GENETIC ENGINEERING

•Many people are unaware that humans have been practicing genetic engineering since the
ancient times. Selective breeding or classical breeding in agricultural crops and livestock has
actually altered the genetic make- up of these organisms over the centuries in such a way
that they no longer resemble their non-domesticated relatives. This practice has been
common long before genes were discovered. What is Biotechnology? By 2007, the human
genome has been fully sequenced. The ability to sequence DNA has been reliant on
technological advancements. The modification of organisms or their genetic components to
produce useful goods is known as biotechnology. For biotechnology, a variety of skills and
procedures have been developed: • Creation of genetically modified organisms, or genetic
engineering • Protein expression and analysis • DNA amplification • DNA fingerprinting
What is Genetic Engineering? - Genetic Engineering is a process of altering the genes that
are found in all living organisms. It involves the transfer of genes or parts of DNA from one
organism to another. Organisms whose genes are altered or modified for a specific purpose
are called transgenic organisms. - Genetic engineering includes classical breeding which is
considered as the traditional way of genetic engineering which practices the mating of
organisms with desirable qualities and Recombinant DNA technology (rDNA), a modern
technique of genetic engineering. Recombinant DNA technology is the joining together of
DNA molecules from two different species. The recombined DNA molecule is inserted into a
host organism to produce new genetic combinations that are of value to science, medicine,
agriculture, and industry Genetic Engineering Techniques The process of genetic engineering
is true to all organisms to be modified: 1. Identification of the organism that contains a
desirable gene. 2. Extraction of the entire DNA from the organism. 3. Isolation of the gene by
removing it from the rest of the DNA. One way to do this is by using a restriction enzyme.
This enzyme searches for specific nucleotide sequence where they will "cut" the DNA by
breaking the bonds at this location. The process of genetic engineering is true to all
organisms to be modified: 4. Preparing the target DNA A circular piece of DNA called a
plasmid is removed from a bacterial cell. Special proteins are used to cut the plasmid ring to
open it up. 5. Insertion of DNA into plasmid The host DNA that produces the wanted protein
is inserted into the opened plasmid DNA ring. Then special cell proteins help close the
plasmid ring. 6. Insertion of plasmid back into cell The circular plasmid DNA that now
contains the host gene is inserted back into a bacteria cell. The plasmid is a natural part of
the bacteria cell. The bacteria cell now has a gene in it that is from a different organism,
even from a human. This is what is called recombinant DNA technology. 7. Plasmid
multiplication The plasmid that was inserted into the bacteria cell can multiply to make
several copies of the wanted gene. Now the gene can be turned on in the cell to make
proteins. 8. Target cells reproduction Many recombined plasmids are inserted into many
bacteria cells. While they live, the bacteria's cell processes turn on the inserted gene and the
protein is produced in the cell. When the bacterial cells reproduce by dividing, the inserted
gene is also reproduced in the newly created cells. 9. Cells produce proteins The protein that
is produced can be purified and used for a medicine, industrial, agricultural, or other uses.
