Unit 10 - Biotechnology

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Biotechnology


I.
Introduction to

Biotechnology


Biotechnology is the application of scientific and engineering principles to process biological materials for
goods and services. Historically, biotechnology took place in the form of win
e and beer
-
making, cheese
making, and bread making (all use microorganisms to create the final product). This was before the molecular
mechanisms were understood


but now, we understand the molecular mechanisms behind the methods, and
have further refine
d the field of biotechnology.


In its broadest definition,
biotechnology

is the application of biological techniques and engineered
organisms to make products or modify plants and animals to carry desired traits. This definition also extends to
the use

of various human cells and other body parts to produce desirable products. The term
bioindustry
refers
to the cluster of companies that produce engineered biological products and their supporting businesses.








Basic Classification of Bioindu
stry Firms



Therapeutics
: products that cure or reduce the incidence of disease.



Diagnostics
: products that test for the presence of various health or disease states.



Agricultural
: products related to crop and livestock production, including genetic engin
eering,
veterinary activities, and food processing.



Bioremediation and new high technology applications
: bioremediation involves using biotechnology
-
designed organisms to clean up oil or other spills. Researchers are creating new sources of energy and
lin
king biotechnology with microelectronics, nanotechnology and other high technology industries.



Bioindustry suppliers
: specialized materials, equipment or services for other bioindustry firms. Such
products include reagents (substances used in chemical rea
ctions),



S
pecialized software, and technical instruments for gene splicing.

C
urrent developments in each of these categories will be regrouped by the field they affect: human health
related products; animal and plant products; food processing;
and
enviro
nmental cleanup and energy.









The Biotechnology Tree





2

1.
Human Health
-
Related Products

Biotechnology is researching a broad range of human
-
health
-
related products. This
unit

will focus only on
pharmaceuticals and genetic engineering.



Diagnostic product
s
-


Biotechnology products

have made it easier to detect and diagnose illnesses

and
harmful microbes
.

By using such methods as biosensors, PCR and DNA probes, many different thing
s

can be detected. Examples include:
pregnancy testing
,
screening techniques to protect the blood su
pply
against contamination by AIDS and the hepatitis B and C viruses
, to help enhance the safety of the food
supply by detecting dangerous microbes and toxins produced that can harm us
.



Pharmaceuticals

-


microbes are used to mass
-
produce

remarkable new me
dical treatments and
applications for improving human health. The Food and Drug Administration has approved preventive
agents or treatments for:

Acute Growth Deficiency


Cystic Fibrosis


Hepatitis B (vaccine and therapeutic)


AIDS
-
related Kaposi's sarcom
a


Anemia


Hairy cell leukemia


Diabetes mellitus


Venereal warts


Acute myocardial infarction (heart attack)


Kidney transplant rejection


Well over 1,000 clinical trials of new drugs and biological agents are currently under way. A majority of
these
are for cancer or cancer
-
related conditions. About one out of seven of the trials are for drugs to
treat AIDS or HIV
-
related conditions.



Gene Therapy

-

Gene therapy is
the use of biotechnology to change an individual’s DNA so that
defective genes are cor
rected.
Introduction of selected genes into a patient's cells can potentially cure or
ease the vast majority of disorders.

More than 4,000 conditions [such as cystic fibrosis, cancer, heart
disease, AIDS, arthritis and senility] result from impairment of

one or more genes
.
Insertion of a
healthy gene from one person to another to treat a health problem is also possible. In 1989, the first
human gene transplant was authorized by a United States District Court in Washington, D.C. Since
then, scientists ha
ve demonstrated that it is possible to insert a healthy human gene into the cells in a
cystic fibrosis patient's lungs. The implanted gene produces an essential protein to replace one that is
defective in cystic fibrosis patients. A second approach is to u
se
a
genetically
-
altered common cold virus
to act as a carrier of the healthy gene into the body. This approach has been successfully tested in the
laboratory. In October of 1995 the first clear gene therapy success was claimed by scientists. Two
children
suffering from an inherited gene that left them without an immune system received normal
genes. One patient's response showed clear improvement.



Vaccines

-


The cost and availability of potential future vaccines may depend on biotechnology research.
Biotec
hnology is used in two ways in the development of these vaccines: to first sequence the
genome
of human pathogens and parasites

so that specific antigens can be identified in these organisms; and
second, to create the vaccine itself
.

One promising new ty
pe of vaccine is the “veggie vaccine”, in
which a plant (such as a fruit or vegetable) is engineered to contain the vaccine for the organism.
Examples include
bananas that vaccinate against cholera, potatoes that vaccinate against Hepatitis B.






