cows and goats).
2000	First pigs cloned: PPL Therapeutics clones the first pigs, engineered to create organs for human transplant.
2000	First transgenic pigs cloned: Infigen clones the first transgenic pigs, as a potential source of organs and tissues for transplant for humans.
2000	The Raelian sect claimed it would clone a human within the year.
2001	Endangered animals cloned by somatic cell nuclear transfer.
2001	A patent was approved to the University of Missouri for a technology for cloning mammals; the University then licensed the patent to Massachusetts company Biotransplant. Critics were troubled since the patent application did not prohibit humans, and specifically mentioned human eggs. Others challenged that the patent was for just the process and not the product, although the patent says it covers 'cloned products'.
2001	Human clones banned in England: the Human Reproductive Cloning Act was passed, barring the implanting of cloned embryos in the womb.
2001	Researchers produced the first clone of an endangered species: a type of Asian ox known as a guar. Sadly, the baby guar, which had developed inside a surrogate cow mother, died just a few days after its birth.
2002	US President Bush set up the Council on Bioethics to advise him on issues such as stem cell research and cloning.
2002	Cloning to make body parts: Advanced Cell Technology proclaimed that cells from cloned cow embryos were employed to cultivate kidney-like organs.
2002	CC (Carbon Copy), the first cloned pet: CC is not a phenotypic copy of the animal she was cloned from. A number of people have expressed interest in having their deceased pets cloned in the hope of getting a similar animal to replace the dead one. But as demonstrated by CC the cloned cat, a clone may not turn out precisely like the original pet whose DNA was employed to create the clone.
2003	Cloning approved for research purposes: the Kentucky House Judiciary Committee banned reproductive cloning but permitted cloning for research purposes. The bill would make the shipping or use of cloned embryos for reproductive purposes a crime punishable by 10–20 years in prison, as well as requiring those conducting cloning research to record it with the state Cabinet for Health Services 30 days before commencement of research.
2003	An endangered type of ox, called the Banteg, was successfully cloned.
2006	A pig was developed to generate omega-3 fatty acids via the incorporation of a roundworm gene.
2007	Primate (rhesus monkey) embryonic stem cells developed by somatic cell nuclear transfer.
2013	Human embryonic stem cells developed by somatic cell nuclear transfer.

1.6.2.1.1. Reproductive cloning

This involves constructing an egg using genetic material from another source. The egg then develops into an embryo, before being planted in a female host's uterus to continue to develop. Reproductive cloning produces copies of whole animals.

1.6.2.1.2. DNA/gene cloning

DNA cloning is a simpler method, whereby DNA is extracted from a host then replicated using plasmids. Even individual genes can be cloned in this manner. Gene cloning produces copies of genes or segments of DNA. A simple demonstration of gene cloning is shown in figures 1.2 and 1.3.

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1.6.2.1.3. Therapeutic cloning

This type of cloning is similar to reproductive cloning, except that stem cells are extracted from the embryo and used to treat the host. This type of cloning has many medical benefits for treating all sorts of diseases but is highly controversial because of the destruction of the embryo following stem cell extraction. A simple demonstration of therapeutic cloning is shown in figure 1.4. Therapeutic cloning produces embryonic stem cells for experiments aimed at creating tissues to replace injured or diseased tissues. The inner cell mass (ICM) is the source of embryonic stem cells. The embryo is destroyed by separating it into individual cells for the collection of ICM cells. Stem cells are found in adults, but the most promising types of stem cells for therapy are embryonic stem cells.

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In the context of cell replacement therapy, therapeutic cloning holds huge potential for de novo organogenesis and the permanent treatment of incurable diseases such as Parkinson's disease, Duchenne muscular dystrophy and diabetes mellitus, as shown by in vivo studies. Major obstacles obstructing advancement in therapeutic cloning are tumorigenicity, epigenetic reprogramming, mitochondrial heteroplasmy, interspecies pathogen transfer and low oocyte availability. Moreover, therapeutic cloning is also often tied to ethical considerations concerning the source, destruction and moral status of IVF embryos based on the argument of potential. Legislative and funding issues also need to be addressed. Future considerations would include a distinction between therapeutic and reproductive cloning in legislative formulations.

1.6.2.1.4. Applications

Reproductive cloning may enable researchers to make copies of animals with potential benefits for the fields of medicine and agriculture. Since the announcement of Dolly the sheep by Scottish researchers, other sheep have been genetically modified to produce milk that contains a human protein essential for blood clotting. This research was conducted to derive the protein from the milk for humans whose blood does not clot properly. This, along with many other studies, exemplifies the possibilities of cloned animals for testing of new drugs and treatment strategies. The main motivation for using cloned animals for drug testing is that they are all genetically identical. This means their responses to the drugs should be uniform rather than variable as seen in animals with different genetic make-ups.

