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BIOTECHNOLOGY: PRINCIPLES AND PROCESSES

Q.1 Define biotechnology (as per European federation of


Biotechnology…EFB).
Ans. The integration of natural science and organisms, cells and
molecular analogues for products & services.

Q2. List out the core techniques that enabled the birth of
Biotechnology.
Ans. 1. Genetic engineering: These techniques aim at changing the
structure of genetic material(DNA/RNA) and introducing it
into a specific host thus bringing about change in the
phenotype of the host organism.
2. Microbe- free conditions for the production of antibiotics,
vaccines and enzymes etc (in this the growth of only desired
microbe is favoured ).

Q3. How is sexual reproduction advantageous over asexual


reproduction? What are its limitations? How can we overcome
that?
Ans. Sexual reproduction leads to re-combination and variations.
Some of them may be very beneficial to the organism. But the
limitation is that it may also lead to accumulation of certain
undesirable genes and elimination of desirable genes. Genetic
engineering can help us to overcome this problem. (this
includes recombinant DNA, gene cloning and gene transfer)
Q4. What are the 3 basic steps in making a GMO?
Ans. 1. Identification of DNA with desirable genes.
2. Introduction of this desired DNA into a specific host.
3. Maintenance of this desired DNA in the host and transfer
to its progeny.
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Q5 Write short notes on various tools used in Recombinant DNA
technology. Explain each tool in detail.
Ans The tools used in Bio-tech are- Restriction enzymes, cloning
vectors, competent host.
1) RESTRICTION ENZYMES:
1. These are molecular scissors which can cut DNA at a specific site
2. Each such enzymes is very specific about the sequence in DNA,
Eg: Hind II can cut DNA at a specific sequence of 6 basic pairs.
3. These restriction enzymes which are also called as NUCLEASES
are of two types. 1. exonucleases and 2. Endonuclease. (
exonucleases can cut the nucleotides from the end while
endonucleases can cut even the middle nucleotides with specific
sequence)
4. Each restriction enzyme can cut DNA at a palindromic site.
Eg: GAATTC. After the two ends are cut the over hanging ends
are called the sticky ends, which form hydrogen bonds with their
complimentary cut counterparts. DNA ligase catalyses the
joining of such two sticky ends.
5. After the DNA is cut into small fragments with the help of
endonucleases, the fragments are now separated by a process
called GEL ELECTROPHORESIS.
6. During this process the –vely charged DNA will move towards
anode through a gel medium.( agarose gel is commonly used)
Finally the fragments of DNA are separated depending on their
size. (Smaller fragments move to the farthest.)
7. Separated fragments must be stained to be visualized. Ethidium
bromide dye is used for this purpose and then they are exposed
to UV radiation. Now the bright orange colored bands of DNA
are visible.
8. These DNA fragments are now cut from agarose gel and then
extracted. This step is called ELUTION.
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2) CLONING VECTORS:
1. Plasmids (extra chromosomal DNA of bacteria) and
bacteriophages (viruses) are commonly used vectors since they
have the ability to multiply fast and produce very high copy
number.
2. If a foreign DNA (cut from an organism, with desirable trait) is
linked to these plasmid, it multiples along with it and soon
several copies of that foreign DNA are made. This new DNA is
now called as recombinant DNA. (rDNA)
3. COMPETENT HOST:
To make the host accept the rDNA is called making it
"competent". The problem in this process is that DNA is
Hydrophilic and cannot pass through the cell membrane which
allows fat soluble substances only. To overcome this problem, the
following practices are followed:
A) Bacteria are treated with divalent cations such as Ca2+ , which
enables the rDNA to pass through the membrane followed by
heat shock method.
By incubating the cells with rDNA on ice and then exposing to
heat shock (420) and putting them back on ice. This treatment
helps bacteria pick up the r DNA
B) Through micro injection method host can be made competent.
In this directly the r DNA is injected into the nucleus of animal
cell.
C) In another method using biolistics or gene gun the DNA is
coated on gold or tungsten micro-pellets with high velocity and
the plant cells are bombarded with these pellets.
D) Using disarmed pathogen: these are the pathogens which while
infecting the cells, transfer desirable rDNA into the host cell.
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Q6. What features facilitate the cloning process into a vector?
Ans. I) ORIGIN OF REPLICATION(ori): The piece of DNA has a
specific region from where the replication starts, this is
called ori. This piece also controls the number of copy
numbers of DNA produced. If the origin supports high copy
number, such vector is more preferred to recover more
copies from target DNA.
II) SELECTABLE MARKER: The vector we selected should be
able to identify a transformant from a non-transformant. It
should eliminate the non-transformant. Selectable markers
are those with desirable qualities like antibiotic
resistance(resistance to ampicillin, chloromphenicol etc)
which are not found with normal strains of E.coli.
Transformation is a procedure through which a piece of DNA
is introduced in a host bactrial.
III) CLONING SITES: The vector should have only a single
recognition site which makes linking of DNA easy. Eg. pBR
322 is the suitable cloning vector of E.coli in which Bam HI
site of Tetracycline resistance allows ligation of foreign
DNA. The recombinant gene plasmids lose tetracycline
resistance due to insertion of foreign DNA but can still be
selected out from non-recombinants ones by plating the
transformants on ampicillin medium. The recombinant
grow on Ampicillin but not on tetracycline . Non
recombinants will grow on both the media(ampicillin and
tetracycline) This is an example of insertional inactivation
where insertion of a foreign gene inactivated the
tetracycline resistance gene. As the above procedure is a
cumbersome one, a visible process can be undertaken to
identify non-transformants from the transformants. In this
a coloured complex enables us to identify the transformants
and non-transformants.
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Use of chromogenic substrate to identify transformants and non
transformants in insertional inactivation: As rDNA is inserted within
the coding sequence of an enzyme α galactosidase. This will
inactive the enzyme. The colonies with this inserted DNA can not
produce any colored compound while those without any insertion
will produce blue colored colonies. Hence the recombinants can
easily be identified from those without the r DNA.

