BCC. XII. BIOTECHNOLOGY PRINCIPLES.

Biotechnology: deals with the techniques of using live organisms or enzymes from organisms to produce
products and processes useful to humans.
Definition of biotechnology according to European Federation Of Biotechnology (EFB) – “the integration of
natural science and organisms, cells and their parts” thereof and molecular analogues for products and
services.
Bioinformatics: management of biological information using computer & statistical techniques
Genetic engineering: The simple addition, deletion, or manipulation of a CHARACTER in an organism to create
a desired change.
Recombinant DNA: the hybrid DNA formed of fusion of DNA fragments of two individuals belonging to same
or different species
Gene Therapy: Modifying DNA of an organism to repair defects or create desired changes
Transformation: natural or artificial genetic alteration of a cell.
Cloning: The gene introduced in a host replicates and multiplies itself in the recipient cell. This process is
known as cloning.
PALINDROME SEQUENCE: the palindrome in DNA is a sequence of base pairs that reads same on the two
strands when orientation of reading is kept the same.

Q. biotechnology - Two core techniques.

The two core techniques that enabled birth of modern biotechnology are:
➢ Genetic engineering (G.E): Techniques to alter the chemistry of genetic material (DNA and RNA), to
introduce these into host organisms and thus change the phenotype of the host organism

➢ Maintenance of sterile (microbial contamination-free) ambience in chemical engineering processes to

enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture of
biotechnological products like antibiotics, vaccines, enzymes, etc.

Q. NUCLEASES:

➢ A restriction enzyme (or restriction endonucleases) recognizes a specific base pair sequence in DNA
called a restriction site and cleaves the phosphodiester backbones of DNA within the sequence.
➢ Restriction enzymes are widely found in prokaryotes and provide protection to the host cell by
destroying foreign DNA that makes entry to it. It acts as a part of defense mechanism.
➢ Restriction enzymes belong to a larger class of enzymes called nucleases.
➢ They are of two types:
➢ Exonucleases remove nucleotides from the ends of the DNA
➢ endonucleases make cuts at specific positions within the DNA.

Q. restriction enzymes AS A TOOL-

➢ RESTRICTION ENDONUCLEASES Acts as molecular scissors/chemical scalpels.
➢ They recognize short sequences of DNA as targets for cleavage.
 ➢ Different enzymes recognize different specific sequences each ranging from 4-8 base pairs. This specific
base sequence is known as – PALINDROMIC RECOGNITION SEQUENCE.
➢ The palindrome in DNA is a sequence of base pairs that reads same on the two strands when
orientation of reading is kept the same. A palindromic recognition site reads the same on the reverse
strand as it does on the forward strand.
5' —— GAATTC —— 3'
3' —— CTTAAG —— 5'
➢ Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but
between the same two bases on the opposite strands. This leaves single stranded portions at the ends.
➢ There are overhanging stretches called sticky ends on each strand. These are named so because they
form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates
the action of the enzyme DNA ligase.
➢ 900 R.E’s have been isolated from over 230 strains of bacteria. Named by a 3 or 4 letter abbreviation
that identifies their origin.
➢ EcoRI – E. coli organism. R – strain. Roman no. indicates the order in which the enzymes were isolated
from that strain of bacteria.

Q. What would happen if the restriction enzymes do not cut the DNA at specific recognition sequences?
If the restriction enzymes do not cut the DNA at the specific sites, the DNA fragment obtained will have no
sticky ends, and hence, the construction of recombinant DNA would be difficult.

Q. What do “Eco”, “R” and “I” refer to in the enzyme EcoRI?

“Eco” refers to the species from which it is taken, “R” refers to the particular strain, and “I” states that it was
the first enzyme isolated from this strain.
Q. ELECTROPHORESIS / SEPARATION AND ISOLATION OF DNA FRAGMENTS: The cutting of DNA by restriction
endonucleases results in the fragments of DNA. These fragments can be separated by a technique known as
gel electrophoresis.
➢ AGAROSE GEL - DNA fragments are –vely charged & are separated by forcing them to move towards
the anode under an electric field through an Agarose gel. Agarose natural polymer from sea weeds.
➢ The DNA fragments separate (resolve) according to their size through sieving effect provided by the
agarose gel. The smaller the fragment size, the farther it moves.
➢ STAINING - The separated DNA fragments can be visualized only after staining the DNA with ethidium
bromide followed by exposure to UV radiation. Bright orange colored bands of DNA APPEAR.
➢ Elution - The separated bands of DNA are cut out from the agarose gel & extracted from the gel piece.
➢ The DNA fragments purified in this way are used in constructing recombinant DNA by joining them
with cloning vectors.