Creation of Genetically Modified Organisms (Genetic Engineering) •Genetic engineering, or
the direct manipulation of genes for practical goals, relies heavily on recombinant DNA
methods. History of GMO Development Classical Breeding •Done through the insertion of
genetic material and must be followed up by gene insertion. •The insertion process does not
occur in nature; therefore, a gene "gun," a bacterial "truck," inserts the genetic material into
the host plant cells. •This genetic material inserts itself into the chromosomes of the host
plant. •Engineers must also insert a "promoter" gene from a virus as part of the package to
make the inserted gene express itself. Genetic Engineering •It focuses on the mating of
organisms with desirable characteristics. •May develop new plant varieties in the selection
process and seek to achieve expression of genetic material present within a species. •It
employs processes that occur in nature, i.e., sexual and asexual reproduction. Creation of
Genetically Modified Organisms (Genetic Engineering) •The major goal of genetic
engineering or Recombinant DNA technology is to genetically modify a living organism by
inserting the desired gene into a cloning vector to form a recombinant DNA which is then
inserted into the host organism for the expression of the gene of interest. •Nucleotide
sequences from two separate sources, usually two species, are joined in vitro into the same
DNA molecule in recombinant DNA. Scientists produce gene-sized fragments of DNA in
identical duplicates, a process known as DNA cloning, to work directly with specific genes to
be merged. •In the laboratory, bacteria and their plasmids can be used to clone DNA
fragments. •Plasmids are tiny circular DNA molecules that replicate independently from the
bacterial chromosome in bacteria. •A cloning vector, which is a DNA molecule that can
transfer foreign DNA into a host cell and replicate there, is the original plasmid. •Foreign
DNA is put into a plasmid, which is then inserted into a bacterial cell as a recombinant
plasmid. •Cloning of the plasmid, which includes the foreign DNA, occurs during
reproduction in the bacterial cell. As a result, numerous copies of a single gene are
produced. Restriction enzymes from bacteria cleave DNA molecules at restriction sites,
which are defined as certain DNA sequences. Recombinant DNA is the method of joining two
or more DNA molecules to create a hybrid. The general outline of recombinant DNA are as
follows; 1. Cutting 2. Selection 3. Ligation 4. Transfer 5. Selection Process 6. Sequencing of
the gene These are some ways in which these plasmids may be introduced into host
organisms; 1. Biolistic. In this technique, a "gene gun" is used to fire DNA-coated pellets on
plant tissues. Cells that survive the 'bombardment', and are able to take up the expression
plasmid coated pellets and acquire the ability to express the designed protein. 2. Plasmid
insertion by Heat Shock Treatment. Heat Shock Treatment is a process used to transfer
plasmid DNA into bacteria. The target cells are pre-treated before the procedure to increase
the pore sizes of their plasma membranes. This pretreatment (usually with CaCl2) is said to
make the cells "competent" for accepting the plasmid DNA. After the cells are made
competent, they are incubated with the desired plasmid at about 4°C for about 30min. The
plasmids concentrate near the cells during this time. Afterwards, a "Heat Shock" is done on
the plasmid-cell solution by incubating it at 42°C for 1 minute then back to 4°C for 2 minutes.
The rapid rise and drop of temperature is believed to increase and decrease the pore sizes in
the membrane. The plasmid DNA near the membrane surface are taken into the cells by this
process. The cells that took up the plasmids acquire new traits and are said to be
"transformed". 3. Electroporation. This technique follows a similar methodology as Heat
Shock Treatment, but, the expansion of the membrane pores is done through an electric
"shock". This method is commonly used for insertion of genes into mammalian cells. The
general outline of recombinant DNA are as follows; After the process of recombinant DNA,
these plasmids or gene copies will now be introduced to its host organisms to confer upon
them the desired trait. A gene for pest resistance for example, as shown from the image
above, may be isolated, cloned and inserted into plant cell. Alternatively, bacterial cells may
express the inserted gene in order to produce protein products. Some important human
proteins like hormones and enzymes are produced by this technique. Genetic modification is
not only for plants. Recombinant bacterial cells with human genes can be used in order to
produce human proteins like insulin to be used by people with type I diabetes, human
growth hormones which is taken to cure stunted growth, and tissue plasminogen activator
which dissolves blood clots among patients who had heart attack. How is Recombinant DNA
made? •A restriction enzyme produces many cuts resulting in restriction fragments. The
most useful restriction enzymes cut DNA in a staggered pattern, resulting in fragments with
"sticky ends" that bind to complementary sticky ends of other fragments. DNA ligase is an
enzyme that binds restrictionfragments together and secures the connections. Reproductive
Cloning of Mammals: Sheep Pigs Cats Deer Horses Dogs Mice Wild goats Gray wolves The
cloning of mammals from adult cells has been achieved in several mammalian species in the
past few years, indicating that the genome of at least some adult cells can be reprogrammed
to support complete development. Reproductive Cloning of Mammals The cloning of
mammals from adult cells has been achieved in several mammalian species in the past few
years, indicating that the genome of at least some adult cells can be reprogrammed to
support complete development. Advantage Cloning of Mammals Gene cloning allows for
making a large number of copies of the desired gene in a matter of a few hours. The cloned
gene can be used for DNA sequencing studies. For the production of large quantities of a
desired protein by cloning into an expression vector. To cure disease using gene therapy.
