Ch.10 MICROBES IN HUMAN WELFARE
IMPORTANT CONCEPTS AND DEFINNITIONS1. Fermentors – Production of beverages and antibiotics on an industrial scale, requires
microbes in very large vessels called fermentors .
growing
2. Antibiotics- Antibiotics are chemical substances, which are produced by some microbes and can
kill or retard the growth of other (disease-causing) microbes.
3. Primary treatment : The treatment step of sewage for physical removal of particles – large and
small – from the sewage through filtration and sedimentation , is called . Primary treatment .
4. Secondary treatment or Biological treatment : After primary treatment .the primary effluent is
passed into large aeration tanks where it is constantly agitated mechanically and air is pumped
associated with fungal filaments to form mesh like structures).
5. BOD (biochemical oxygen demand) -BOD refers to the amount of the oxygen that would be
consumed if all the organic matter in one liter of water were oxidised by bacteria
6. Activated sludge- Once the BOD of sewage or waste water is reduced significantly, the
effluent is then passed into a settling tank where the bacterial ‘flocs’ are allowed to sediment.
This sediment is called activated sludge.
7. Anaerobic sludge digesters- A small part of the activated sludge is pumped back into the
aeration tank to serve as the inoculum. The remaining major part of the sludge is pumped into
large tanks called anaerobic sludge digesters.where anaerobica bacteria and the fungi digest the
sludge.
8. Methanogens- Certain bacteria, which grow anaerobically on cellulosic material, produce large
amount of methane along with CO2 and H2. These bacteria are collectively called methanogens,
9. Biocontrol refers to the use of biological methods (using biological agents for natural predation )
for controlling plant diseases and pests , rather than using chemicals .
10. Biofertilisers- In agriculture Biofertilisers are organisms that enrich the nutrient quality of the
soil.
11. Organic farming –Agriculture by using only biofertilisers is called organic farming because
there are problems of pollution , associated with the overuse of chemical fertilizers. The main
sources of biofertilisers are bacteria, fungi and cyanobacteria.
12. Mycorrhiza- Fungal symbiotic association with plants which helps plants to absorb phosphorus
from soil , is called mycorrhiza. Many members of the genus Glomus form mycorrhiza..
13. Cyanobacteria -Cyanobacteria are autotrophic microbes widely distributed in aquatic and
terrestrial environments many of which can fix atmospheric nitrogen, e.g. Anabaena, Nostoc,
Oscillatoria, etc.
IDENTICAL TERMS1. Fermented Beverages- Wine and beer are produced without distillation after fermentation so they are called as
fermented beverages.
Distillation Beverages- Whisky, brandy and rum are produced by distillation of the fermented broth that’s
why they are called “Distillation Beverages.”
2. Acetobactor aceti- A bacterium which produces acetic acid commercially.
Lactobacillus- A bacterium which grows in milk and convert it into curd by producing lactic acid.
3. Primary Sludge- After primary treatment all solids that settle down like soil, small pebbles, settled debris
etc.is called primary sludge.
Activated Sludge- During secondary treatment the ‘flocs’ (masses of bacteria associated with fungal
filaments to form mesh like structures) are allowed to sediment. This sediment is called activated sludge.
4. Endomycorrhiza- Intimated fungal association with certain woody plants is endomycorrhiza.
Ectomycorrhiza- superficial fungal association with certain woody plants is endomycorrhiza.
5. Biocontrol - A method of controlling pests that relies on natural predation rather than introduced
chemicals.
Biofertilizers- Biofertilisers are organisms that enrich the nutrient quality of the soil.
IMPORTANT POINTS, PRINCIPLES AND THEORYMICROBES IN HOUSEHOLD PRODUCTSMicro-organisms such as Lactobacillus and others commonly called lactic acid bacteria (LAB) grow in milk
and convert it to curd. LAB produce acids that coagulate and partially digest the milk proteins. It also
improves its nutritional quality by increasing vitamin B12. In our stomach too, the LAB play very beneficial
role in checking diseasecausing microbes.
The dosa and idli is also fermented by bacteria. The dough, which is used for making bread, is fermented
using baker’s yeast (Saccharomyces cerevisiae). A number of traditional drinks and foods are also made
by fermentation by the microbes. ‘Toddy’, a traditional drink of some parts of southern India is made by
fermenting sap from palms.
Microbes are also used to ferment fish, soyabean and bamboo shoots to make foods.
Cheese, is one of the oldest food items in which microbes were used. The large holes in ‘Swiss cheese’
are due to production of a large amount of CO2 by a bacterium named Propionibacterium sharmanii. The
‘Roquefort cheese’ are ripened by growing a specific fungi on them, which gives them a particular flavour.
MICROBES IN INDUSTRIAL PRODUCTSIin industry, microbes are used to synthesise a number of products valuable to human beings. Beverages
and antibiotics are some examples. Production on an industrial scale, requires growing microbes in very
large vessels called fermentors .
Fermented BeveragesMicrobes like yeast are used for the production of beverages like wine, beer, whisky, brandy or rum.