In 2008, the FDA decided that meat and milk from cloned animals, such as cattle, pigs and goats, are as safe as those from non-cloned animals. This decision means that researchers are now free to use cloning methods. These methods can be used to make copies of animals with desirable agricultural traits, such as high milk production or lean meat. However, because cloning is still very expensive, it will likely take many years until food products from cloned animals actually appear in supermarkets. Moreover, cloning can also be utilized to create clones to build populations of endangered, or possibly even extinct, species of animals. During the era of genetic engineering most of the research has been focused towards transgenesis using rDNA and cloning as a basic tool for the procurement and maximum utilization of elite traits.

1.6.2.2. DNA fingerprinting

Different individuals carry different alleles. Most alleles useful for DNA fingerprinting differ on the basis of the number of repetitive DNA sequences they contain. If DNA is cut with a restriction enzyme that recognizes sites on either side of the region that varies, DNA fragments of different sizes will be produced. A DNA fingerprint is made by analyzing the sizes of DNA fragments produced from a number of different sites in the genome that vary in length. The more common the length variation at a particular site and the greater the number of sites analyzed, the more informative the fingerprint. A simple demonstration of DNA fingerprinting is illustrated in figure 1.5.

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The techniques used in DNA fingerprinting also have applications in paleontology, archaeology, various fields of biology and medical diagnostics. In biological classification, it can help show evolutionary change and relationships on the molecular level, and it has the advantage of being useable even when only very small samples, such as tiny pieces of preserved tissue from extinct animals, are available. In criminal investigations, the DNA fingerprint of a suspect's blood or other body material is compared to that of the evidence from the crime scene to see how closely they match. The technique can also be used to establish paternity. DNA fingerprinting is generally regarded as a reliable forensic tool when done properly, but some scientists have called for wider sampling of human DNA to ensure that the segments analyzed are indeed highly variable for all ethnic and racial groups. It is possible to create false genetic samples and use them to misdirect forensic investigators, but if those samples have been produced using gene amplification techniques they can be distinguished from normal DNA evidence.

1.6.2.2.1. Applications

Personal identification. This is the idea of keeping everyone's DNA on a computer as a bar code. This concept has been discussed and has been decided to be impractical and very expensive. It is very unlikely to become a system in general use. Photo identification cards and social security numbers, for instance, are much more efficient methods of identification and are not likely to change.

Paternity and maternity: This is also a well-known application of DNA fingerprinting. This is the test used to find out who is the father of a baby or child. Every individual has a variable number tandem repeat (VNTR) pattern which is inherited from their parents. The pattern in each individual is different but it is similar enough to reconstruct the parents' VNTR. This method can also be used to ascertain the real biological parents of an adopted child or determine legal nationality. Individuals should be careful when using a test like this because it may have surprising results that could cause distress.

Criminal identification and forensics. This is a very famous field of DNA fingerprinting. It has become popularly known because of the hit TV series CSI: Crime Scene Investigation. It is a very important use of DNA fingerprinting because it can prove an individual's innocence or guilt of committing a crime. To be used, a sample of DNA has to be obtained from the scene of the crime and matched with the suspect in question. The two pieces of DNA are then compared through VNTR patterns.

Diagnosis and cures for inherited diseases. DNA fingerprinting can also be used to detect and cure genetically inherited diseases. Using DNA fingerprinting one can detect genetic diseases such as cystic fibrosis, hemophilia, Huntington's disease and many others. If the disease is detected at an early age it can be treated and there is a greater chance that it can be defeated. Some couples who are carriers of a disease seek out genetic counsellors who can use a DNA fingerprint to help them understand the risks of having an affected child and give them information and assistance. The fingerprints can be used by researchers to look for patterns that specific diseases have and try to figure out ways that they can cure them.

1.6.2.3. rDNA technology

Restriction enzymes are enzymes that cut DNA only at particular sequences (figures 1.6 and 1.7). Different restriction enzymes have different recognition sequences. This makes it possible to create a wide variety of different gene fragments. To harness the power of rDNA technology, human insulin produced by bacteria plasmids is used to replicate rDNA. Plasmids are small circles of DNA found in bacteria which replicate independently of the bacterial chromosome. Pieces of foreign DNA can be added within a plasmid to create a recombinant plasmid. Replication often produces 50–100 copies of a recombinant plasmid in each cell. The route to the production of human insulin by bacteria is shown in figure 1.7.

