Biology investigatory project class 12

Genetic engineering

1. BIOLOGY INVESTIGATORY PROJECT

“Genetic Engineering-An Overview”


SUBMITTED BY
Name : Arajit Kumar Pati
Class : XII
BOARD ROLL NO. : 12642500

TERM 1(2021-2022)







DAV PUBLIC SCHOOL
CHANDRASEKHARPUR , BHUBANESWAR-21

2. DEPARTMENT OF BIOLOGY,

DAV PUBLIC SCHOOL,
CHANDRASEKHARPUR , BHUBANESWAR-21

Certificate

This is to certify that the investigatory project report entitled, “Genetic
Engineering – An Overview” submitted by Master Arajit Kumar Pati of
class XII, Roll. Number – 11, in partial fulfillment of All India Senior
School Certificate examination in Biology (Term 1) for the session 2021-
22 was carried out under our supervision and guidance.









Ms. Nibedita Pati Mr. Subha Dipta Jena
PGT in Biology PGT in biology




Examiner

3. ACKNOWLEDGEMENT

I would like to convey my sincere thanks to Dr. Keshaba Chandra
Satapathy, Principal, DAV Public School Chandrasekharpur,
Bhubaneswar for his kind co-operation, valuable suggestions and guidance
to carry out this work.

I am highly thankful to my biology teachers Mr. Subha Dipta Jena and
Ms. Nibedita Pati PGTs in Biology for their invaluable guidance, constant
support and timely suggestions which has helped me to complete this
project work in time.

I would also like to express my gratitude to my parents and friends for
their unstinted support and co-operation throughout this project work.

Last but not the least, I am immensely indebted to the almighty for his
blessings and grace without which I could not have undertaken the task
and my efforts would never have been a success.






Arajit Kumar Pati

4. CONTENT

Sl. No. Topic Page No.
1) INTRODUCTION 1
2) OBJECTIVE 1
3) TERMINOLOGIES AND
PROCEDURES
2-8
3.1) DNA Cloning 2-6
3.2) Screening and isolation of DNA 6
3.3) Insertion of gene to hosts 7
3.4) DNA sequencing 8
3.5) Retrieval of foreign gene products 8
4) RESULT, ANALYSIS AND
INTERPRETATION
8
5) DISCUSSION 9
6) CONCLUSION 10
7) REFERENCES 10

5. INTRODUCTION
What is genetic engineering ?
Genetic engineering refers to those areas of study which
employ technology to modify or manipulate the genetic
material of organisms for commercial, medical and
research purposes.

It is an evolving area which focuses on exclusive
genetic research, proper diagnosis and treatment of
diseases. It employs recombinant DNA technology to
engineer radically different genetic material with desired
efficacy. Recombinant DNA is imperative to our understanding of the structure, function
and regulation of genes to produce desired outcomes.

OBJECTIVE
The project aims to develop an understanding of recombinant DNA technology and its
implication in the field of genetic engineering. It also focuses on the increasing
contribution of genetic engineering in other fields such as medicine, gene therapy,
vaccination and immunization programs, disease control, etc. Thus, this project can help
us appreciate that :-

➢ Genetic engineering has transformed our lives by bringing in better medication and
care.
➢ It has developed our understanding about the elementary molecules that inherit life
from one generation to the next.

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6. TERMINOLOGIES AND PROCEDURES
Some commonly used terminologies and procedures in recombinant DNA technology are
as follows:-

DNA cloning :
It refers to the production of identical copies of a single fragment of DNA, such as a gene.
In practice, this procedure is carried out by inserting a DNA fragment into a small DNA
molecule and then allowing this molecule to replicate inside simple living cell such as a
bacterium.

Restriction digestion :
DNA extracted from donor organism is cut into small fragments for cloning. Special
molecules called restriction enzymes(e.g., EcoR1 & BamH1) are able to cleave the DNA
at specific target sequences (palindromic nucleotide sequences). Hence the desirable
fragment (gene), with a promoter, can be obtained by a suitable restriction enzyme whose
target sequences flank the gene. Some aspects are discussed here under :

Overhanging / Sticky ends :-
Restriction enzymes are peculiar in making
staggered cuts i.e., they leave single
stranded overhangs at the site of cleavage.
Often, these overhangs are called “sticky
ends”, as they can pair with other
overhangs having complementary
sequences. This complimentarity is
achieved when the same restriction
enzyme is used for both the plasmid and
the donor DNA.

Vector :
Obligatory to the process of DNA cloning are small independently replicating molecules
called DNA vectors (carriers). They stage the foreign DNA and multiply its counts through
replication. Since vectors have an unique ORI, the need of vectors for replication is very
pronounced. Some commonly used vectors along with their properties have been
tabulated.