Q. A mixture of the fragmented DNA was run on an agarose gel. The gel was stained with ethidium bromide
but no bands were observed. What would be the cause?
This may be due to the following reasons:
➢ The DNA must have degraded by nucleases.
➢ The electrodes are placed in the opposite direction in the gel assembly. Consequently, the DNA
molecules move out of the gel.
Maybe ethidium bromide was not added sufficiently and hence DNA was not visible

Q. TOOL II. Cloning vectors:

Vectors are DNA molecules which carry a foreign DNA fragment to be cloned. Plasmids & Bacteriophages e
Common vectors. They can replicate in the bacterial cells independent of the control of bacterial chromosomal
DNA.
Features required for cloning vectors: A cloning vector is a carrier DNA molecule in which a desired DNA
fragment can be integrated in such a way that the carrier molecule does not lose its capacity for self-
replication.
➢ COPY NUMBER - Bacteriophages (virus) have very high copy numbers of their genome within the
bacterial cells. Plasmids have either low copy number (1-4/cell) or a high copy number (10-100/cell).
Once a selected DNA piece is linked to either bacteriophage or Plasmid DNA, we can multiply its
number to the copy number of bacteriophages/plasmids.
➢ ORIGIN OF REPLICATION: a particular sequence in a genome at which replication is initiated. Any
foreign DNA linked to this sequence can be made to replicate within the host cell. This a particular
sequence is also responsible for controlling the copy number of the linked DNA. In order to recover
 many copies of the desired DNA, it should be cloned in a vector whose origin support high copy
number.
➢ Selectable marker: Selective markers are antibiotic resistant genes which help in identifying and
eliminating non-transformants and selectively permitting the growth of transformants. Genes encoding
resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline, kanamycin are used as
markers for E.coli. Normal E.coli cells do not carry resistance against any of these antibiotics.
➢ Cloning sites:
• vector should have very few, preferably one site for the commonly used restriction enzymes.
• The plasmid pBR322 has origin of replication and two antibiotic resistance genes – ampR & tetR.
• ampR gene has Pst I recognition site while tetR gene has recognition sites for Hind III, Sal I.
• Same restriction endonuclease (e.g Bam H I) is used to cut both the foreign DNA as well as the plasmid.
• Foreign DNA and the Plasmid are sealed together with ligase.

➢ Screening / Insertional Inactivation: Selectable markers help in identifying and eliminating non-
transformants & selectively permitting the growth of transformants. E.g. antibiotic resistance gene, β-
galactosidase gene, gene expressing Green Fluorescent Protein (GFP)

❖ antibiotic resistance genes as selective markers in cloning vectors:

o Bam H I cut the tetracycline resistance gene, as a result the tetR gene gets inactivated. Now it
becomes easy to select recombinants (transformants) from non-recombinants by plating them
on ampicillin medium and then tetracycline medium. The recombinants will grow in ampicillin
medium but not in medium containing tetracycline.
❖ β-galactosidase genes as selective markers in cloning vectors:
o Selection of recombinants due to inactivation of antibiotic gene is a complex procedure as it
requires simultaneous plating on two plates having different antibiotics. Therefore, alternative
selectable markers have been developed on the basis of their ability to produce colour in the
presence of a chromogenic substrate.
o In this, a recombinant DNA is inserted within the coding sequence of β-galactosidase. This
results into inactivation of the enzyme, which is referred to as insertional inactivation.
o The presence of a chromogenic substrate gives blue coloured colonies if the plasmid in the
bacteria does not have an insert.
o Presence of insert results into INSERTIONAL INACTIVATION of the β-galactosidase gene and
the colonies do not produce any colour,
o Such colonies are identified as recombinant colonies.
 ➢ Vectors for plants and animals:

