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Principles and process of Biotechnology : Notes and Study Materials -pdf

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Principles and process of Biotechnology : Practice Paper 1

11. BIOTECHNOLOGY: PRINCIPLES & PROCESSES

· Biotechnology is the technique of using live organisms or their enzymes for products & processes useful to humans.

· The European Federation of Biotechnology (EFB) defines Biotechnology as ‘the integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services’.

Biotechnology deals with:

– Microbe-mediated processes (making curd, bread, wine etc).

– In vitro fertilization (test-tube baby programme).

– Synthesis and using of a gene.

– Preparation of DNA vaccine.

– Correcting a defective gene.

PRINCIPLES OF BIOTECHNOLOGY

Core techniques of modern biotechnology

· Genetic engineering: The technique in which genetic material (DNA & RNA) is chemically altered and introduced into host organisms to change the phenotype.

· Bioprocess engineering: Maintenance of sterile ambience in chemical engineering processes for growing desired microbe/eukaryotic cell for the manufacture of antibiotics, vaccines, enzymes etc.

Basic steps in genetically modifying an organism

a) Identification of DNA with desirable genes: Traditional hybridisation leads to inclusion and multiplication of undesirable genes along with desired genes. In genetic engineering, only desirable genes are introduced.

b) Introduction of the identified DNA into the host: A vector DNA such as plasmid is used to deliver an alien piece of DNA into the host organism.

c) Maintenance of introduced DNA in the host and transfer of the DNA to its progeny: A piece of alien DNA has no the sequence called Origin of replication (ori) needed for starting replication. So, it cannot multiply itself in the progeny cells of the organism. Hence alien DNA is integrated into the recipient genome (it has ori). It multiplies & inherits along with host DNA.

· The process of joining and inserting a foreign piece of DNA into a host organism to produce new genetic combinations is called recombinant DNA technology.

· First recombinant DNA (rDNA) was produced by Stanley Cohen & Herbert Boyer (1972).

· They isolated an antibiotic resistance gene (piece of DNA) from a plasmid of Salmonella typhimurium. It was linked with a plasmid vector and transferred into E. coli. As a result, the gene was expressed & multiplied in E. coli.

TOOLS OF RECOMBINANT DNA TECHNOLOGY

1. Restriction Enzymes (‘molecular scissors’)

– The enzymes that cut DNA at specific sites into fragments.

– They belong to a class of enzymes called nucleases.

– In 1963, two enzymes responsible for restricting growth of bacteriophage in E. coli were isolated. One enzyme added methyl groups to DNA. The other (restriction endonuclease) cut DNA.

– More than 900 restriction enzymes have been isolated from over 230 strains of bacteria.

Naming of the restriction enzymes:

– First letter indicates genus. The second two letters indicate species of prokaryotic cell from which they were isolated.

E.g. EcoRI comes from E. coli RY 13 (R = the strain. Roman numbers = the order in which the enzymes were isolated from that strain of bacteria).

Types of Restriction enzymes:

· Exonucleases: They remove nucleotides from the ends of the DNA.

· Endonucleases:

– They cut at specific positions within the DNA. E.g. EcoRI.

– They bind to specific recognition sequence of the DNA and cut the two strands at specific points.

– The first restriction endonuclease is Hind II. It cuts DNA molecules by recognizing a specific sequence of 6 base pairs. This is called the recognition sequence for Hind II.

– Restriction endonuclease recognizes a specific palindromic nucleotide sequences in the DNA. It is a sequence of base pairs that read the same on the two strands in 5‘ → 3‘ direction and in 3‘ → 5‘ direction. E.g. Palindromic nucleotide sequence for EcoRI is

5‘ —— GAATTC —— 3‘

3‘ —— CTTAAG —— 5‘

Steps in formation of recombinant DNA by EcoRI

– Restriction enzymes cut the strand a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands. This leaves single stranded overhanging stretches at the ends. They are called sticky ends. They form H-bonds with their complementary cut counterparts. This stickiness facilitates action of the enzyme DNA ligase.

– When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of sticky-ends and these are joined together by DNA ligases.

2. Cloning Vector

– It is a DNA molecule that can carry a foreign DNA segment and replicate inside the host cells.

E.g. Plasmids, bacteriophages etc.

– Plasmids are autonomously replicating circular extra-chromosomal DNA of bacteria. Some plasmids have only 1-2 copies per cell. Others have 15-100 copies per cell.

– Bacteriophages (high number per cell) have very high copy numbers of their genome within the bacterial cells.

– When the cloning vectors are multiplied in the host, the linked piece of DNA is also multiplied to the numbers equal to the copy number of the vectors.

Features required for cloning into a vector

a. Origin of replication (ori)

– This is a sequence where replication starts.

– A piece of DNA linked to ori can replicate within the host cells. This also controls the copy number of linked DNA. So, for getting many copies of the target DNA, it should be cloned in a vector whose origin support high copy number.

b. Selectable marker (marker gene)

– It is a gene that helps to select the transformants and eliminate the non-transformants.

– If a piece of DNA is introduced in a host bacterium, it is called transformation. Such bacterium is transformant. If transformation does not take place, it is non-transformant.

– Selectable markers of E. coli include the genes encoding resistance to antibiotics like ampicillin, chloramphenicol, tetracycline, kanamycin etc. Normal E. coli cells have no resistance against these antibiotics.

c. Cloning sites

– These are the recognition sites for restriction enzymes.

– To link the alien DNA, the vector needs a single or very few recognition sites.

– More than one recognition sites generate several fragments. It complicates the gene cloning.

– Ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.

E.g. In vector pBR322, foreign DNA is ligated at Bam H I site of tetracycline resistance gene. As a result, recombinant plasmid is formed. If ligation does not occur, it is called non-recombinant plasmid.