3

2.
An
imal Products

Genetic engineering may improve an animal's economic value. Genetically engineered hormones, transfer of
genes from other species, and introduction of human genes to produce specified substances are all being used
today for this purpose. Expe
riments with transplantation of animal organs to humans are under way.



Transgenic Animals
-

A transgenic animal contains genes which have been inserted from another
species into the egg to generate a particular scientifically useful or marketable charact
eristic. Offspring
include the desired trait. The vast majority of transgenic animals being produced today are laboratory
mice with genes inserted from many different species (including humans). Typical is the Oncomouse,
which is genetically modified to de
velop malignant tumors useful for human cancer research. Recently
developed transgenic farm animals include cattle, chickens, pigs, rabbits, sheep, fish, and goats.
Transgenic cattle and swine have recently been developed to produce human growth hormone.
S
heep
have been engineered to produce blood clotting factors in their milk (for hemophiliacs); cows can be
engineered to produce various proteins in their milk; goats have been engineered to produce silk in their
milk; and many more.






Gene

Pharming"

-

The

term "gene pharming," playing on the words
farming

plus
pharmaceutical
,
refers to the production of biologically active drugs using genetically altered animals. For example,
Genzyme Transgenics has purchased a farm in western Massachusetts to breed geneti
cally altered goats
which produce human therapeutic and diagnostic proteins in their milk. Gene pharming is expected to be
a less costly production method than traditional cell culture methods. In August of 1995, the FDA issued
guidelines for medicines der
ived from the milk of genetically altered animals.







Organ Transplants

-

There are not enough donors of human organs to meet the need. Researchers hope
that genetic engineering will soon make it possible to alter the pig so that it can become a routine so
urce
of organs for transplant into humans. Research to reduce the chances for rejection of transplanted animal
organs is close to human clinical trials. The pharmaceutical industry is interested in developing the
technology and is investing in the research
.







Cloning



Cloning is the production of multiple identical organisms in the laboratory.
One reason for
cloning animals would be to mass
-
produce many identical animals that have the same desirable traits as
the original animal. Example: suppose that o
ne cow produces an extraordinarily large amount of milk;
cloning this cow would create many of these cows!

Or to produce numerous fast racehorses.

3.
Plant Products

The world’s increasing ability to grow subsistence crops has increased food supplies over

the past two decades,
yet undernutrition remains a serious problem in developing countries because of population expansion. We will
need to increase global food production by another 50% in the next 50 years to stay even with global population
growth! T
his depends on improving the crops we now use.

Genetic manipulation to improve plants is as old as farming itself. W
e

have traditionally created plants with
desirable traits through selective breeding. But this is a slow and tedious process. Microbes
offer us tools to
speed incorporation of valuable characteristics into existing plants and, in some cases, provide valuable traits
from the microbes’ own genetic pool.



Agrobacterium tumefaciens



this is a bacterium that infects plants, causing a disease.

It does this by



inserting some of its DNA into the plant cell it is infecting. Scientists learned that they could eliminate



the bacterium’s disease
-
producing capabilities without altering its ability to insert DNA into the plant



4


cell. This is now a useful tool for inserting foreign genes into plants. Using this bacterium, scientists


can make plants virus resistant, pesticide resistant plants, herbicide resistant plants, plants with increased



storage life, a
nd plants with increased vitamins and nutrition.



Bacillus thuringiensis



called

Bt

for short,
this is a bacterium that
creates

its own

natural insecticides
that are friendly to the environment.
The toxin produced by this bacteri
um

can be concentrated fo
r use
as a natural spray
,

or the gene for this toxin production can be genetically
-
engineered into a plant, so that
the plant produces its own toxin!

4.
Food Pro
duction Industry

Food
-
processing research currently focuses on growth and fermentation by yeas
t and bacteria. These methods
are well known technologies used in cheese and bread
-
making. Biotechnology applications include producing
fermentation starter cultures with specific taste, texture or other characteristics; creating plant tissue for the
produ
ction of plant
-
derived ingredients (starches for example); and improving waste management (such as oil
or other waste digesting bacteria).



Fermentation

-

Fermented foods use microbes to convert the original food into a fermented product by
the use specif
ic microbes. These microorganisms use the original product for growth and reproduction,
and in the process they excrete byproducts into the environment surrounding themselves and the food.
These byproducts plus the part of the original product that is not
consumed is the fermented food.
Fermented foods include meat, fish, fruits, and vegetables. They may be fermented separately or in
conjunction with each other to produce the desirable end product.