Gene therapy replaces a faulty gene or adds a new gene in an attempt to cure disease or
improve the body's ability to fight disease. Disadvantage Cloning of Mammals Clones often
suffer from premature aging, genetic abnormalities, immune deficiencies, and shorter
lifespans APPLICATION OF GENETIC ENGINEERING •Gene Cloning A process by which large
quantities of a specific, desired gene or section of DNA may be cloned or copied once the
desired DNA has been isolated. Gene Cloning 1. The gene or DNA that is desired is isolated
using restriction enzymes. 2. Both the desired gene and a plasmid are treated with the same
restriction enzyme to produce identical sticky ends. 3. The DNAs from both sources are
mixed together and treated with the enzyme DNA ligase to splice them together. 4.
Recombinant DNA, with the plasmid containing the added DNA or gene, has been formed. 5.
The recombinant plasmids are added to a culture of bacterial cells. Under the right
conditions, some of the bacteria will take in the plasmid from the solution during a process
known as transformation. 6. As the bacterial cells reproduce (by mitosis), the recombinant
plasmid is copied. Soon, there will be millions of bacteria containing the recombinant
plasmid with its gene. 7. The introduced gene can begin producing its protein via
transcription and introduced translation. APPLICATIONS OF GENETIC ENGINEERING 1.
Medical Applications Diagnosis of Diseases •The identification of human genes in which
mutation has a role in genetic illnesses is one advantage of DNA technology. Scientists can
use PCR with primers corresponding to cloned disease genes to identify many human
genetic illnesses, then sequence the amplified result to look for the disease-causing
mutation. Human Gene Therapy •The altering of a patient's genes is known as gene therapy.
Gene therapy has a lot of promise for curing diseases caused by a single faulty gene. Genes
are delivered into certain types of cells, such as bone marrow, using vectors. Gene therapy
poses ethical concerns, such as whether or not human germ- line cells should be treated to
rectify a genetic abnormality in future generations. Pharmaceutical Products Synthesis of
Small Molecules for Use as Drugs •Imatinib is a tiny chemical that prevents the over
expression of a leukemia causing receptor. Protein-base pharmaceuticals can be mass-
produced on a vast scale. Protein Production in Cell Cultures Protein Production by "Pharm"
Animals and Plants Pharmaceutical Products Protein Production by "Pharm" Animals and
Plants Genes from one species are inserted into the genome of another animal to create
transgenic animals. Transgenic animals are pharmaceutical "factories," producing vast
quantities of ordinarily rare medicament compounds. Plants called "pharma" are being
created to produce human proteins for medical purpose. 2. Forensic Evidence and Genetic
Profiles •Analysis of tissue or bodily fluids can reveal an individual's unique DNA sequence,
or genetic profile. Genetic profiles can be used to identify human remains and give evidence
in criminal and paternity proceedings. Southern blotting can be used to assess genetic
profiles utilizing RFLP analysis. •The use of genetic markers known as short tandem repeats
(STRs), which are differences in the number of repeats of specific DNA sequences, is much
more sensitive. To amplify and then identify STRs of various lengths, PCR and gel
electrophoresis are utilized. It's extremely unlikely that two persons who aren't identical
twins share the same STR markers. 3. Environmental Cleanup •Microorganisms' metabolism
can be altered through genetic engineering. Some genetically modified bacteria can harvest
minerals from the environment or breakdown potentially harmful trash. To replace fossil
fuels, biofuels employ crops including corn, soybeans, and cassava. 4. Agricultural
Applications •Agricultural productivity and food quality are being improved using genetic
engineering. •Animal Husbandry. The selective breeding process is sped up through genetic
engineering of transgenic animals. Beneficial genes can be passed down from one variety to
the next. Genetic Engineering in Plants. •A variety of crop plants have been genetically
modified to produce desirable features, according to agricultural specialists. For delivering
new genes into plant cells, the Ti plasmid is the most often employed vector. Many valuable
genes have been transferred through genetic engineering in plants, including those for
herbicide resistance, insect resistance, salt tolerance, and better nutritional value of crops.