· Restriction sites: Hind III, EcoR I, BamH I, Sal I, Pvu II, Pst I, Cla I.
· ori
· Antibiotic resistance genes: ampR and tetR.
· Rop: codes for the proteins involved in the replication of plasmid.

– When a foreign DNA is inserted within a gene of bacteria, that gene is inactivated. It is called insertional inactivation. Here, the recombinant plasmids lose tetracycline resistance due to insertion of foreign DNA.

– When the plasmids are introduced into E. coli cells, 3 types of cells are obtained:

o Non-transformants: They have no plasmid. So they are not resistant to either tetracycline or ampicillin.

o Transformants with non-recombinant plasmid: They are resistant to both tetracycline & ampicillin.

o Transformants with recombinant plasmid: They are resistant only to ampicillin.

– Recombinant plasmids can be selected out from non-recombinant ones by plating transformants on ampicillin medium. Then the transformants are transferred on tetracycline medium.

– The recombinants grow in ampicillin medium but not on tetracycline medium. But, non-recombinants grow on the medium containing both the antibiotics.

– Thus, one antibiotic resistance gene helps to select the transformants. The inactivated antibiotic resistance gene helps to select recombinants.

– But this type of selection of recombinants is a difficult procedure because it needs simultaneous plating on 2 plates having different antibiotics. So, alternative selectable markers have developed based on their ability to produce colour in presence of a chromogenic substrate.

– In this, a recombinant DNA is inserted into the coding sequence (gene) of an enzyme, b-galactosidase. So, the gene is inactivated (insertional inactivation). Such colonies do not produce any colour. These are identified as recombinant colonies.

– If the plasmid in bacteria have no an insert, it gives blue coloured colonies in presence of chromogenic substrate.

d. Vectors for cloning genes in plants & animals

Genetic tools of some pathogens can be transformed into useful vectors for delivering genes to plants & animals. E.g.

· Agrobacterium tumefaciens (a pathogen of many dicot plants) can deliver a piece of DNA (T-DNA) to transform normal plant cells into a tumor. These tumor cells produce the chemicals required by the pathogen.

The tumor inducing (Ti) plasmid of A. tumefaciens is modified into a cloning vector which is not pathogenic but can use mechanisms to deliver genes of interest into plants.

· Retroviruses in animals can transform normal cells into cancerous cells. So, they are used to deliver desirable genes into animal cells.

3. Competent Host (For Transformation with Recombinant DNA)

– Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. So bacterial cells are made ‘competent’ to take up alien DNA or plasmid as follows:

– Treat bacterial cells with a specific concentration of a divalent cation (e.g. calcium) → DNA enters the bacterium through pores in cell wall → Incubate the cells with recombinant DNA on ice → Place them briefly at 42⁰C (heat shock) → Put them back on ice → Bacteria take up recombinant DNA.

Other methods to introduce alien DNA into host cells

· Micro-injection: In this, recombinant DNA is directly injected into the nucleus of an animal cell.

· Biolistics (gene gun): In this, cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA. This method is suitable for plants.

· ‘Disarmed pathogen’ vectors: They infect the cell and transfer the recombinant DNA into the host. E.g. A. tumefaciens.

PROCESSES OF RECOMBINANT DNA TECHNOLOGY

1. Isolation of the Genetic Material (DNA)

– Treat the bacterial cells/plant or animal tissue with enzymes like lysozyme (bacteria), cellulase (plants), chitinase (fungus) etc. The cell is broken releasing DNA & other macromolecules (RNA, proteins, polysaccharides & lipids).

– RNA is removed by treating with ribonuclease. Proteins are removed by treatment with protease. Other molecules are removed by appropriate treatments.

– When chilled ethanol is added, purified DNA precipitates out as a collection of fine threads in the suspension.

2. Cutting of DNA at Specific Locations

– Purified DNA is incubated with the restriction enzyme. As a result, DNA digests. These DNA fragments are separated by a technique called gel electrophoresis.

– Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion. DNA is negatively charged. So it moves towards the anode. DNA fragments are separated according to their size through sieving effect of the agarose gel (a polymer extracted from sea weeds). The smaller sized fragment moves farther.

– The process is repeated with the vector DNA also.

– DNA fragments can be seen as bright orange coloured bands when they are stained with ethidium bromide and exposed to UV radiation.

– DNA bands are cut out from agarose gel. It is called elution. The cut-out gene of interest and cut vector are mixed and ligase is added. It creates recombinant DNA.

3. Amplification of Gene of Interest using PCR

– Polymerase Chain Reaction (PCR) is the synthesis of multiple copies of the gene of interest in vitro using 2 sets of primers & the enzyme DNA polymerase.

– Primers are small chemically synthesized oligonucleotides that are complementary to the regions of DNA.

Steps of PCR:

· Denaturation: It is the heating of target DNA (gene of interest) at high temperature (94⁰C) to separate the strands. Each strands act as template for DNA synthesis.

· Annealing: It is the joining of the two primers (at 52⁰C) at the 3’ end of the DNA templates.

· Extension: It is the addition of nucleotides to the primer using a thermostable DNA polymerase called Taq polymerase. It is isolated from a bacterium, Thermus aquaticus. It remains active in high temperature during the denaturation of double stranded DNA.

Through continuous replication, the DNA segment is amplified up to 1 billion copies.

The amplified fragment can be used to ligate with a vector for further cloning.

4. Insertion of Recombinant DNA into Host Cell

– Using any methods, the ligated DNA is introduced into recipient (host) cell / organism. They take up DNA from its surrounding.

– If a recombinant DNA bearing ampicillin resistant gene is transferred into E. coli cells, the host cells become ampicillin-resistant cells.

– If the transformed cells are spread on agar plates containing ampicillin, only transformants will grow. Untransformed recipient cells will die.

5. Obtaining the Foreign Gene Product

– The aim of recombinant DNA technology is to produce a desirable protein.