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7. Vectors Properties Diagrams
Plasmids • Small circular DNA
molecules present in
bacterial cytoplasm
• Replicable
• Have specific sequences
called polylinkers to
recognize restriction
enzymes. Foreign DNA
is spliced with the cut
open plasmids


Viral phages
(e.g.,
bacteriophage)
• Lambda phage is known
to infect bacteria (E.Coli)
• Central part of the
lambda genome is
suitable for restriction
endo nuclease activity


BACs
(Bacterial
Artificial
Chromosomes)
• Vectors based on F-
factor(fertility factor)
plasmids of E.Coli
• Carry large amounts of
DNA


YACs (Yeast
Artificial
Chromosomes)
• Vectors based on
autonomously replicating
plasmids of baker’s yeast.
Segregate properly like
any yeast chromosome
• Carry the largest inserts
of all


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8. Recombinant DNA :
The combination of the plasmid and
the foreign DNA is called recombinant
DNA. It is recombinant in the sense
that DNA from two different species
are integrated to form a single whole. It
is the artifact of genetic engineering.

Splicing using DNA ligase :
Although recombinant DNA
associations are formed spontaneously,
although the ends are paired, the sugar
phosphate backbone is not sealed. An
enzyme called DNA ligase
accomplishes this task of sealing the two
segments, forming a continuous double
stranded helix.

Once recombinant DNA is synthesized, it is inserted into a competent bacterium(say)
for its multiplication. Competency of host can be achieved in two ways :

Bacteria are treated with specific concentrations of calcium ions and then incubated
periodically on ice, followed by heat stock. This increases the efficiency of DNA entry.

Electroporation :
Here, a suspension of bacteria and foreign DNA is exposed to high voltage electric
discharge, which creates pores in the cell membrane. These pores are large enough to allow
DNA entry into the cell.

DNA libraries :
The original mixture of transformed bacterial cells is
spread out on the surface of a growth medium in a flat
Petri dish. Each cell undergoes successive rounds of cell
division and visible colonies form. These colonies are
called DNA clones. The collection of clones on a Petri
dish, containing hundreds of DNA fragments, is called a
DNA library. It is of two types :- genomic library
(containing entire genomes of organisms) and cDNA
library (containing coding sequences of DNA, derived
from mature mRNA transcripts).
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9. Not all bacteria in the solution contain the desired gene/plasmid. That which bacteria have
incorporated the recombinant DNA can be realized by the following :-

Markers :
A marker is a gene introduced next to the gene of interest in the plasmid. This gene confers
a trait (often visible to naked eye) suitable for artificial selection of the transformed bacteria
(transformants) among others (non-transformants).

Blue white screening :

PRE-REQUISITES

Mutant -Galactosidase ➢ E.Coli

LacZ sequence peptide chain
(from vector)


➢ X-Gal Blue pigment
Herein, the plasmid containing lacZ sequence is interrupted by inserts from foreign DNA.
This means that the bacteria transformed with recombinant DNA should contain a non-
functional -Galactosidase enzyme. The bacterial colonies are then grown in a medium
containing X-gal(substrate). Consequently, bacterial colonies with the engineered plasmid
wouldn’t show any color(white), whereas those not containing the desired plasmid would
develop blue color.
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10. Antibiotic resistance gene :
It is a marker inserted adjacent to the
foreign DNA insert in the plasmid, such
that when the bacteria are grown in a
medium containing antibiotics, the
transformants would not die whereas the
non-transformants would die. For E.Coli,
genes encoding resistance to ampicillin,
tetracycline, kanamycin, etc., are
considered useful selectable markers.

Screening and isolation of DNA:
In a genomic library, one has to identify or screen the regions containing the gene of
interest. Screening involves identification of the desired gene clone by virtue of homology
with known sequences. Single stranded probes labeled with radioactive phosphorus are
prepared from the genes with known sequences. Then, a porous nitrocellulose filter is laid
on the plate so that cells from bacterial colonies adhere to it. The cells are ruptured and
the DNA is separated into single strands, all on the membrane. When this is done, the filter
is gently dipped into the probe solution and then subjected to autoradiography, to locate
the DNA hybridized with the probe. Black spots become visible at specific locations on the
membrane, announcing the presence and location of the desired clone.
Back on the Petri dish, the corresponding clones are retrieved and put in a buffered
nutrient solution. Once the colonies grow further, the cells are disrupted using detergent
solutions, releasing nuclei acids along with other bio molecules. Proteins are denatured by
repeated extractions with phenol. RNA is cleaved using specific RNase enzymes. Finally,
DNA is precipitated with chilled alcohol. We say, DNA gets isolated. Further isolation of
desired gene is done using hybridization techniques.

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11. Insertion of gene to hosts :
After DNA cloning, the gene is often inserted into a suitable host for expressing it into the
desirable protein on a large scale. Before inserting it, modifications are done in the foreign
gene to make it compatible to the host machinery. There are various ways to insert a
transcribable DNA into a host.

Micro-injection :
In this method, recombinant DNA is injected directly into an animal cell using a glass
micropipette. It is carried out mechanically using two micro-manipulators.