• FOR PLANTS - “Ti plasmid” of Agrobacterium tumifaciens which is pathogenic to most of the dicot
plants. It naturally transfers T DNA to plants and is responsible for the production of tumor. This has
now been modified into a cloning vector which is no more pathogenic to the plants. It is used to
deliver genes of our interest into a variety of plants.
• FOR ANIMALS AND HUMANS – Disarmed Retrovirus are used to deliver desirable genes.

Q. Can you think and answer how a reporter enzyme can be used to monitor transformation of host cells by
foreign DNA in addition to a selectable marker?
A reporter enzyme can be used to differentiate transformed cells by tracking down the activity of its co-
responding genes (receptor gene). For e.g., (3-galactosidase (Lac Z) activity is not found in transformed cells so
that they appear white in colour. The others, which appear blue in colour, indicate that cells do not carry
foreign DNA.
Q. What would happen if a plasmid without a selectable marker was chosen as a cloning vector?
A selectable marker helps to distinguish transformed cells from the non-transformed ones. If the cloning
vector does not have a selectable, marker, it would be difficult to select the transformants.

Q. TOOL III. Competent host. DNA vector being hydrophilic & a very large molecule, cannot get across cell
membrane. To force a bacterial to take up the rDNA, the bacterial cell must be made competent to take up
DNA. This is done by
➢ Microinjection – direct injection of rDNA into eukaryotic host without the help of vectors
➢ Particle bombardments/ Gene Gun Or Biolistics – Microscopic particles of gold or tungsten are
coated with the DNA of interest and bombarded onto host cells. Vector not required.
➢ Heat shock method: cold CaCl solution + rDNA + Host mix is incubated on ice followed by treatment at
42⁰C & repeat this cycle.
➢ Use of Disarmed Pathogens Vectors (retrovirus), which when allowed to infect the cell, transfer the
rDNA into the host

Q. What does ‘competent’ refer to incompetent cells used in transformation experiments?

“Competent” means the ability of a cell to intake foreign DNA. Alteration in the cell walls allow the foreign
DNA to incorporate into the host cell.

Q. What do you understand by gene cloning?

Gene cloning or DNA cloning is the process in which the gene of interest is copied out of the DNA extracted
from an organism. Steps involved:
Isolation of DNA fragment or gene
Selection of the appropriate vector
The isolated DNA fragment is incorporated into the vector
The recombinant vector is transferred in the host cell
The recombinant host cell is cloned.
Q. process of rDNA technology:

Step 1: Isolation of the genetic material (DNA):

➢ When the bacteria/plant/animal tissues are treated with lysozyme (bacteria), cellulase (plant cells),
chitinase (fungus), DNA is released.
➢ The RNA can be removed by treatment with ribonuclease whereas proteins can be removed by
treatment with protease.
➢ Other molecules can be removed by appropriate treatments
➢ purified DNA ultimately precipitates out after the addition of chilled ethanol. This can be seen as
collection of fine threads in the suspension.

Step 2: Cutting of DNA at Specific Locations:

➢ Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction
enzyme, at the optimal conditions for that specific enzyme.
➢ Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion.
➢ DNA is a negatively charged molecule, hence it moves towards the positive electrode (anode).
➢ The process is repeated with the vector DNA also.
➢ After having cut the source DNA as well as the vector DNA with a specific restriction enzyme, the cut
out ‘gene of interest’ from the source DNA and the cut vector with space are mixed and ligase is added.
➢ This results in the preparation of recombinant DNA.