– If a protein encoding foreign gene is expressed in a heterologous host, it is called a recombinant protein.

– The cells with foreign genes can be grown in laboratory. The cultures are used to extract the desired protein and purify it by using separation techniques.

– The cells can also be multiplied in a continuous culture system. Here, the used medium is drained out from one side while fresh medium is added from the other. It maintains the cells more physiologically active and so produces a larger biomass. It yields more desired protein.

Bioreactors

– These are the vessels in which raw materials are biologically converted to specific products, enzymes etc., using microbial, plant, animal or human cells.

– Bioreactors are used to produce large quantities of products. They can process 100-1000 litres of culture.

– A bioreactor provides the optimal growth conditions (pH,

temperature, substrate, salts, vitamins, oxygen) to get desired product.

– The most commonly used bioreactors are of stirring type (stirred-tank bioreactor).

It is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. The stirrer facilitates even mixing and oxygen availability. Alternatively, air can be bubbled through the reactor.

The bioreactor has

· An agitator system

· An oxygen delivery system

· A foam control system

· A temperature control system

· pH control system

· Sampling ports (for periodic withdrawal of the culture).

6. Downstream Processing

– It is a series of processes such as separation and purification of products after the biosynthetic stage.

– The product is formulated with suitable preservatives. Such formulation undergoes thorough clinical trials and strict quality control testing

CBSE Class 12 Biology Important Questions Chapter 11 – Biotechnology: Principles and Processes

1 Mark Questions

Chapter 11
Biotechnology Principles and Processes

1 Marks Questions
1. A restriction enzyme digests DNA into fragments. Name the technique used to check the progression of this enzyme and separate DNA fragments.
Ans. Gel electrophoresis

2. Name two commonly used vectors in genetic engineering.
Ans. Plasmid and Bacteriophage.

3. Some enzymes are considered as molecular scissors. in genetic engenrring. What is the name assigned to such enzymes?
Ans. Restriction Enzymes.

4. Write conventional nomenclature of EcoRI.
Ans.E. – Escherichia; co – coli; R – Name of Strain; I – order in which enzyme isolated from strain of bacteria.

5. A linear DNA fragment and a plasmid has three restriction sites for EcoRIhow many fragments will be produced from linear DNA and plasmid respectively.
Ans.Number of fragments of linear DNA = 4
Number of fragments of plasmid = 3

6. An extra chromosomal segment of circular DNA of a bacterium is used to carry gene of interest into the host cell. What is the name given to it?
Ans. Plasmid.

7. Identify the recognition sites in the given sequences at which E.coli will be cut and make sticky ends.
5´-GAATTC-3´
3´-CTTAAG-5´
Ans. $5 - G^{↓} A A T T C 3$
$3 - C T T A A_{↑} G 5$

8.Name the substance used as a medium in gel electrophoresis.
Ans.Agarose

9.What is Bioconversion?
Ans.Bioconversion refers to the process by which raw material are converted to specific product by microbial, plant or animal cell.

10.Name the bacterium that yields thermostable DNA polymerase.
Ans.Thermusaquaticus.

11.Which enzymes are known as “molecular Scissors”?
Ans.Restriction Endonuclease.

12.Name the commonly used vector for trans formation in plant cell?
Ans.Agrobacterium tumefacien.

13.Name the technique used for amplification of DNA?
Ans.Polymerase Chain Reaction.

14.Name the enzyme responsible for removal of 5 – phosphate group from nucleic acid?
Ans.Alkaline Phosphates.

15.Who isolated Restriction enzymes for the first time?
Ans.Warner Arber & Hamilton Smith.

16.Why do eukaryotic cells do not contain restriction enzymes?
Ans.Because in eukaryotic cell, DNA is heavily methylated.

17.Why does DNA moves towards anode in gel electrophoresis.
Ans.Because of presence of phosphate group, DNA is negatively charged & $∴$ moves towards anode.

2 Mark Questions

Chapter 11
Biotechnology Principles and Processes

2 Marks Questions
1.Name two main steps which are collectively referred to as down streaming process. Why is this process significant?
Ans.Separation and Purification
This process is essential because before reaching into market, the product has to be subjected for clinical trial and quality control.

2. How does plasmid differ from chromosomal DNA?
Ans.

	Plasmid DNA		Chromosomal DNA
(i) (ii) (iii)	Circular DNA Occurs only in bacterial cells Used as Vector in rDNA technology	(i) (ii) (iii)	Linear DNA Occurs in nucleus of eukaryotic cells and bacterial cell. Not used as vector in rDNA technology.

3. A bacterial cell is shown in the figure given below. Label the part ‘A’ and ‘B’. Also mention the use of part ‘A’ in rDNA technology.

Ans. A- Plasmid, B – Nucleoid
Plasmid is used as vector to transfer the gene of interest in the host cell.

4. Mention two classes of restriction enzymes. Suggest their respective roles.
Ans.Exonucleases and endonucleases

Exonucleases remove nucleotides from the ends of the DNA.
Endonucleases cut DNA at specific sites beween the ends of DNA.

5. In the given process of separation and isolation of DNA fragments, someof the steps are missing, Complete the missing steps –
A : Digestion of DNA fragments using restriction endonucleases
↓
B : ……………………………………………………..
↓
C : Staining with ethidium bromide
↓
D :Visualisation in U.V. light
↓
E : …………………………………………………….
↓
F : Purification of DNA fragments.
Ans.B – Gel Electrophoresis
E – Elution

6.Write any two properties of restriction endonuclease enzymes?
Ans.(i) Each Restriction endonuclease functions by inspecting the length of DNA sequence & bindto DNA at the recognition Sequence.
(ii) It cuts the two strands of DNA at specific point in their sugar – phosphate backbone.