Fermentation involves the
introduction of the desirable mi
crobes into the original product. This may be accomplished by using
starter cultures or by using microbes from a previous fermentation. The environmental conditions
surrounding the microbes and the original product are brought to levels that will support a
nd enhance the
growth of the desirable microbes, while preventing or disrupting the growth of spoilage or pathogenic
microbes that may have been present on the original product. This prevention can be accomplished by
temperature, oxygen level, or by the by
products that are excreted from the desirable microbes
themselves. The original product and the microbes are then left in the favorable conditions for a period
of time so the fermentation can take place. The amount of time depends on the original raw mater
ial, the
type of microbes used in the fermentation process, and the level of fermentation that is desired.




o

Lactocooccus lactis

-

used in dairy fermentation

o

Steptococcus thermophilus

-

used in dairy fermentation

o

Leuconostoc

sp.
-

used in wine making, dai
ry fermentation

o

Pediococcus

sp.
-

meat fermentation, vegetable fermentation, ripening of some cheeses

o

Lactobacillus

sp.
-

meat fermentation, vegetable fermentation, dairy fermentation, sourdough
bread

o

Bifidobacterium

sp.
-

added to dairy products to pro
mote intestinal health.

o

Propiopnibacterium

sp.
-

Swiss cheese

o

Yeasts
-

bread, beer, wine, liquors

o

Molds
-

ripening cheeses, soy sauce

o

Lactobacillus delbruekii
,
an
d
Streptococcus thermophilus

-

making of yogurt





5

5.
Environmental
Cleanup and Energy
A
pplications

Renewable sources of energy have been a national priority since the mid
-
1970s. Environmental cleanup of large
toxic spills and of military bases remains a challenge. Biotechnologists are researching ways of addressing these
needs.



Energy

-

Bio
technology may offer efficient ways to produce renewable energy by using
microorganisms, modified plants, plant material, municipal and animal wastes, and other sources to
produce different types of fuels and gases. Research is underway exploring the use o
f organisms to
enhance the recovery of fossil fuels, to improve coal desulfurization, and to convert coal to gasoline.
Scientists are also looking into producing fermented fuel from biomass.
Commercialization of this
technology will depend on energy compa
nies and others investing in the necessary machinery to use the
products. Relatively little research and development has been done in the U.S. on the environmental
impacts of these crops.



Bioremediation

-

"Bioremediation" involves the use of microorganism
s to degrade various types of
environmental pollution, such as waste oil and heavy metals, to produce environmentally safe
byproducts. This method was used to clean oil spills in the Gulf of Mexico and Prince William Sound,
and might be used to decontamina
te military bases or to remove heavy metals from soil. It might also be
used to clean up nuclear waste. Approval was recently given to release genetically engineered bacteria to
"feast on pollutants in Oak Ridge National Laboratory soil."

We are also look
ing at using bacteria to
degrade wastes in landfills and in sewage systems.




Biomining



Because traditional ways of extracting minerals, such as gold and copper, from ores that
are mined are very harsh on the environment (extracted by extreme heat or toxi
c chemicals), scientists
are looking into using microbes to extract the ores. For example,
Thiobacillus ferooxidans

naturally
oxidizes copper sulfide for it’s energy. As they “chew up” the ore, copper is released in a natural
process!



Microbes: The Bu
ilding Blocks for Biotechnology



In summary, the field of biotechnology would not be possible without our friends the microbes. When
deciding which microbe to use for a particular application, we first must consider the unique properties of that
micr
obe. For example, yeast ferment sugars, so they lend themselves nicely to the fermentation industry.
Bacteria have plasmids that can be manipulated and exchanged easily, so they are useful in creating genetically
-
modified organisms.



II.
Making Recom
binant DNA


Overview of the Process

How does recombinant DNA technology work? The organism under study, which will be used to donate DNA
for the analysis, is called the
donor organism.

The basic procedure is to extract and cut up DNA from a donor
genome in
to fragments containing from one to several genes and allow these fragments to insert themselves
individually into opened
-
up small autonomously replicating DNA molecules such as bacterial plasmids. These
small circular molecules act as carriers, or
vectors
,

for the DNA fragments. The vector molecules with their
inserts are called
recombinant DNA

because they consist of novel combinations of DNA from the donor

6

genome (w
hich can be from any organism) with vector DNA from a completely different source (generally a
bacterial plasmid or a virus). The recombinant DNA mixture is then used to transform bacterial cells, and it is
common for single recombinant vector molecules to

find their way into individual bacterial cells. Bacterial cells
are plated and allowed to grow into colonies. An individual transformed cell with a single recombinant vector
will divide into a colony with millions of cells, all carrying the same recombina
nt vector. Therefore an
individual colony contains a very large population of identical DNA inserts, and this population is called a
DNA
clone
.
A great deal of the a
nalysis of the cloned DNA fragment can be performed at the stage when it is in the
bacterial host. Later, however, it is often desirable to reintroduce the cloned DNA back into cells of the original
donor organism to carry out specific manipulations of gen
ome structure and function. Hence the protocol is
often as follows:


