BIOTECHNOLOGY ASSIGNMENT

Chapter-11 Principles and process

Prepared by Babu rajendra (BRK)

One mark questions

1. Define biotechnology
Biotechnology is branch that deals with using live organisms or enzymes to produce products useful
to humans.

2. Mention the definition given by EFB on biotechnology

‘The integration of natural science and organisms, cells and molecular analogues for products and
services’.

3. Mention the two core techniques that enabled birth of modern biotechnology
a.Genetic engineering and
b. Maintenance of sterile (microbial contamination-free) ambience.

4. Mention the limitation of Traditional hybridisation procedures

Lead to inclusion of undesirable genes along with the desired genes.

5. Mention the advantage of genetic engineering.

Genetic engineering allows us to isolate and introduce only one or a set of desirable genes without
introducing undesirable genes into the target organism.

6. Define origin of replication(Ori site) on DNA

Specific DNA sequence which is responsible for initiating replication.

7. Define gene cloning

Alien DNA is linked with the origin of replication, so that, this alien piece of DNA can replicate and
multiply itself in the host organism. This process of making multiple identical copies of any template
DNA is called gene cloning.

8. Name the scientists who first conducted rDNA technology

Stanley Cohen and Herbert Boyer in 1972

9. Define plasmid.
Autonomously replicating circular extra-chromosomal DNA found only in cytoplasm of bacteria.

10. Which enzyme is called Molecular scissor?

Restriction enzymes

SKCH PU College Biotechnology Chapter 11 Assignment Prepared by Babu Rajendra

 11. In which micro organism Cohen and Boyer isolated antibiotic resistant gene?
Salmonella typhimurium.

12. Define recombinant DNA

New combination of circular DNA created by inserting foreign DNA into plasmid in vitro is known as
recombinant DNA.

13. When was REN first isolated?

In the year 1963 two REN were isolated in Escherichia coli. These two REN restrict the growth of
bacteriophage in E coli.

14. Define Eco RI.

Restriction endonuclease isolated from Escherichia coli RY 13.

15. Expand EFB

European federation for Biotechnology.

16. What are palindromes?

These are groups of letters that form the same words when read both forward and backward,e.g.,
“MALAYALAM”.

17. Define palindrome in DNA.

DNA which can be read in the same way on two strands in both directions.

18. Write the palindromic sequence cut by EcoRI.

5' —— GAATTC —— 3'
3' —— CTTAAG —— 5'

19. Name the stain used to see the DNA fragments in agarose gel after electrophoresis.
Ethedium bromide.

20. Name the technique used to separate DNA fragments based on their size.
Gel electrophoresis

21. Define elution.

Extraction of desired gene from gel after electrophoresis

22. Define insertional inactivation.

Inactivation of Lac gene when a foreign gene is inserted is called insertional inactivation.

23. Define Ti plasmid.

Tumour inducing plasmid found in Agrobacterium tumifacien.
SKCH PU College Biotechnology Chapter 11 Assignment Prepared by Babu Rajendra
 24. Mention the function of T DNA in Agrobacterium tumifacien.
Segment of DNA in Ti plasmid that is transferred to plant genome during infection.

25. Why DNA cannot pass through cell membrane?

Because DNA is a hydrophilic molecule.

26. Mention the chemical used to precipitate purified DNA.

Chilled ethanol.

27. Define Taq polymerase.

DNA polymerase extracted from a bacterium Thermus aquaticus .

28. Define annealing during PCR.

Binding of primers to template DNA strand.

29. When sticky ends in DNA are produced?

When DNA are cut by the same restriction enzyme.

30. Name the non-biological method used to insert desired gene into plant.
Biolistics

31. Mention an alternate selectable marker.

Lac z gene

Two marks questions

1. Mention the three basic steps in genetically modifying an organism.
1. Identification of DNA with desirable genes.
2. Introduction of the identified DNA into the host.
3. Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

2. Mention the functions of different REN

One of the REN Added methyl groups to bacterial DNA, while the other cut bacteriophage DNA.

3. Write the convention for naming REN.

 The first letter of the name comes from the genus.
 Second two letters come from the species of the prokaryotic cell from which the enzyme
 isolated
 The fourth letter is in capital form derived from the Strain of microbes.
 The Roman letter followed is the order of discovery.

4. Explain the types and functions of nucleases.

There are two kinds of nucleases:
SKCH PU College Biotechnology Chapter 11 Assignment Prepared by Babu Rajendra
 1.Exonuclease
2. Endonuclease
Exonuclease removes nucleotides from the free ends of the DNA.
Endonucleases cuts within the DNA.

5. Write a note on selectable markers.

 The genes that are required to identify recombinant from the non-recombinant are called
selectable markers.
 These also help in identifying and eliminating non-transformants and permitting the growth
of the transformants.
 The gene coding resistance to antibiotics such as Ampicilin, Tetracycline are used as selectable
markers for E.coli.

6. Define the method known as biolistics or gene gun.

 It is a non-biological method to insert foreign DNA in to plant genome.
 Micro-particles of gold or tungsten coated with alien DNA.
 Later Gold particles are bombarded to plant cells with high velocity.

7. ‘Taq polymerase is used in PCR’ give reason.