7.What are ‘Selectable marker’? What is their use in genetic engineering?
Ans.A selectable marker is a gene which helps in selecting those host cells which contains thevector& eliminating the non–transformanteg – gene encoding resistance to antibiotics are usefulSelectable markers as they allow Selective growth of transformants only.

8.How can the desired product formed after genetic engineering be produced on a commercial scale?
Ans.The product obtained from genetic engineering is subjected to a series of processes collectivelycalled downstream processing before it made into final processes involved in downstreamprocessing are :- Separation & purification.

9.What is “Insertional Inactivation”?
Ans.If a recombinant DNA is inserted within the coding Sequence of enzyme B–galactosidase. Thisresults into inactivation of enzyme which is referred to as “Insertional Inactivation”. The presenceof chromogenic Substrate gives blue–coloured colonies if the plasmid in bacteria does not have aninsert presence of insert results into insertional inactivation &the colonies do not produce anycolor.

10.What are the two basic techniques involved in modern Biotechnology?
Ans.The two basic techniques involved in modern Biotechnology are:-
a)Genetic Engineering is the technique of altering the nature of genetic material or introduction of it into another host organism to change its phenotype.
b)Techniques to facilitate the growth & multiplication of only the desired microbes or cells in large number under sterile conditions for manufacture

11.Represent diagrammatically the E. coli. Cloning vector $β$ PBR 322.
Ans.

12.Differentiate between plasmid DNA and chromosomal DNA?
Ans.Plasmid DNA is extranuclear DNA, found in protoplasmic whereas chromosomal DNA is the nuclear or genetic DNA which is found within the nucleus.

13.What is the role of enzyme “Ligase” in genetic Engineering?
Ans.Enzyme “Ligase” acts as molecular Suture which helps in joining two pieces of DNA. The Joining process requires ATP as it derive energy to construct phosphodiester bond between cohesive ends.

14.Name the components a bioreactor must possess to achieve the desired product?
Ans.Enzyme “Ligase” acts as molecular Suture which helps in joining two pieces of DNA. The Joining process requires ATP as it derive energy to construct phosphodiester bond between cohesive ends.

15.The following proteins of given molecular weight are Subjected to Get electrophoresis. Write the order of Sequence in which these proteins are isolated in a gel?

S.no.	Proteins	Mol.wt
1.	Albumin	23,000
2.	Keratin	48,000
3.	Myosin	1,25,000
4.	Haemoglobin	84,000
5.	Ribozyme	62,000
6.	Insulin	1,14,000

Ans.The sequence of proteins obtained from top to bottom in a gel:-
Myosin > Insulin >Haemoglobin> Ribozyme > Keratin > Albumin.

16.How is gene Z used as a marker?
Ans.Lac Z gene codes for enzyme Β-galactosidase, if a recombinant DNA is inserted within thecoding sequence of an enzyme Β-galactosidase. This results into inactivation of enzyme. Thebacterial colonies whose plasmid does not have an insert produce blue colour but those with aninsert do not produce any colour.

17.What is Bioreactor? What are the advantages of Stirred tank Bioreactor over Shake flask. Show diagrammatically a simple Stirred tank Bioreactor?
Ans.Bioreactors are large vessels in which raw materials are biologically converted into specificproteins using microbial, plant, animal or human cells. The advantages of Bioreactor over shakeflask are :-
a)It provides optimal conditions for achieving desired product by providing optimum growth conditions eg. temp, pH etc.
b)Small volume of cultures can be withdrawn periodically from bioreactor to test the sample.
c)It has an agitation system, temp control system, from control system & pH control system
d)

3 Mark Questions

Chapter 11
Biotechnology Principles and Processes

3 Marks Questions
1. Since DNA is a hydrophillicmoelcule, it cannot pass through cell membranes. Name and explain the technique with which the DNA is forced into (ii) a bacterial cell (ii) a plant cell (iii) an animal cell.
Ans. (i) Chemical treatment and exposure to cold and high temp.(42°C) alternatively. (Bacterial cell)
(ii) Biolistics or gene gun. (Plant cell)
(iii) Micro-injection. (animal cell) Explanation – Refer page 200, biology Text Book for class XII.

2. How will you obtain purified DNA from a cell?
Ans.Cells are treated with appropriate enzymes to release DNA. Lysozyme (bacteria), cellulase (plant cells), chitinase (fungus).

RNA and proteins are removed by treatment with ribonuclease and protease enzymes respectively.

3. In recombinant DNA technology, vectors are used to transfer a gene of interest in the host cells. Mention any three features of vectors that are most suitable for this purpose.
Ans.(i) Have origin of replication(Ori)
(ii) Have a selectable marker
(iii) Have at least one recognition site.

4. Why is “Agrobacterium–mediated genetic engineering transformation” inplants considered as natural genetic engineering?
Ans.Agrobacterium tumefaciens is a pathogen in many dicot plants. It is able to deliver a piece of DNA (T–DNA) to transform normal plant cell into a tumor and directs these tumor cells to produce the chemicals required by pathogen.

5. Observe the given sequence of nitrogenous bases on a DNA fragment and answer the following question –
5´ – CAGAATTCTTA – 3´
3´ – GTCTTAAGAAT – 5´
(a) Name a restriction enzme which can recognise this DNA sequence.
(b) Write the sequence after digestion.
(c) Why are the ends generated after digestion called sticky ends?
Ans. (a) EcoRI

(c) These are named sticky ends, because they form hydrogen bonds with their complementary cut parts.

6. A selectable marker is used in the section of recombinants on the basis of their ability to produce colour in presence of chromogenic substrate.
(a) Mention the name of mechanism involved.
(b) Which enzyme is involved in production of colour?
(c) How is it advantageous over using antibiotic resistant gene as a selectable marker?
Ans. (a) Insertional inactivation
(b) b-galactosidase.
(c) Selection of recombinants due to inactivation of antibiotics requires simultaneous plating on two plates having different antibiotics. (Refer page 200 NCERT Biology for class XII).