IV) VECTOR FOR CLONING GENES IN PLANTS AND ANIMALS:


1. Agrobactrium tumifaciens is a pathogenic bacterium which
has Ti-DNA in its plasmid that causes tumors in many dicot
plants. These tumor cell produce useful substances for
growth of bacteria.
2. Some retroviruses also have this ability to transform normal
cell into cancerous cell.
3. Now scientists are using these vectors to transfer desirable
genes into specific host. The Ti Plasmid is now used as a
suitable vector which does not produce disease in plants but
delivers genes of our interest in plants.
4. Similarly retroviruses have also been disarmed and used to
deliver desirable genes into animal cells.
5. Thus a foreign gene is ligated to these suitable vectors which
faithfully transfer these genes to the host cells where the
genes multiplies to produce several copies.

Q7. List out various steps in r DNA technology in sequential order.


Ans. Isolation of DNA ---------- Fragmentation of DNA by using
restriction enzymes ---------- Ligation of DNA fragment into a
suitable vector -----------Transferring the rDNA into a host ------
-------- Culturing the host cell in a medium at large scale to
extract the product of our interest.
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Q8. Explain in detail various steps in r DNA technology in sequential
order.
Ans 1) ISOLATION OF DNA/GENETIC MATERIAL:
a) DNA is the universal genetic material with a few exceptional
viruses.
b) DNA is enclosed within a membrane and has RNA, proteins,
polysaccharides and lipids along with it. To obtain pure DNA
requires elimination of all these substances through
hydrolyses.
c) Bacterial cells are treated with lysozyme for degrading their
cell- walls, plant cells to be treated with cellulose and fungal
cells with chitinase.
d) RNA can be removed by treating with RNAase treatment and
proteins can be removed by using proteases. Now chilled
ethanol is added to obtain purified DNA which precipitates
out. It can be separated by a process called SPOOLING.

2. CUTTING OF DNA AT SPECIFIC LOCATION:


A) This cutting can be done by INCUBATING purified DNA with
RESTRICTION ENZYMES. Agarose gel electrophoresis is done
to check the digestion process.
B) DNA, being negatively charged is attracted towards anode.
This process is repeated with vector DNA also. Now ligation of
foreign DNA with the plasmid DNA is done with the help of
enzyme ligase.

3. AMPLIFICATION OF GENE OF INTEREST BY PCR:


A) Its a polymerase chain reaction to amplify DNA/RNA.
B) By using heat stable/ thermo stable DNA polymerase
obtained from Thermus aquaticus bacterium, the double
strand is denatured and the two strands of DNA are
separated.
C) Using a primer (which is a short stretch of oligonucleotide)
and DNA polymerase enzyme, complementary DNA strands can
be made. Thus repeated process results in the synthesis of several
copies of the original DNA.(from 1 segment 1 billion copies can be
made).
D) The fragments of DNA may be ligated to a vector for further cloning if
necessary.
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4. INSERTION OF r DNA INTO THE HOST CELL/ ORGANISM:


A) First step is to make the host cell competent to receive the rDNA
B) The ligated DNA is now introduced into the host cell.Eg: the DNA
containing Ampicillin resistant factor may be introduced into the
host cell. To check its functional validity we spread these
transformed cells on agar plates containing Ampicillin and see
whether they could grow. Selectable markers (transformants)
will be selected from non transformants.
5. OBTAINING THE FOREIGN GENE PRODUCT:
A) The rDNA when introduced into a suitable host cell (may be a
bacterium), multiples to produce several copies (every time the
bacterium multiples, a new copy of the transformed gene is
produced ).
B) Now the protein of our interest can be produced on large scale .
This protein is then extracted , purified and marketed. This
procedure requires frequent change of culture medium .
C) As the protein production cannot be done on large scale using
volume cultures we can overcome this problem by using
"BIOREACTORS". If any protein encoding gene is expressed in a
heterologous host it is called as recombinant protein.

Q9. Explain the term bioreactors. What are its various types? How
do they function?
Ans. 1) It is a huge vessel where raw materials are biologically
converted into specific products. (eg: enzymes can be made
from plant/animal cells at a large scale).
2) A bioreactor provides optimal conditions for achieving the
desired product. Factors like temp, pH etc are controlled.
Substrate, salts, vitamins and oxygen are provided for
achieving the desired product.
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STRUCTURE AND FUNCTION OF A BIOREACTOR:


1. It is a cylindrical tank with a curved base where the reactors
content are mixed.
2. The stirrer also provides oxygen supply throughout.
3. The main systems of bioreactor are agitator system, oxygen
delivery system,foam control system, temp. control system, pH
control system, sampling ports (so that small volume of the
culture can be drawn periodically)

BIOREACTORS ARE OF TWO TYPES:


Simple and sparged stirred tank bioreactors. The advantage with
sparged stirred reactor is that the sterile air bubbles that are
sparged increase the oxygen transfer area by increasing the
surface area.

DOWNSTREAM PROCESSING:
The product obtained from the bioreactor is to be tested before it
is marketed as a "finished product".For this, the product should
be separated and purified. This process is called
"DOWNSTREAMING PROCESS". Suitable preservatives will be
added (called formulation). Then the product is subjected to
clinical trials. Strict quality control testing for each product is
required before marketing the product.

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