Step 3: Amplification of gene of interest using PCR- PCR produces identical copies of short DNA sequence in
three stages:-
A. Denaturation - heat at 90ᴼ - 95ᴼC to break hydrogen bond holding the two strands of DNA.
B. Annelaing (hybridizing) – oligonucleotide primers complementary to the either end of the target sequence
are aligned at 50-65ᴼC.
C. Extension – DNA polymerase obtained from Thermus aquaticus is added to synthesize complementary
strands in 5’-3’ direction.
Step 4: Introduction of rDNA into the host cell/organism
➢ Insertion of Recombinant DNA into the Host Cell/Organism There are several methods of introducing
the ligated DNA into recipient cells.
➢ Recipient cells after making them ‘competent’ to receive, take up DNA present in its surrounding.
➢ So, if a recombinant DNA bearing gene for resistance to an antibiotic (e.g., ampicillin) is transferred
into E. coli cells, the host cells become transformed into ampicillin-resistant cells.
➢ If we spread the transformed cells on agar plates containing ampicillin, only transformants will grow,
untransformed recipient cells will die.
➢ due to ampicillin resistance gene, one is able to select a transformed cell in the presence of ampicillin.
➢ The ampicillin resistance gene in this case is called a selectable marker.

Step 5: obtaining foreign gene product - BIOREACTORS

➢ If any protein encoding gene is expressed in a heterologous host, is called a recombinant protein.
➢ After having cloned the gene of interest and having optimized the conditions to induce the expression
of the target protein, one has to consider producing it on a large scale.
1. The cells with cloned genes of interest may be grown on a small scale in the laboratory. The cultures
may be used for extracting the desired protein and then purifying it by using different separation
techniques.

2.The cells can also be multiplied in a continuous culture system wherein the used medium is drained out
from one side while fresh medium is added from the other to maintain the cells in their physiologically
most active log/exponential phase. This type of culturing method produces a larger biomass leading to
higher yields of desired protein.
 3.BIOREACTORS: Small volume cultures cannot yield appreciable quantities of products. To produce in
large quantities, bioreactors having capacity 100-1000 litres of culture are required.
➢ bioreactors are the vessels in which raw materials are biologically converted into specific
products, individual enzymes, etc., using microbial plant, animal or human cells.
➢ It provides the optimal conditions for achieving the desired product by providing optimum
growth conditions (temperature, pH, substrate, salts, vitamins, oxygen)
➢ Two types of bioreactors are – stirred tank reactor and sparged stirred tank reactor
➢ STIRRED-TANK REACTOR - is usually cylindrical or with a curved base to facilitate the mixing of
the reactor contents. The stirrer facilitates even mixing and oxygen availability throughout the
bioreactor.
➢ TYPICAL BIOREACTOR HAS –
o an agitator system,
o an 02 delivery system
o a foam control system,
o a temperature control system,
o pH control system
o Sampling ports – for the periodical collection of small volumes of the culture.

Step 6: downstream processing:

➢ After completion of the biosynthetic stage, the product has to be subjected through a series of
processes before it is ready for marketing as a finished product.
➢ The processes include separation and purification together known as downstream processing.
➢ The product has to be formulated with suitable preservatives.
➢ Such formulation has to undergo thorough clinical trials as in case of drugs.
➢ Strict quality control testing for each product is also required.
➢ The downstream processing and quality control testing vary from product to product.
Q. Besides better aeration and mixing properties, what other advantages do stirred tank bioreactors have
over shake flasks?
Shake flasks are used for growing and mixing the desired materials on a small scale in the laboratory. A large
scale production of desired biotechnological product is done by using ‘bioreactors’. Besides better aeration
and mixing properties, the bioreactors have following advantages
(i) Small volumes of cultures are periodically withdrawn from die reactor for sampling.
(ii) It has a foam control system, pH control system and temperature control.

Q. What would happen when one grows a recombinant bacterium in a bioreactor but forget to add
antibiotic to the medium in which the recombinant is growing?
In the absence of an antibiotic mixture of both transformants and non-transformants will be produced. It
would result in the production of poor quality yield.

Q. How is the copy number of plasmid vector and yield of the recombinant protein related to each other?
The copy number of plasmid vector is directly related to the yield of recombinant protein. Higher the copy
number of vector plasmid, greater is the copy number of gene and consequently, the yield of the recombinant
protein is in higher amounts.