7.Mention the important properties which a good vector must possess?
Ans.The important properties which a good vector must possess are :-
i) Size :- The vector must have small size so that it is easier to purify & isolate.
ii) Origin of replication :- This is a sequence of base pairs where replication starts. Any piece of DNA linked to this sequence can be made to replicate within its host cell & thus, controls the copy number of linked DNA.
iii) Selectable Marker :- A marker is a gene which helps in selecting those host cells which contain the vector & eliminating the non – transformants Common Selectable marker include gene encoding resistance to antibiotics.
iv) Cloning Sites :- The vector Should have a few or at least one unique recognition site to link the foreign / alien DNA. Presence of a particular recognition site enables the particular restriction enzyme to cut the vector.

8.Describe any three vectors less method of introducing the rDNA into a competent host cell?
Ans. i) Transformation :- In order to force bacteria to take up the plasmid, the bacterial cell must first be made competent to take up DNA. This is done by treating them with specific concentration of divalent cationeg. Ca2+ which increases the efficiency with which DNA enters the bacterium through pores in its cell wall Recombinant DNA can then be forced into such cells by incubating the cells with recombinant DNA on ice, followed by placing them at 420 C& then putting them back into ice. This enables the bacteria to take up the recombinant DNA.
ii) Microinjection :- recombinant DNA is directly injected into the nucleus of an animal cell using a micro – needle of tip with diameter (~ 4mm)
iii) Biolistics / Gene gun :- cells are bombarded with high velocity micro – particles of gold or tungsten coated with DNA.

9.Why is Agrobacterium mediated genetic transformation described as Natural Genetic engineering in plants?
Ans.Agrobacterium tumefacien, anatural pathogen of Several dicot plants is able to deliver a piece of DNA known as “t – DNA” to transform normal plant cell into a tumor & direct gene transfer transform tumor cells to produce chemicals required by pathogen . The tumor inducing (Ti) plasmid of
Agrobacterium tumefacien has now been modified into a cloning vector which is no more pathogenic to plant but is still able to use the mechanism to deliver genes of our interest into a variety of plants Since Agrobacterium tumefacien has the natural ability to donate a part of its DNA to the plant during infection. This property of Agrobacterium is exploited and a gene of interest is ligated into T-DNA so that it automatically gets transformed into plant cell thus, Agrobacterium tumefacien is known as “Natural Genetic Engineer” of plants.

10.Mention the important tools required for genetic engineering technology?
Ans.The process of genetic engineering is accomplished only when we have following key tools :-
a)Restriction enzymes:- Restriction enzymes are a group of endonucleases which cut the DNA at Specific position anywhere in its length. Each restriction endonuclease functions by inspecting the length of DNA & binds to DNA at the recognition Sequence.
b)Cloning Vector:- The DNA molecule which carry the desired DNA Segment of an organism & transfer it to cell or DNA of another organism is called cloning vector.
c)Desired foreign DNA:- The segment of DNA containing genes having desired characters & which are being transferred into genome of another cell with the help of vector is called foreign DNA.

5 Marks Questions

Chapter 11
Biotechnology Principles and Processes

5 Marks Questions
1. The development of bioreactors is required to produce large quantities of products.
(a) Give optimum growth conditions used in bioreactors.
(b) Draw a well labelled diagram of simple stirred – tank bioreactor.
(c) How does a simple stirred – tank’ bioreactor differ from sparged stirred – tank’ bioreactor?
Ans. (i) Temperature, pH, susbtrates, salts, vitamins and oxygen.
(ii) (a) simple stirred–tank bioreactor

(iii) The stirrer facilitates even mixing and oxygen availability throughout simple–stirred tank bioreactor, whereas in case of sparged stirred-tank bioreactor, air is bubbled throughout the reactor for proper mixing.

2. In the given figure, one cycle of polymerase chain reaction (PCR) is shown-

(a) Name the steps A, B and C.
(b) Give the purpose of each of these steps.
(c) State the contribution of bacteriumThermusaquaticus in this process.
Ans. (a) Denaturation – Heat denatures DNA to separate complementary strands.
(b) Annealing : Primers hybridises to the denatured DNA strands.
(c) Extension : Extension of primers resulting in synthesis of copies of target DNA sequence. Enzyme Tag polymerase is isolated from the bacterium Thermusaquaticus. This enzyme induces denaturation of double stranded DNA at high temperature.

3. Study the figure of vector pBR322 given below in which foreign DNA is ligated at the Bam H1 site of tetracyline resistance gene.

Answer the following questions :
(a) Mention the function of rop.
(b) What will be the selectable marker for this recombinant plasmid and why?
(c) Explain transformation.
Ans. (a) ‘rop’ codes for the proteins involved in the replication of plasmid
(b) Selectable marker – ampicillin resistance gene. It will help distinguishing trAns.formants from non-trAns.formants after plating them on ampicillin containing medium.
(c) Transformation – It is the phenomenon by which the DNA isolated from one type of cell and introduced into another type and is able to bring about some of the properties of former to the later.

4.Describe the various steps involved in Recombinant DNA technology with the help of a well labeled. Diagram?
Ans. i) Identification of DNA with desirable Genes:- Other molecules in the target cell can beremovedby appropriate treatment & purified DNA ultimately precipitates out after addition ofchilled ethanol.
ii) Cutting the DNA at specific location :- After having cut the source DNA as well as vector DNA with Specific restriction enzyme, the cut out “gene of interest” from the source DNA & the cut vector with space are mixed & ligase is added.
iii)Insertion of Recombinant DNA into host cell :- Recipient cells after making them competent to receive takes up DNA in its surrounding. Recombinant DNA is introduced into suitable host cell by vector – based or vector – less method.

iv)Selection & Screening :- If a recombinant DNA bearing gene for resistance to an antibiotic is trAns.ferred into E-coli the host – cell become trAns.formed into ampicillin – resistant cells. Due to this amp gene one is able to select a trAns.formed cell in the presence of ampicillin. This amp r gene is called selectable marker.
v)Obtaining the foreign Gene product :- After having cloned the gene of interest & having optimized the conditions to induce expression of the target protein, one has to consider producing it on large scale.