Recombinant DNA •Recombinant DNA technology is a technique that changes the
phenotype of an organism (host) when a genetically altered vector is introduced and
integrated into the genome of the organism. So, the process involves the introduction of a
foreign piece of DNA structure into the genome, which contains our gene of interest. And at
last, it has to be maintained in the host and carried forward to the offspring (Shinde et.al.
2018). •The primary tools of recombinant DNA technology are bacterial enzymes called
restriction enzymes. Each enzyme recognizes a short, specific nucleotide sequence in DNA
molecules and cuts the molecules' backbones at that sequence. The result is a set of double-
stranded DNA fragments with single-stranded ends, called "sticky ends. •Sticky ends are not
really sticky; however, the bases on the sticky ends form base pairs with the complementary
bases on other DNA molecules. Thus, the sticky ends of DNA fragments can be used to join
DNA pieces originating from different sources. •The recombinant DNA molecules have to be
made to replicate and function genetically within a cell to be useful. One method for doing
this is to use plasmid DNA from bacteria. Small DNA fragments can be inserted into the
plasmids, which are then introduced into bacterial cells. •As the bacteria reproduce, so do
the recombinant plasmids. The result is a bacterial colony in which the foreign gene has
been cloned. Applications of Recombinant DNA Technology 1. Production of Transgenic
Plants •By utilizing the tools and techniques of genetic engineering, it is possible to produce
transgenic plants or genetically modified plants. Many transgenic plants have been
developed with better qualities like resistance to herbicides, insects, or viruses or with the
expression of male sterility, etc. 2. Production of Transgenic Animals •By the use of rec DNA
technology, desired genes can be inserted into the animal so as to produce the transgenic
animal. The method of rec DNA technology aids the animal breeders to increase the speed
and range of selective breeding in the case of animals. 3. Production of Hormones •By the
advent of techniques of rec DNA technology, bacterial cells like E.coli are utilized for the
production of different fine chemicals like insulin, somatostatin, somatotropin, and
endorphin. Human Insulin Hormone, i.e., Humulin, is the first therapeutic product that was
produced by the application of rec DNA technology. 4. Production of Vaccines •Vaccines are
the chemical preparations containing a pathogen in an attenuated (or weakened) or inactive
state that may be given to human beings or animals to confer immunity to infection. •A
number of vaccines have been synthesized biologically through recDNA technology; these
vaccines are effective against numerous serious diseases caused by bacteria, viruses, or
protozoa. These include vaccines for polio, malaria, cholera, hepatitis, rabies, smallpox, etc.
The generation of DNA vaccines has revolutionized the approach to the treatment of
infectious diseases. DNA-vaccine is the preparation that contains a gene encoding an
immunogenic protein from the concerned pathogen. 5. Biosynthesis of Interferon
•Interferons are the glycoproteins that are produced in very minute amounts by the virus-
infected cells. Interferons have antiviral and even anti-cancerous properties. By the recDNA
technology method, the gene of human fibroblasts (which produce interferon's in human
beings) is inserted into the bacterial plasmid. These genetically engineered bacteria are
cloned and cultured so that the gene is expressed and the interferons are produced in
relatively high quantities. This interferon, so produced, is then extracted and purified. 6.
Production of Antibiotics •Antibiotics produced by microorganisms are very effective against
different viral, bacterial, or protozoan diseases. Some important antibiotics are tetracycline,
penicillin, streptomycin, novobiocin, bacitracin, etc. The recDNA technology helps in
increasing the production of antibiotics by improving the microbial strains through
modification of genetic characteristics. 7. Production of Commercially Important Chemicals
•Various commercially important chemicals can be produced more efficiently by utilizing the
methods of rec DNA technology. A few of them are the alcohols and alcoholic beverages
obtained through fermentation, organic acids like citric acid, acetic acid, etc., and vitamins
produced by microorganisms. 8. Application in Enzyme Engineering •As we know that the
enzymes are encoded by genes, so if there are changes in a gene, then definitely the enzyme
structure also changes. Enzyme engineering utilizes the same fact and can be explained as
the modification of an enzyme structure by inducing alterations in the genes which encode
for that particular enzyme. 10. Gene Therapy •Gene therapy is undoubtedly the most
beneficial area of genetic engineering for human beings. It involves the delivery of specific
genes into the human body to correct the diseases. Thus, it is the treatment of diseases by
transfer and expression of a gene into the patients' cells so as to ensure the restoration of a
normal cellular activity. HISTORY OF LIFE ON EARTH Conditions on Early Earth made the
Origin of Life Possible •On early Earth, chemical and physical processes may have formed
very primitive cells in a series of stages: 1. Small organic compounds are synthesized
abiotically. 2. These tiny molecules are joined together to form macromolecules. 3.