Q. Stages involved in biotechnology/rDNA technology:

• Generation of DNA fragments and selection of the desired piece of DNA
• Insertion of the selected DNA into a cloning vector i.e. plasmid, to create a recombinant DNA
• Introduction of the recombinant vectors into the host cells (bacteria)
• Multiplication and selection of clones containing the recombinant molecules
• Expression of the gene to produce the desired product.
Q. What would be the molar concentration of human DNA in a human cell? Consult your teacher.
The molar concentration of DNA in human cell is 2 mg/ml of cell extract.

Q. Why are proteases added while isolating the DNA?

Proteases degrade the proteins so that they do not interfere with the downstream DNA treatment.

Q. If the “denaturation” step is missed during PCR, what would be its effect on the entire process?
If the denaturation process is missed, there will no separation of DNA strands, the primers will not anneal to
the template strands and eventually, amplification of DNA will not occur.

Q. Can you recall meiosis and indicate at what stage a recombinant DNA is made?
Recombinant DNA is formed due to crossing over between non-sister chromatids of homologous
chromosome. It occurs during pachytene stage of prophase of meiosis I

Q. How is Ti plasmid of Agrobacterium tumefaciens modified to convert it into a cloning vector?

The Ti plasmid is a tumour-inducing plasmid. The genes responsible for its pathogenic nature are either
removed or altered so that it does not harm the plants and only delivers the gene of interest.

Q. Do biomolecules such as DNA, proteins exhibit biological activity in anhydrous conditions?

No, biomolecules do not exhibit biological activity in anhydrous conditions. That is why life does not sustain
without water.
Distinguish:

CHROMOSOMAL DNA PLASMID DNA

Large or circular Small and circular DNA.
Genomic DNA Extra chromosomal DNA
Genes are necessary for growth development and Not necessary for general functions. Genre regulate
reproduction special functions such as antibiotic resistance,
nitrogen fixation etc.
one copy of DNA is present One to many copies are present.
Found in both prokaryotes and eukaryotes prokaryotes

EXONUCLEASES ENDONUCLEASES
cuts nucleic acids from the middle cuts at terminal ends
releases single nucleotides oligonucleotides
forms sticky ends forms sticky or blunt ends
DNA POLYMERSE II BAMHI
Q. Collect 5 examples of palindromic DNA sequences by consulting your teacher. Better try to create a
palindromic sequence by following base-pair rules.
Palindrome nucleotide sequences in the DNA molecule are groups of bases that form the same sequence
when read both forward and backward. Five examples of palindromic DNA sequences are as follows:
(i) 5′-—————GGATCC——————3’
3′—————CCTAGG—————–5′
(ii) 5’—————AAGCTT——————3′
3′——————TTCGAA —————-5′
(iii) 5′—————–ACGCGT—————–3′
3′——————TGCGGA————– 5′
(iv) 5′———-ACTAGT————3′
3′——————TGATCA————5′
(iv) 5′—————AGGCCT—————3′
3′——————TCCGGA————–5′
Q. From what you have learnt, can you tell whether enzymes are bigger or DNA is bigger in molecular size?
How did you know?
Both DNA and enzymes are macromolecules. DNA is a polymer of deoxyribonucleotides and enzymes are
polymers of amino acids.
DNA is bigger in molecular size as compared to proteins because synthesis of proteins is regulated by a small
segment of DNA, called genes and also a large number of proteins can be synthesised by a DNA molecule.
Q. Can you list 10 recombinant proteins which are used in medical practice? Find out where they are used as
therapeutics (use the internet).
(i) Human insulin – Diabetes
(ii) ) Human growth hormone – Dwarfism cure
(iii) Blood clotting factor Y1H/IX-Haemophilia
(iv) TPA (tissue plasminogen activator) – Heart attack/strokes
(v) PDGF (platelet derived growth factor) – Stimulates wound healing.
(vi) Interferon – Treatment of viral infection.
(vii) Interlinking – Enhances immune reaction,
(viii) Hepatitis B vaccine – Prevention of infectious disease.
(ix) Herpes Vaccine – Prevention of infectious disease.
(x) DNase I – Treatment of cystic fibrosis.
Q. Name a recombinant vaccine that is currently being used in the vaccination program.
The hepatitis-B vaccine.