5.Expand PCR? Describe the different Steps involved in this technique?
Ans. PCR stands for polymerase chain reaction.It is a technique for amplification of gene of interest
or to obtain multiple copies of DNA of interest.
The PCR requires primers, taq polymerase, target sequence, DNA sample &deoxyribonucleotides.
PCR includes number of cycles for amplifying DNA of interest invitro. Each cycle has three steps :-
a)DENATURATION:- The first step is denaturation of SNA sample in a reaction mixture to 940 c. During this step, DNA strand gets separated.
b)RENATURATION / ANNEALING:- The temperature is allowed to cool down to 500c to allow two oligo-nucleotide primers to anneal to complementary sequence in DNA molecule.
c)EXTENSION:- The temperature is raised to 750c. At this temperature, taq – polymerase initiates DNA Synthesis at 3-OH end of primer.

6.What are Restriction enzyme? Why do bacteria have these restriction enzymes. Show diagrammatically a restriction enzyme its recognition & the product it produces?
Ans. Restriction enzymes are endonucleases which recognize a specific sequence within DNA andcut the DNA within that sequence at a specific point. In bacteria, these restriction enzymes operatea modification restriction system which modifies & cuts the foreign DNA entering into the bacterial cell& thus, provides immunity to bacterial cell.
Name of Restriction enzyme- EcoRISubstrate DNA on which it acts

Biotechnology: Principles and Processes Class 12 Biology MCQs

1. Biolistics (gene gun) is suitable for
(a) introducing rDNA into plant cells
(b) introducing rDNA into animal cells
(c) disarming the pathogen vectors
(d) DNA fingerprinting.

Answer

Answer: a

2. In genetic engineering experiments, restriction enzymes are used for
(a) viral DNA
(b) bacterial DNA
(c) eukaryotic DNA
(d) any type of DNA.

Answer

Answer: b

3. The DNA fragments produced by the use of restriction endonucleases can be separated by
(a) polymerase chain reaction
(b) gel electrophoresis
(c) density gradient centrifugation
(d) any of the above.

Answer

Answer: b

4. Plasmids in bacterial cells are
(a) extra-chromosomal DNA, which cannot replicate
(b) extra-chromosomal DNA, which can . self-replicate
(c) extra DNA associated with the genome
(d) extra DNA, associated with the genome, but cannot replicate.

Answer

Answer: b

5. The DNA polymerase enzyme used in PCR is obtained from
(a) Thermus aquaticus
(b) Escherichia coli
(c) Agrobacterium tumefaciens
(d) Salmonella typhimurium.

Answer

Answer: a

6. While isolating DNA from bacteria, which of the following enzymes is not used? [NCERT Exemplar]
(a) Lysozyme
(b) Ribonuclease
(c) Deoxyribonuclease
(d) Protease

Answer

Answer: a

7. Significance of ‘heat shock’ method in bacterial transformation is to facilitate [NCERT Exemplar]
(a) binding of DNA to the cell wall
(b) uptake of DNA through membrane transport proteins
(c) uptake of DNA through transient pores in the bacterial cell wall
(d) expression of antibiotic resistance gene

Answer

Answer: c

8. Which of the following steps are catalysed by Taq polymerase in a PCR reaction? [NCERT Exemplar]
(a) Denaturation of template DNA
(b) Annealing of primers to template DNA
(c) Extension of primer end on the template DNA
(d) All of the above

Answer

Answer: c

9. Which of the given statement is correct in the context of observing DNA separated by agarose gel electrophoresis? [NCERT Exemplar]
(a) DNA can be seen in visible light
(b) DNA can be seen without staining in visible light
(c) Ethidium bromide stained DNA can be seen in visible light
(d) Ethidium bromide stained DNA can be seen under exposure to UV light

Answer

Answer: d

10. ‘Restriction’ in Restriction enzyme refers to [NCERT Exemplar]
(a) cleaving ofphosphodiester bond in DNA by the enzyme
(b) cutting of DNA at specific position only
(c) prevention of the multiplication of bacteriophage in bacteria
(d) all of the above

Answer

Answer: c

11. The role of DNA ligase in the construction of a recombinant DNA molecule is [NCERT Exemplar]
(a) formation of phosphodiester bond between two DNA fragments
(b) formation of hydrogen bonds between sticky ends of DNA fragments
(c) ligation of all purine and pyrimidine bases
(d) none of the above

Answer

Answer: b

12. In an experiment, recombinant DNA bearing ampicillin-resistance gene is transferred into E.coli cells. The host cells are then cultured on a medium containing ampicillin. The result will be
(a) both transformants and non-transformants cannot survive.
(b) both transformants and non-transformants can survive.
(c) transformants only and not the non-transformants can survive.
(d) transformants cannot survive, but non-transformants can not.

Answer

Answer: c

13. The _______ in a vector helps in identifying the transformants and eliminating the non-transformants.

Answer/Explanation

Answer:
Explaination: Selectable marker.

14. When the enzyme _______ is inactivated in E.coli, the transformants do not produce any colour in the presence of a chromogenic substrate.

Answer/Explanation

Answer:
Explaination: β-galactosidase.

15. _______ is the method of bombarding high velocity microparticles of gold or tungsten coated with DNA, into plant cells.

Answer/Explanation

Answer:
Explaination: Biolistics/gene gun.

16. To isolate DNA from fungal cells for biotechnology experiments, enzyme _______ is necessary.

Answer/Explanation

Answer:
Explaination: Chitinase.

17. _______ is used as cloning vector for transformation in plant cells.