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Genetically modified plants have generated a large amount of interest in recent years and continue to do so. In spite of this, the general public remains largely uninformed of what a genetically modified (GM) plant actually is or what merits and demerits the technology has to offer, particularly with a view to the range of applications for which it can be used. From the first generation of GM crops, two major areas of concern have appeared, specifically danger to the environment and hazards to human health. Since GM plants are steadily being established in the European Union (EU) there is the possibility of increased public concern regarding potential health issues. While it is now routine for the press to espouse 'health campaigns', the information they publish is often unpredictable and unreliable compared to the available scientific proof. It is essential to understand the rDNA-technology-mediated production of transgenic plants (figure 1.8).

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1.6.2.4. Stem cell therapy

A stem cell is an undifferentiated, dividing cell that gives rise to a daughter cell like itself and a daughter cell that becomes a specialized cell type (figure 1.9).

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1.6.2.4.1. Applications

Stem cells can be used to study development, i.e. they may help us understand how a complex organism develops from a fertilized egg. In the laboratory, scientists can follow stem cells as they divide and become increasingly specialized, making skin, bone, brain and other cell types. Identifying the signals and mechanisms that determine whether a stem cell chooses to carry on replicating itself or differentiates into a specialized cell type, and into which cell type, will help us understand what controls normal development. Some of the most serious medical conditions, such as cancer and birth defects, are due to abnormal cell division and differentiation. A better understanding of the genetic and molecular controls of these processes may yield information about how such diseases arise and suggest new strategies for therapy. This is an important goal of stem cell research.

Stem cells have the ability to replace damaged cells and treat disease. This property is already used in the treatment of extensive burns, and to restore the blood system in patients with leukemia and other blood disorders. Stem cells may also hold the key to replacing cells lost in many other devastating diseases for which there are currently no sustainable cures. Today, donated tissues and organs are often used to replace damaged tissue, but the need for transplantable tissues and organs far outweighs the available supply. Stem cells, if they can be directed to differentiate into specific cell types, offer the possibility of a renewable source of replacement cells and tissues to treat diseases including Parkinson's, stroke, heart disease and diabetes. This prospect is an exciting one, but significant technical hurdles remain that will only be overcome through years of intensive research.

Stem cells can be used to study disease. In many cases it is difficult to obtain cells that are damaged by a disease and to study them in detail. Stem cells, either carrying the disease gene or engineered to contain disease genes, offer a viable alternative. Scientists could use stem cells to model disease processes in the laboratory, and better understand what goes wrong.

Stem cells could provide a resource for testing new medical treatments. New medicines could be examined for safety on specialized cells generated in large numbers from stem cell lines—reducing the requirement for animal testing. Various types of cell lines are already used in this way. Cancer cell lines, for example, are used to screen potential anti-tumor drugs.

There now follows an overview of the most recent discoveries in biotechnology.

Genetic modification is an alternative for incorporating new traits or producing elite varieties, particularly in crops and trees. These superior varieties of plants have socio-economic and environmental advantages. However, complex and lengthy EU protocols are delaying their introduction to the market [69]. Considering GM development, scientist's state that Europe has not acquired a leading position in the global GM market and that Europe is lagging behind in worldwide GM developments, and they call for a more technically authentic decision-making process.

In another study scientists have produced microbes that cannot 'run away from home'. Swarmbots are the microbes engineered by rDNA technology from genetically engineered bacteria, and could be useful in various fields. Current reports suggest a platform technology (microbial swarmbot) which employs a spatial arrangement to regulate the growth dynamics of engineered bacteria. This scheme can stop GM organisms from escaping into the surrounding environment and additionally it can often encourage colonies of bacteria to react to changes against surrounding environment [70].

Current researchers have also discovered an effective method of gene transfer which involves culturing and transfecting of cells with genetic material on an array of carbon nanotubes. This innovation has overcome the drawbacks of other gene editing techniques. In this study researchers were more focused on the structure of regulatory sequences in DNA is packaged in a cell [71].

Recent transfection approaches have considerable limitations, as most of them are time consuming, cytotoxic, have inefficient introduction of test molecules into target cells and are limited by the size of the genetic cargo. Golshadi et al [71] developed a novel approach of inserting genes and biomolecules into tens of thousands of mammalian cells. This was achieved using hollow carbon nanotubes, manufactured by template-based nanofabrication processes, to achieve rapid high-efficiency transfer with low cytotoxicity, which will ultimately overcome the molecular weight limits of recent approaches. In addition to nucleic acids this approach can be utilized to deliver drugs or proteins. We have hypothetically demonstrated this approach in figure 1.10.