Because

the donor DNA was cut into many different fragments, most colonies will car
ry a different
recombinant DNA (that is, a different cloned insert). Therefore, the next step is to find a way to select the clone
with the insert containing the specific gene in which we are interested. When this clone has been obtained, the
DNA is isolat
ed in bulk and the cloned gene of interest can be subjected to a variety of analyses, which we shall
consider later in the chapter. Notice that the cloning method works because individual recombinant DNA
molecules enter individual bacterial host cells, and

then these cells do the job of amplifying the single molecules
into large populations of molecules that can be treated as chemical reagents.

The term
recombinant DNA

must be distinguished from the natural DNA recombinants that result from
crossing
-
over b
etween homologous chromosomes in both eukaryotes and prokaryotes. Recombinant DNA in
the sense being used in this chapter is an unnatural union of DNAs from different organisms. Some geneticists
prefer the alternative name
chimeric DNA
,
after the mythological Greek monster Chimera. Through the ages,
the Chimera has stood as the symbol of an impossible biological union, a combination of parts of different
animals. Likew
ise, recombinant DNA is a DNA chimera and would be impossible without the experimental
manipulation that we call
recombinant DNA technology.

Step 1:
Isolating DNA

The first step in making recombinant DNA is to isolate donor and vector DNA.
Donor DNA

is th
e DNA you
are going to insert into the bacterial plasmid.
The procedure used for obtaining
vector DNA

(the DNA from the
bacterium that you are inserting the desired DNA into)
depends on the nature of the vector. Bacterial plasmids
are commonly used vector
s, and these plasmids must be purified away from the bacterial genomic DNA.


Step 2:
Cutting DNA

The breakthrough that made recombinant DNA technology possible was the discovery and characterization of
restriction enzymes.

Restriction enzymes are produce
d by bacteria as a defense mechanism against phages.
The enzymes act like scissors, cutting up the DNA of the phage and thereby inactivating it. Importantly,

7

restriction enzymes do not cut randomly; rather, they cut at specific DNA target sequences, which
is one of the
key features that make them suitable for DNA manipulation.

Let's look at an example: the restriction enzyme
Eco
RI (from
E. coli
) recognizes the following six
-
nucleotide
-
pair sequence in the DNA of any organism:


The enzyme
Eco
RI cuts within this sequence but in a pair of staggered cuts between the G and the A
nucleotides.


This staggered cut leaves a pair of identical single
-
stranded “sticky ends.” The ends are called
sticky

because
they can hydrogen bond (stick) to a complementary sequence. Production of these sticky ends is another feat
ure
of restriction enzymes that makes them suitable for recombinant DNA technology. The principle is simply that,
if two different DNA molecules are cut with the same restriction enzyme, both will produce fragments with the
same complementary sticky ends,
making it possible for DNA chimeras to form. Hence, if both vector DNA
and donor DNA are cut with
Eco
RI, the sticky ends of the vector can bond to the sticky ends of a donor
fragment when the two are mixed.

Dozens of restriction enzymes with different seq
uence specificities have
now been identified
.

Joining DNA

Most commonly, both donor DNA and vector DNA are digested with the use of a restriction enzyme that
produces sticky ends and then mixed in a test tube to allow the sticky ends of vector and donor D
NA to bind to
each other and form recombinant molecules. When the two populations are mixed, DNA fragments from the
two sources can unite, because double helices form between their sticky ends. However, the backbones can be
sealed by the addition of the en
zyme
DNA ligase
.

Amplifying recombinant DNA

The
resulting

recombinant DNA enters a bacterial cell by transformation. After it is in the host cell, the plasmid
vector is able to replicate because plasmids normally have a replication origin. However, now tha
t the donor
DNA insert is part of the vector's length, the donor DNA is automatically replicated along with the vector. Each
recombinant plasmid that enters a cell will form multiple copies of itself in that cell. Subsequently, many cycles
of cell division

will take place, and the recombinant vectors will undergo more rounds of replication. The
resulting colony of bacteria will contain billions of copies of the single donor DNA insert. This set of amplified
copies of the single donor DNA fragment is the DNA

clone
.