Answer/Explanation

Answer:
Explaination: Agmbacterium.

18. Downstream processing involves separation and _______ .

Answer/Explanation

Answer:
Explaination: Purification.

19. _______ is the process of extraction of DNA from the separated bands of DNA in agarose gel.

Answer/Explanation

Answer:
Explaination: Elution.

20. _______ gene in the E.coli vector, pBR 322 codes for enzymes/proteins involved in the replication of the plasmid.

Answer/Explanation

Answer:
Explaination: Rop.

21. _______ are the E.coli enzymes that remove the nucleotides from the ends of DNA strands.

Answer/Explanation

Answer:
Explaination: Exonucleases.

22. _______ is an autonomously replicating, circular, extra-chromosomal DNA in bacterial cells.

Answer/Explanation

Answer:
Explaination: Plasmid.

23. Match the items in Column I with those in Column II.

Column I	Column II
A. Sea weeds	1. Gel electrophoresis
B. Staining of DNA	2. Source of Agarose
C. Separation of DNA fragments	3. Isolation of DNA from the gel
D. Elution	4. Ethidium bromide

Answer/Explanation

Answer:
Explaination: A – 2, B – 4, C – 1, D – 3

24. Match the Column l with the Column II.

Column I	Column II
A. Competent host	1. Separation and purification
B. Cloning vector	2. Culturing of cells in large volumes
C. Downstream processing	3. Taq polymerase
D. PCR	4. Divalent cation (Ca2+)
E. Bioreactor	5. pBR 322
	6. Gel electrophoresis

Answer/Explanation

Answer:
Explaination: A – 4, B – 5, C – 1, D – 3, E – 2

25. Since DNA fragments are positively charged they move towards the anode. [True/False]

Answer/Explanation

Answer:
Explaination: False.

26. Agarose, the most commonly used matrix in gel electrophoresis is obtained from fungi. [True/False]

Answer/Explanation

Answer:
Explaination: False.

27. The DNA fragments separate according to their size through agarose gel during electrophoresis. [True/False]

Answer/Explanation

Answer:
Explaination: True.

28. The cloning vector pBR 322 has three antibiotic-resistance genes. [True/False]

Answer/Explanation

Answer:
Explaination: False.

29. The vector DNA and foreign DNA are cut by the same restriction endonuclease. [True/False]

Answer/Explanation

Answer:
Explaination: True.

Directions (Q30 to Q32): Mark the odd one in each of the following groups.
30. Cellulose, Lysozyme, Chitinase, Endonuclease

Answer/Explanation

Answer:
Explaination: Endonuclease.

31. Hind III, EcoRI, Sal I, Rop

Answer/Explanation

Answer:
Explaination: Rop.

32. Denaturation, Elution, Annealing, Extension

Answer/Explanation

Answer:
Explaination: Elution.

33. How has European Federation of Biotechnology (EFB) defined biotechnology?

Answer/Explanation

Answer:
Explaination: The European Federation of Biotechnology defines biotechnology as the integration of natural science and organisms, cells or parts thereof and molecular analogues, for products and services; it encompasses both .traditional and modem molecular biology.

34. Name the technique that is used to alter the chemistry of genetic material (DNA/RNA) to obtain the desired result. [Delhi 2016C]

Answer/Explanation

Answer:
Explaination: Genetic engineering.

35. What is meant by gene cloning?

Answer/Explanation

Answer:
Explaination: Gene cloning refers to the process in which a numer of identical copies of a gene of interest are made by introducing the gene into an appropriate host using a suitable Vector.

36. Why is it not possible for an alien DNA to become part of a chromosome anywhere along its length and replicate normally? [AI 2014]

Answer/Explanation

Answer:
Explaination: The alien DNA has to be linked to a specific sequence of DNA, called origin of replication, on the chromosome; this origin of replication is responsible for initiation of replication.

37. Name the specific sequence of DNA in a plasmid that the ‘gene of interest’ ligates with, to enable it to replicate. [AI 2013C]

Answer/Explanation

Answer:
Explaination: Origin of replication (Ori)

38. Write the two components of the first artificial recombinant DNA molecule constructed by Cohen and Boyer. [Foreign 2014]

Answer/Explanation

Answer:
Explaination: An antibiotic-resistance gene and the plasmid of Salmonella typhimurium.

39. Name the two enzymes that are essential for constructing a recombinant DNA. [Delhi 2017C]

Answer/Explanation

Answer:
Explaination: Restriction endonuclease and DNA ligase.

40. In the year 1963, two enzymes responsible for restricting the growth of bacteriophage in E.coli were isolated. How did the enzymes act to restrict the growth of the bacteriophage? [AI 2011C]

Answer/Explanation

Answer:
Explaination:
– One of them (exonucleases) added methyl groups to DNA.
– The other (endonucleases) cut the DNA at specific points within the DNA.

41.Name the enzyme that helps to join DNA fragments. [AI 2017C]

Answer/Explanation

Answer:
Explaination: DNA ligase

42. Mention the role of Restriction enzyme in Recombinant DNA technology. [Delhi 2017C]

Answer/Explanation

Answer:
Explaination: They act as molecular scissors to cut DNA at specific locations.

43. What does ‘R’ stand for, in the restriction endonuclease, EcoRI?

Answer/Explanation

Answer:
Explaination: R stands for RY 13, the strain of E.coli bacterium.

44. A plasmid and a DNA sequence in a cell need to be cut for producing recombinant DNA. Name the enzyme that acts as molecular scissors to cut the DNAs.

Answer/Explanation

Answer:
Explaination: Restriction endonucleases.

45. Suggest a technique to a researcher who needs to separate fragments of DNA. [Delhi 2016]

Answer/Explanation

Answer:
Explaination: Gel electrophoresis.