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Wrapping of histone by nuclear DNA to form nucleosomes limits the binding of transcription factors to gene regulatory sequences. Iwafuchi et al used low and high levels of micrococcal nuclease digestion to evaluate the nucleosomal configuration in mouse liver and finally discovered that MNase-accessible nucleosomes are present more at liver-specific enhancers than at promoters and ubiquitous enhancers (figure 1.11). [72]. They have concluded that as pioneer factor FoxA displaces linker histone H1, nucleosomes are not solely repressive to gene regulation when they are retained with, and exposed by, pioneer factors [72].

In another study, a comprehensive molecular investigation (transcriptional mapping of human embryo) of the embryo's first week of development has been carried out [73]. In this study transcriptional mapping of human embryo development, including the sequenced transcriptomes of 1529 individual cells from 88 human preimplantation embryos, are presented. They report that there are considerable differences in embryonic development between humans and mice.

The Hydra (freshwater polyp) is capable of restructuring a complete creature from any piece of its body. It has been observed that Hydra remains alive when all its neurons have gone. Hydra constantly differentiate a sophisticated nervous system made of interstitial stem cells. Wenger et al describe the impact of the loss of neurogenesis in Hydra by performing transcriptomic profiling at five positions along the body axis [74]. They have concluded from their study how epithelial cells alter their genetic program by overexpressing a sequence of genes, of which some are related to various nervous functions. These epithelial cells improve their sensing ability when neurogenesis is compromised. This unknown plasticity may represent the potential of epithelial-like cells in early Planulozoa development [74].

Schaumberg et al have taken genetic engineering to a more advanced level. Currently, various researchers a are developing modular, programmable genetic circuits that control specific plant functions [75]. Plant synthetic biology ensures enormous scientific benefits, containing the possible advancement of a sustainable bio-based economy by the prognostic design of synthetic gene circuits. These circuits are generated from quantitatively characterized genetic parts; nevertheless, this approach has considerable obstacles in work with plants as it requires time for stable transformation. Schaumberg co-workers described the quantitative characterization of genetic parts and circuits for plant synthetic biology, describing how genetic circuits control specific plant functions [75].

Another breakthrough was based on the determination of microbial growth using GM fluorescing E. coli cells. This approach works in a similar way to the method that is adopted to determine population levels of animals in the particular environments [76]. The growth dynamics of microbes is characteristically determined after observing them directly, or when death is rare. In mammalian gut microbiota, neither of these conditions holds, thus typical tactics cannot exactly determine the microbial growth under an in vivo environment. To determine microbial growth dynamics, Myhrvold et al explored a novel approach, by using distributed cell division counting (DCDC), that uses the precise segregation at cell division of genetically encoded fluorescent particles [76]. DCDC can allow the measurement of microbial growth during antibiotic therapy, gut dysbiosis, infection, or other situations relevant to human health. GM fluorescing E. coli measures gastrointestinal microbe growth rates (the population) that lives inside mammalian gastrointestinal tracts.

Another major breakthrough was achieved in the field of optogenetics, in regulating the movement of proteins. Protein regulation in eukaryotes is a very complex process. One of the key mechanisms for protein regulation is active nucleocytoplasmic transport. LEXY is a potential optogenetic toolbox with various applications in synthetic and cell biology. With the help of optogenetic tools it is possible to control the import of nuclear protein and several reports are already available on this. Optogenetics is the approach to control well-defined events within specific cells of living tissue by using genetics and optics. In 2016 Niopek et al, for the first time, evidenced an approach for spatiotemporal regulation of the export of a tagged protein using a light-inducible nuclear export system (LEXY) [77]. In this study light can be utilized to regulate the import/export of proteins from the cell nucleus and also the activity of proteins in mammalian cells [77] (see figure 1.12).