Molecules are packaged into protocells. 4. Self-replicating molecules arose from the origins
of self- replicating molecules. Synthesis of Organic Compounds on Early Earth •Earth, like the
rest of the solar system, formed around 4.6 billion years ago. Before 4.2 to 3.9 billion years
ago, the Earth was likely bombarded by rocks and ice, which evaporated water and
prevented seas from forming. •Water vapor and chemicals generated by volcanic eruptions
are likely to have been present in Earth's early atmosphere (nitrogen, nitrogen oxides,
carbon dioxide, methane, ammonia, hydrogen, hydrogen sulfide). •In the early twentieth
century, A. J. Oparin and I. Oparin, the early atmosphere, according to B. S. Haldane, was a
decreasing environment. Stanley Miller and Harold Urey conducted lab experiments in 1953
that demonstrated the possibility of abiotic synthesis of organic compounds in a reducing
environment. •However, the evidence for a reduction in the early atmosphere is still lacking.
The first organic chemicals may have been produced near volcanoes or deep- sea vents,
rather than in the atmosphere. Miller-Urey tests show that organic molecules might have
originated in a variety of environments. Abiotic Synthesis of Macromolecules •Simple
chemicals have been used to create RNA monomers spontaneously. When little organic
molecules are concentrated on hot sand, clay, or rock, they polymerize. The Fossil Record
•The fossil record illustrates shifts in Earth's life history. Sedimentary rocks are the richest
source of fossils because they are deposited in layers called strata. Few people have been
discovered, and even fewer have been petrified. •The fossil record is skewed in favor of
species that have been extinct for a long time: it has been around for a long time; were
numerous and broad and there were some hardest parts. •Fossils are evidence of organisms
that lived in the past. They can be actual remains like bones, teeth, shells, leaves, seeds,
spores or traces of past. In addition, these are activities such as animal burrows, nests and
dinosaur footprints or even the ripples created on a prehistoric shore. •Medusa effect is an
exceptional preservation, fine details such as original color and individual muscle fibers are
retained, features often visible in electron microscopes. Six Ways of Fossilization 1.
Unaltered preservation - Small organism or part trapped in amber, hardened plant sap 2.
Permineralization/ Petrification - The organic contents of bone and wood are replaced with
silica, calcite or pyrite, forming a rock-like fossil. 3. Replacement - hard parts are dissolved
and replaced by other minerals, like calcite, silica, pyrite, or iron 4. Carbonization or
Coalification - The other elements are removed and only the carbon remained. 5.