46. Name the material used as matrix in gel electrophoresis and mention its role. [AI 2014C]

Answer/Explanation

Answer:
Explaination: Agarose; it provides the sieving effect for the DNA to resolve according to their size.

47. Why do DNA-fragments move towards the anode during gel electrophoresis? [CBSE 2018C, Delhi 2011C; HOTS]

Answer/Explanation

Answer:
Explaination: DNA-fragments are negatively charged and hence, move towards the anode.

48. What is the role of ethidium bromide during agarose-gel electrophoresis of DNA fragments?

Answer/Explanation

Answer:
Explaination: The gel is stained by ethidium bromide, to view the separated DNA bands when exposed to UV light.

49. Which main technique and instrument is used to isolate DNA from a plant cell? [CBSE Sample Paper 2013, 2014]

Answer/Explanation

Answer:
Explaination:
– Centrifugation is the technique.
– Centrifuge is the instrument.

50. Name two commonly used vectors for genetic engineering.

Answer/Explanation

Answer:
Explaination: Plasmids and bacteriophages.

51. Why are engineered vectors preferred these days?

Answer/Explanation

Answer:
Explaination:
(i) Engineered vectors facilitate easy linking of foreign DNA.
(ii) They help in selection of recombinants.

52. Mention the uses of cloning vectors in biotechnology. [Delhi 2011]

Answer/Explanation

Answer:
Explaination:
(i) Cloning vectors are used to make multiple copies of the desired DNA/ gene.
(ii) They are used to transfer the gene of interest to the host cell.

53. Why is ‘plasmid’ an important tool in biotechnology experiments? [AI2013C]

Answer/Explanation

Answer:
Explaination: Since plasmids can replicate within the bacterial cell independently of the genomic DNA, any alien DNA ligated to it will also multiply, i.e. it is used as a vector as well as in gene cloning.

54. Why is it essential to have a ‘selectable marker’ in a cloning vector? [AI 2010; HOTS]

Answer/Explanation

Answer:
Explaination: It helps in identifying the recombinants from the non-recombinants.

55. Why are antibiotic-resistance genes used as markers in E.coli? [HOTS]

Answer/Explanation

Answer:
Explaination: It is because the normal E. coli cells do not have resistance to any such antibiotics.

56. Name two antibiotic-resistance genes in the pBR 322 of E.coli plasmid.

Answer/Explanation

Answer:
Explaination: Ampicillin-resistance gene and tetracyclin- resistance gene.

57. A plasmid without a selectable marker was chosen as vector for cloning a gene. How does this affect the experiment? [HOTS]

Answer/Explanation

Answer:
Explaination: In the absence of a selectable marker, it will not be possible to identify the recombinants from the non-recombinants.

58. State what happens when an alien gene is ligated at Sal I site of pBR 322 plasmid. [Delhi 2013C]

Answer/Explanation

Answer:
Explaination: The transformant loses the tetracycline- resistance.

59. State what happens when an alien gene is ligated at Pvu I site of pBR 322 plasmid. [AI 2013C]

Answer/Explanation

Answer:
Explaination: The transformant loses ampicillin-resistance.

60. How is Agrobacterium tumefaciens able to transform a normal plant cell into a tumour? [Delhi 2013C]
Or
Biotechnologists refer to Agrobacterium tumefaciens as a natural genetic engineer of plants. Give reasons to support the statement. [AI 2011]

Answer/Explanation

Answer:
Explaination: When it infects a plant cell, it delivers a part of its DNA, called ‘T-DNA’ (or Tumour- inducing (Ti) plasmid) into the plant cell and transforms it into a tumour cell.

61. How can retroviruses be used efficiently in biotechnology experiments in spite of them being disease causing? [AI 2013C]

Answer/Explanation

Answer:
Explaination: Retroviruses are disarmed (diseases-causing gene is removed/inactivated) and used as vectors to deliver the recombinant/alien DNA into animal cells.

62. Name the commonly used vector for transformation in plant cells.

Answer/Explanation

Answer:
Explaination: Agrobacterium tumefaciens.

63. How does an alien DNA gain entry into a plant cell by ‘biolistic’ method? [Foreign 2013]

Answer/Explanation

Answer:
Explaination: The cells are bombarded with high velocity micro-particles of gold or tungsten, coated with the alien DNA.

64. Mention the type of host cells suitable for the gene guns to introduce an alien DNA. [Delhi 2014]

Answer/Explanation

Answer:
Explaination: Plant cells.

65. Name the host cells in which micro-injection technique is used to introduce an alien DNA. [Foreign 2014]

Answer/Explanation

Answer:
Explaination: Animal cells.

66. Why is the enzyme cellulase needed for isolating genetic material from plant cells and not from animal cells? [Delhi 2013, 2010; HOTS]

Answer/Explanation

Answer:
Explaination: Cellulase is used to digest the cell wall (cellulose) of plant cells; animal cells have no cell wall and hence, it is not needed.

67. How can bacterial DNA be released from the bacterial cell for biotechnology experiments? [Delhi 2011]

Answer/Explanation

Answer:
Explaination: The bacterial cell has to be treated with lysozyme, to digest the cell wall to release DNA.

68. To use the restriction enzyme to cut the DNA, the DNA must be in a pine form, i.e. free from proteins and RNAs associated with it. How is it achieved?

Answer/Explanation

Answer:
Explaination:
– Proteins are removed by using proteases.
– RNAs are removed by using ribonucleases (RNases).

69. Write the names of the enzymes that are used for isolation of DNA from bacterial and fungal cells, respectively for Recombinant DNA technology. [AI, Foreign 2014]

Answer/Explanation

Answer:
Explaination:
– Lysozymes for bacterial cells.
– Chitinase for fungal cells.

70. Write the importance of the bacterium Thermus aquaticus in polymerase chain reaction. [Delhi 2013C]

Answer/Explanation

Answer:
Explaination: It is the source of thermostable DNA- polymerase enzyme.