Taq polymerase is a DNA polymerase that functions at very high temperature. Hence it is used in
PCR.

8. Write a note on downstream processing.

 A series of process done to product after its synthesis in bioreactor before marketing is
called downstream process.
 The process includes separation, purification, and addition of preservatives.

9. Draw a neat labeled diagram of pBR322.

10. Draw a neat labelled diagram of gel

Electrophoresis

SKCH PU College Biotechnology Chapter 11 Assignment Prepared by Babu Rajendra

Three marks questions
1. Give the diagrammatic representation of rDNA technology.

2. Mention the steps in formation of rDNA by the action of REN –EcoR1.

1.Isolation of the Genetic material (DNA)
2. Amplification of DNA by PCR
3. Insertion of desired DNA into plasmid and Releasing of recombinant DNA into Bacterium
4. Obtaining the Foreign Gene product or Recombinant product:
5. Downstream processing

3. Write a note on alternate selectable markers.

 The gene other than antibiotic resisting gene that is used to confirm the presence of foreign
gene is called alternate selectable markers.
 The commonly used alternate selectable marker is Lac z gene.
 The foreign DNA is inserted in to lac z gene that codes an enzyme called β-galactosidase. This
results in inactivation of the gene. It is called insertional inactivation.
 This enzyme converts chromatogenic substrate (X-Gal) into blue color product( Galactose
and 5-bromo+4 chloro indigo)
 The non-recombinant produce enzyme and give blue colored colonies.
 The recombinant unable to produce β-galactosidase and does not produce blue colored
colonies after addition of chromatogenic substrate i.e. X-Gal.

SKCH PU College Biotechnology Chapter 11 Assignment Prepared by Babu Rajendra

 4. Explain the Vectors for cloning genes in plants and animals.
In plants
a. Biological method – By using a bacterium Agrobacterium tumifacien.
b. Non-biological method – Biolistics or gene gun method.
In Animals
a.Biological method – By using Retro virus
b. Non biological method- Microinjection.

5. Explain how a bacterium is made competent to take up rDNA.

 The bacterial cell is treated with calcium, which increases the efficiency of DNA up take by
the bacteria.
 Recombinant DNA and the bacterial cells are incubated in ice, followed by placing them
briefly at 42oC (heat shock) and then putting them back in ice.

6. Explain the mechanism involved in Gel electrophoresis

 The DNA fragments are made to move in a matrix in an electric field.
 The DNA fragments are negatively charged hence they move from cathode to anode.
 DNA fragments separate according to their size through sieving effect provided by the
agarose gel.
 Hence the smaller DNA fragments move farther than smaller fragments.
 The separated fragments are seen by staining them with Ethidium bromide and later by
exposing to UV radiation.

7. What are bioreactors? Mention the commonly used bioreactors in rDNA technology.
 Larger vessels which are designed to culture genetically modified organisms to produce our
desired product from raw material.
 There are two types of stirred type of bioreactors
a. Simple stirred-tank bioreactor
b.Sparged stirred tank bioreactor.

8. Mention the components of commonly used bioreactor.

1. Agitator system.
2. An oxygen delivery system
3. A foam control system.
4. A temperature control system
5. pH control system
6. Sampling ports so that small volumes of the culture can be withdrawn periodically

Five marks questions

1. Explain the features of cloning vectors.
There are three features of cloning vectors. They are
a. Origin of replication -This is the sequence where the replication starts
SKCH PU College Biotechnology Chapter 11 Assignment Prepared by Babu Rajendra
 b. Selectable markers-The genes that are required to identify recombinant from the non-recombinant
are called selectable markers.
c. Cloning site-The gene in vector in which our desired gene or alien DNA is inserted is called cloning
site. Cloning site is in palindromic site resent in any one of the antibiotic resistance
genes.

2. Explain Separation and isolation of desired DNA.

 Bacterial cell wall digested by Lysozyme.
 Plant cell wall is digested by cellulase and pectinase.
 Fungal cell wall is digested by chitinase.
 RNA of the cellular content is digested by ribonuclease.
 Proteins are removed by Proteases.
 Purified DNA is precipitated by adding chilled ethanol.
 The precipitated DNA is separated and removed by spooling.
 The extracted DNA is treated with restriction endonucleases.
 Due to action of this enzyme the DNA is broken down into large number of fragments.
 From these DNA fragments the desired DNA is separated by electrophoresis.

3. Explain PCR with suitable diagram.

a. Denaturation
1.The target DNA is heated to high temperature (940c)
2. It results in the separation of the two strands.
b. Primer Annealing
1. Two primers are added.
2. The primers anneal to template strand.
c. Extension Of Primers
1. In this step an enzyme called Taq DNA polymerase, DNA nucleotides and Mg++ are added.
2. In the presence of taq polymerase, nucleotides and Mg++ DNA synthesis takes place.
3.The cycle is repeated to produce more number of copies.
SKCH PU College Biotechnology Chapter 11 Assignment Prepared by Babu Rajendra
 4. Draw and label the following
a. Simple stirred- tank bioreactor
b. Sparged stirred-tank bioreactor

5. Give diagrammatic representation for formation of Recombinant DNA by the action of Restriction
endonuclease

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SKCH PU College Biotechnology Chapter 11 Assignment Prepared by Babu Rajendra