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Tissue engineering is an emerging area that has been attracting considerable attention from researchers. In an effort to create artificial cartilage tissue, researchers from Umea University have used cartilage cells from cow knee joints [78]. This novel approach can be utilized to develop healthy cartilage tissue, and further researchers could use the findings to develop a treatment, or cure, for osteoarthritis using stem-cell-based tissue engineering. In this study, scientists have investigated how cells (primary bovine chondrocytes), signaling molecules and the artificial support material for the cartilage-like tissues (neotissues) can collectively work to encourage tissue regeneration at an injured joint site. This approach offers treatment against osteoarthritis by using healthy cartilage tissue which contains stem cells [78]. The next breakthrough was made by researchers at the Harvard Wyss Institute for Biologically Inspired Engineering and Harvard Medical School, which was based on R-bodies (retractable protein polymer) polymeric protein inclusions synthesized inside the cytoplasm of bacteria. One of the most important features of these proteins that they show structural variation at different pH. At high pH R-bodies resemble a coil of ribbon whereas at lower pH they undergo a conformational change and convert themselves into pointy hollow tubes. These hollow tubes are capable of puncturing through membranes, rupturing the cell membrane and releasing material present inside. This could offer applications in new drug delivery and other applications in biotechnology and medicine [79].

Cellular reprogramming is an excellent approach to renew the capacity of selected tissue to synthesize useful biochemicals inside the body. Scientists have invested many years to replace the beta cells (insulin-producing pancreatic cells) that are lost in diabetes. In 2016, Ariyachet et al showed the potential of antral stomach cells (cells derived from the lower stomach) after reprogramming to produce functional insulin-secreting cells [80]. In this study Ariyachet and co-workers separated tissue from mice and after reprogramming cultured them into 'mini-organs'. These mini-organs produced insulin when transplanted back into the animals under in vivo conditions (see figure 1.13) [80].

In another study scientists have argued for the compulsory labeling of genetically modified foods, which is essentially required to let the community, especially consumers, to know what they are eating. This is based on a broad evaluation of the global scientific and legal frameworks associated with genetically modified organisms [81]. Certain producers and manufacturers are afraid of a negative consumer response to GM labelled products.

The CRISPR-Cas approach is often used for targeting, regulating, and editing genomic sequences, it is a potential technique for producing genetically modified animals. Yoshimi et al developed two new gene modification methods: lsODN (long single-stranded oligodeoxynucleotide) and 2H2OP (two-hit two-oligo with plasmid). These approaches use clustered, regularly interspaced, short palindromic repeats (CRISPR) Cas systems (associated proteins) and single-stranded oligodeoxynucleotides (ssODN) [82].

Opiates have been used from historical times as pain-relieving agents and are mainly derived from opium poppies. Modification of the alkaloid biosynthetic pathway to increase the productivity of these alkaloids is difficult as regulation of biosynthetic pathways in plants is complex. Therefore, current researchers used a step-wise fermentation approach using engineered strains of E. coli to trigger the production of thebaine up to a 300-fold increase. According to these researchers, this enhancement is may be due to the presence of strong activity of enzymes related to thebaine synthesis from (R)-reticuline in E. coli. These developments in opiate production using genetically modified E. coli system signify a key step towards alternative opiate production systems.

Emerging novel proteins can be a lead source in the drug delivery industry. The recent discovery of R-protein in the cytoplasm of bacteria that live inside Paramecia (tiny aquatic organisms) can possibly be utilized to deliver drugs [84]. To understand this phenomenon, it is essential to understand the role of R-bodies in Paramecia, as shown in figure 1.14. Recently researchers have identified that the response of R-bodies present in Paramecia can be manipulated in a pH-dependent manner. These proteins are capable of selectively rupturing membrane compartments, thus may be important for programming cellular compartmentalization [84].

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Another work synthesized genetically encoded biosensors for intracellular concentration of a target metabolite. These biosensors are synthesized from transcription factors conjugated with a fluorescent substance to track product formation. By tracking pathways researchers are engineering microbes in highly creative factories to produce products such as fine chemicals, therapeutics and biofuels [85].

Intestinal parasites infect more than one billion people worldwide, resulting in malnutrition and developmental issues [86]. Currently two benzimidazoles and two nicotinic acetylcholine receptor agonists are approved by the WHO. They are available on the market, however, the development of resistant strains against these agents urgently required new anthelminthics. Researchers made efforts to develop Bacillus thuringiensis (Bt) crystal (Cry) proteins to treat intestinal nematode infections in humans. Cry proteins have been considered as safe for humans. We have used these proteins from over 50 years as crop insecticides.

For the production of these insecticidal proteins (Bt proteins), researchers inserted genes in the non-toxic bacteria Lactococcus lactis (figure 1.15). This can further help in integration of this protein in to dairy products, or in the form of probiotics to further deliver the protein to the intestines of affected people.