Recrystallization - Hard parts are converted to more stable minerals or small crystals turn
into larger crystals 6. Authigenic preservation - Molds and casts are formed after most of the
organism have been destroyed or dissolved How Rocks and Fossils Are Dated •Knowing the
age of a fossil can help a scientist establish its position in the geologic time scale and find its
relationship with the other fossils. There are two ways to measure the age of a fossil: relative
dating and absolute dating. Relative Dating. •This type of age-determining manner is based
upon the study of layer of rocks. It does not tell the exact age: only compare fossils as older
or younger, depends on their position in rock layer. Fossils in the uppermost rock layer/
strata are younger while those in the lowermost deposition are oldest. Law of Superposition:
•if a layer of rock is undisturbed, the fossils found on upper layers are younger than those
found in lower layers of rocks. However, because the Earth is active, rocks move and may
disturb the layer making this process not highly accurate. Law of Original Horizontality:
•Deposition of rocks happen horizontally tilting, folding, or breaking happened recently. Law
of Cross-cutting relationships: •If an igneous intrusion or a fault cut through existing rocks,
the intrusion/fault is YOUNGER than the rock it cuts through. Absolute Dating. •This type of
age-determining manner determines the actual age of the fossil. Through radiometric
dating, using radioactive isotopes carbon-14 and potassium-40. Considers the half-life or the
time it takes for half of the atoms of the radioactive element to decay. The decay products of
radioactive isotopes are stable atoms. Key events in life's history include the origins of
single-celled and multi-celled organisms and the colonization of land •The Archaean,
Proterozoic, and Phanerozoic eons are represented in the geologic record. Multicellular
eukaryotic life is included in the Phanerozoic. Paleozoic, Mesozoic, and Cenozoic are the
three eras of the Phanerozoic. Extinction events in the fossil record correspond to major
borders between geological divisions. Each of the five big extinction events resulted in the
extinction of more than half of the Earth's species. 1. Ordovician-Silurian Extinction: 440
million years ago (species made extinct: 85%) The first mass extinction on Earth happened
during a time when animals like corals and shelled brachiopods A quick beginning of mass
glaciation enveloped the southern supercontinent, Gondwana, near the end of the
Ordovician epoch. Glaciation on this magnitude trapped large amounts of the world's water
and substantially dropped global sea levels, robbing many species of their essential habitats,
damaging food chains, and reducing reproductive success. 2. Late Devonian extinction: ~ 365
million years ago (species made extinct: 75%) The Devonian epoch, also known as the "age
of fish," saw the emergence and collapse of many prehistoric marine species. Although
creatures had begun to emerge on land by this period, the majority of life still swam in the
waters. That is, until vascular plants like trees and flowers triggered a second global
extinction. These blooms effectively created massive "dead zones," which are areas where
algae deplete oxygen in the water, killing marine life and disrupting marine food networks. 3.
Permian-Triassic extinction: ~ 253 million years ago (species made extinct: 96% marine life;
70% terrestrial life) This extinction event, known as the "Great Dying," is the largest to have
ever occurred on Earth. It wiped out 90 percent of all species on the globe and destroyed
land-based reptiles, insects, and amphibians. A period of intense volcanism was responsible
for this disastrous occurrence. At the time, the world was made up of only one
supercontinent, Pangaea, which some scientists say contributed to a lack of movement in
the world's oceans, resulting in a worldwide pool of stagnant water that only served to
increase carbon dioxide levels. The amount of oxygen in the ocean has also decreased as sea
temperatures have risen. Corals were among the worst- affected marine life forms, and it
took 14 million years for the ocean reefs tos recover their former splendor. 4. Triassic-
Jurassic extinction: ~ 201 million years ago (species made extinct: 80%) The Triassic period
saw the emergence of new and diverse living forms, including dinosaurs, which began to
populate the planet. Sadly, a number of volcanoes erupted at the same period. Although the
cause of the fourth mass extinction is unknown, experts believe it was caused by extensive
volcanic activity in an area of the world now covered by the Atlantic Ocean. Alternative
explanations for the mass extinction include rising carbon dioxide levels releasing trapped
methane from permafrost, which would have resulted in a similar chain of events. 5. K-Pg
extinction: ~ 66 million years ago (species made extinct: 75%) The Cretaceous-Paleogene
extinction, also known as "the day the dinosaurs died," is the most well-known of all major
extinction events. The extinction is also known as the K-T extinction, and scientists refer to it
as the "K-Pg extinction" since the letter "C" stands for the Cambrian, a prior geological age.
The letter "K" comes from the word "Kreide," which means "Cretaceous" in German.
According to scientists, the majority of extinctions on Earth at the time would have occurred
within months of the impact. Many animals that could fly, burrow, or dive to the deepest
depths of the oceans, on the other hand, survived. For example, modern-day birds are the
sole real offspring of dinosaurs, with over 10,000 species considered to have descended
from impact survivors. Mass Extinctions •Most species that have ever existed are now
extinct, according to the fossil record. Changes in a species' environment can lead to
extinction. •At times, the pace of extinction has skyrocketed, resulting in a mass extinction.
Disruptive global environmental changes are the cause of mass extinction. Each of the five
big extinction events resulted in the extinction of more than half of the Earth's species