When plants are considered as hosts, two methods exist.

Brute force strategy :
This method involves use of gene gun or biolistics to shoot gold/tungsten atoms coated with
DNA molecules towards plant cells. This results in random incorporation of the foreign
DNA into chromosomes. However, it may danage plant cells and the foreign DNA may
get incorporated into mitochondrial matrix or chloroplast stroma.

Disarmed pathogens as vectors :
Agarobacterium Tumefaciens are gall (tumor) inducing bacteria in plants. The tumor cells,
in turn, produce opine as an energy substrate for these bacteria. Their Ti(Tumor inducing)
plasmid has a section of DNA, called ‘T-DNA’, which is capable to replicate itself into the
plant cells. Once it is inside the cell, it uses plant machinery to transcribe and further
translate itself into tumor causing products. However, this segment of the plasmid can be
customized by insertional inactivation, so that the bacteria instead act like a messenger of
desired genes into the host plant cells. An implication of having ‘T-DNA’ is, there is no
extra need of DNA isolation from the amplified plasmids in DNA clones.

Engineering a Ti plasmid
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12. DNA sequencing :
Once the desired gene is cloned and expressed, its sequence can be found out. The
nucleotide sequence is the most fundamental level of knowledge of a gene or genome. It is
the blueprint that contains the instructions for building an organism, and no understanding of
genetic function or evolution could be complete without obtaining this information.

Retrieval of foreign gene products :
In almost all recombinant technologies, the ultimate aim is to produce a desirable protein.
For producing proteins on a large scale, cultures of host cells are grown and the protein is
extracted and purified. The cultures may be grown in a nutrient medium in laboratories or
in a continuous culture system(where the medium is recycled time and again) in industries.
In industries, vessels such as bioreactors are used for commercial production of specific
gene products. These provide optimal growth conditions to the host cells. After
biosynthesis of the products, they are subjected to separation and purification, collectively
known as downstream processing. Strict quality control testing of each product and of each
batch ensures the credibility of the product.

RESULT, ANALYSIS AND INTERPRETATION
Nowadays, manipulating genes for the commercial production of different biomolecules is
not a herculean task. This is inherent in the wide ranging diversity of restriction endo-
nucleases, ligases, vectors, donor and host cells. Genes are so steady and universal across
organisms that, one can exploit genetic machinery of any feasible organism to express the
information of another organism. In fact, with the help of databases storing sequences of
entire genomes of organisms, we can prepare any gene just by gluing nucleotides together
as per its sequence. The striking revolution of genetic engineering can be appreciated by
observing the trend in the cost of DNA sequencing per genome across two decades.
In context of the above graph, the bright future of genetic engineering is quite evident.
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13. DISCUSSION
The implications of biotechnology are wide ranging. Firstly, there has been an increasing
trend of commercialization of gene products. Especially, when these products include,
antibodies, proteins, biofuels, industrially important enzymes(e.g., zymase, invertase),
artificial fibers, agro-based chemicals, growth factors and hormones which confer
genetically superior characteristics. Genetic engineering produces proteins that offer
advantages over traditional proteins isolated from other biological sources. These
advantages include :
➢ High purity
➢ High specific activity
➢ Steady supply
➢ Batch-to-batch consistency

Again, it can’t be hidden, the mega objectives of Recombinant DNA technology which
include :
➢ Identifying, isolating and modifying new genes and the proteins they encode
➢ Re-expressing genes in other hosts or organisms
➢ Correcting endogenous gene defects
➢ Identification of mutations
➢ Performing structure and function analyses on proteins
➢ Manufacturing large quantities of specific gene products such as hormones, vaccines,
and other biological agents of medical interest.

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14. CONCLUSION
“OUR OWN GENOMES CARRY THE STORY OF EVOLUTION, WRITTEN IN
DNA, THE LANGUAGE OF MOLECULAR GENETICS, AND THE NARRATIVE
IS UNMISTAKABLE.”

Genetic engineering is a relatively new field in molecular biology and along with it, it has
also brought many other fields to the forefront such as bioinformatics, gene therapy,
hybridization, DNA fingerprinting and molecular paleontology. All these have been
possible solely because of the continuous efforts of scientists belonging to different
disciplines of science, who have worked in the neighborhood past to unravel the secrets
and the potential of the molecular basis of inheritance. Secrets still remain, but enough has
been discovered to gather interest, to dig more and to discover more. In future, the science
of life is going to be lively to engineer life!

REFERENCES
• https://www.slideshare.net/NishantUpadhyay6/biotechnology-principles-and-
processes-investigatory-project
• https://www.1000sciencefairprojects.com/Biology/recombinant-dna-technology.php
• https://www.britannica.com/science/recombinant-DNA-technology
• https://en.wikipedia.org/wiki/Blue%E2%80%93white_screen
• www.google.co.in
• Class 12 NCERT textbook



Thank you

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