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Another major breakthrough in the year 2015 was that the Joint Research Centre (JRC) developed a new database called the bioinformatics pipeline or JRC GMO-Amplicons, which includes the more than 240 000 DNA sequences emerging in GMOs [87]. This database will offer a complete source for the detection of DNA target sequences and also assist in checking the presence of GMOs in food, feed and the environment. This record is the broadest source of information available and can help in developing approaches for identifying GMOs in food and feed [88]. DiCarlo et al, in their study 'Safeguarding CRISPR-Cas9 gene drives in yeast' described an effective safeguarding mechanisms for working with gene drives and unveiled a first-of-its-kind approach for reversing the changes they spread [88].

It was observed that heat shock response is a type of stress which is important in understanding the biogenesis and degradation of proteins inside the body and it is equally important to understand cellular death [89]. It is well-known that aging can increase the risk for protein conformational disease. It was observed that during the onset of reproductive maturity HSR declines quickly. The suppression was because of the increase in H3K27me3 marks at stress gene loci. In adult cells, genes start aging by turning off cell stress responses [89]. These responses protect the cell by keeping potential proteins folded and functional. During early maturity germ line stem cells throw the switch and individual cells retain their reproductive capability [89]. Based on similar work, Yang et al investigated the minimal set of gene functions required to sustain life in E. coli. This study covers the set of genes required to sustain life [90]. By using a comparative genomics-based core proteome, minimal gene lists have been suggested.

Embryonic stem cell development is an area of heightened attention today. Recently, several scientists discovered an approach to control embryonic stem cell differentiation with the help of beams of light. This approach allows them to be transformed into neurons in reaction to an accurate external sign. In another study, the complete pathway of endocytosis was described by a team of researchers. In particular, they studied how cells perform endocytosis by absorbing molecules which can result in rapid embryonic healing. Considering this factor, the outcomes of the study can be employed to design better treatments for wounds in adults [92].

Researchers have engineered the cassava plant to offer higher levels of vitamin B6 in its storage roots and leaves. This could help guard millions of people in Africa against serious deficiencies [93].

Several reports are available on target-based mutagenesis mediated by Cas9 (RNA-guided DNA endonuclease enzyme). Woo et al [94] reported genomic editing in plants without insertion of foreign DNA into cells. They introduced the Cas9 protein to guide RNA into protoplasts of several plants and achieved targeted mutagenesis at frequencies of up to 46%. This study may alleviate regulatory concerns related to genetically modified plants, as the researchers used the Cas9 protein and did not insert foreign DNA.

Several reports were published in 2012 and while it is not possible to describe all of these, some promising research cannot be skipped, such as the research based on the revolutionary tool for editing DNA (CRISPR), which could replace all medicine with better treatments. This research was discovered by Jennifer A Doudna, a biochemist at UC Berkeley, and her collaborator, Emmanuelle Charpentier of the Helmholtz Centre.

In general, CRISPR allows scientists to change DNA and it could allow scientists to cure diseases. This technology came from a project studying how bacteria fight against viruses. Some bacteria have an adaptive immune response called CRISPR that allows then to detect viral DNA and destroy it (figure 1.16). One of the parts of the CRISPR system, called Cas9, is able to seek out, cut and eventually degrade viral DNA in a specific way. Doudna and colleagues realized that they could harness its functions as a genetic engineering tool. Currently this technology is being used in many parts of the world which Their work suggests that this tool will perhaps fundamentally change both medicine and agriculture. A number of studies have repaired defective DNA in mice, e.g. treating genetic disorders. Furthermore, plant-based researchers have used CRISPR to manipulate genes in crops, raising hopes that it could lead to a better food supply. In 2014, MIT Technology Review called CRISPR 'the major biotech discovery of the century' [95].

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Now pharmaceutical companies are showing interest in taking CRISPR to the next level to produce new drugs in the form of therapeutic proteins. Using CRISPR-based technology can create animal models, treat blood related disorders (easy to deliver in the blood), correct mutations and have certain clinical applications which will be seen in future. However, CRISPR technology raises many ethical issues, so discussion continues on validation and safety concerns. In January 2016, Novartis declared that it would be using Doudna's CRISPR technology for its study into cancer treatments. It intends to manipulate the genes of immune cells so that they will attack tumors (figure 1.17). Microbes have been using CRISPR to alter their own DNA for millions of years, and currently they continue to do so all over the planet, from the bottom of the sea to the recesses of our own bodies. They utilize it as a complicated immune system, permitting them to learn to identify their enemies. Now researchers are discovering that microbes use CRISPR for other purposes as well (figure 1.17).

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