The same yeast Saccharomyces cerevisiae used for bread-making and commonly called brewer’s yeast,
is used for fermenting malted cereals and fruit juices, to produce ethanol.
Wine and beer are produced without distillation whereas whisky, brandy and rum are produced
by distillation of the fermented broth.
AntibioticsAnti is a Greek word that means ‘against’, and bio means ‘life’, together they mean ‘against life’ (in the
context of disease causing organisms); whereas with reference to human beings, they are ‘pro life’ and
not against.
Antibiotics are chemical substances, which are produced by some microbes and can kill or retard the
growth of other (disease-causing) microbes.
Alexander Fleming while working on Staphylococci bacteria, once observed a mould growing in one of his
unwashed culture plates around which Staphylococci could not grow. He found out that it was due to a
chemical produced by the mould and he named it Penicillin after the mould Penicillium notatum. Its full
potential as an effective antibiotic was established by Ernest Chain and Howard Florey. This antibiotic
was extensively used to treat American soldiers wounded in World War II. Fleming, Chain and Florey
were awarded the Nobel Prize in 1945, for this discovery.
Antibiotics have greatly improved our capacity to treat deadly diseases such as plague, whooping cough
(kali khansi ), diphtheria (gal ghotu) and leprosy (kusht rog), which used to kill millions all over the globe.
Today, we cannot imagine a world without antibiotics.
Chemicals, Enzymes and other Bioactive MoleculesAspergillus niger (a fungus) produces citric acid,
Acetobacter aceti (a bacterium) produces acetic acid;
Clostridium butylicum (a bacterium) produces butyric acid
Lactobacillus (a bacterium) produces lactic acid.
Yeast (Saccharomyces cerevisiae) is used for commercial production of ethanol.
Enzymes like Lipases are used in detergent formulations and are helpful in removing oily stains from the
laundry.
The bottled juices are clarified by the use of pectinases and proteases.
Streptokinase produced by the bacterium Streptococcus and modified by genetic engineering is used as
a ‘clot buster’ for removing clots from the blood vessels of patients who have undergone myocardial
infraction leading to heart attack.
Cyclosporin A, that is used as an immunosuppressive agent in organ-transplant patients, is produced by
the fungus Trichoderma polysporum.
Statins produced by the yeast Monascus purpureus have been commercialised as blood-cholesterol
lowering agents. It acts by competitively inhibiting the enzyme responsible for synthesis of cholesterol.
MICROBES IN SEWAGE TREATMENTThe municipal waste-water including human excreta is also called sewage. It contains large amounts of
organic matter and microbes. Many of which are pathogenic.
This cannot be discharged into natural water bodies like rivers and streams directly .Before disposal,
hence, sewage is treated in sewage treatment plants (STPs) to make it less polluting. Treatment of waste
water is done by the heterotrophic microbes naturally present in the sewage. This treatment is carried out
in two stages:
Primary treatment : These treatment steps basically involve physical removal of particles – large and
small – from the sewage through filtration and sedimentation. These are removed in stages; initially,
floating debris is removed by sequential filtration. Then the grit (soil and small pebbles) are removed by
sedimentation. All solids that settle form the primary sludge, and the supernatant forms the effluent. The
effluent from the primary settling tank is taken for secondary treatment.
Secondary treatment or Biological treatment : The primary effluent is passed into large aeration tanks
,where it is constantly agitated mechanically and air is pumped into it. This allows vigorous growth of
useful aerobic microbes into flocs (masses of bacteria associated with fungal filaments to form mesh like
structures). While growing, these microbes consume the major part of the organic matter in the effluent.
This significantly reduces the BOD (biochemical oxygen demand) of the effluent. BOD refers to the
amount of the oxygen that would be consumed if all the organic
matter in one liter of water were oxidised by bacteria. The sewagewater is treated till the BOD is reduced.
The BOD test measures the rate of uptake of oxygen by micro-organisms in a sample of water and thus,
indirectly, BOD is a measure of the organic matter present in the water. The greater the BOD of waste
water, more is its polluting potential. Once the BOD of sewage or waste water is reduced significantly, the
effluent is then passed into a settling tank where the bacterial ‘flocs’ are allowed to sediment. This
sediment is called activated sludge. A small part of the activated sludge is pumped back into the aeration
tank to serve as the inoculum.
The remaining major part of the sludge is pumped into large tanks called anaerobic sludge digesters.
Here, other kinds of bacteria, which grow anaerobically, digest the bacteria and the fungi in the sludge.
During this digestion, bacteria produce a mixture of gases such as methane, hydrogen sulphide and
carbon dioxide. These gases form biogas and can be used as source of energy as it is inflammable.
MICROBES IN PRODUCTION OF BIOGASBiogas is a mixture of gases (containing predominantly methane) produced by the microbes and which
may be used as fuel.
Certain bacteria, which grow anaerobically on cellulosic material, produce large amount of methane along
with CO2 and H2. These bacteria are collectively called methanogens, and one such common bacterium
is Methanobacterium.
These bacteria are found in the anaerobic sludge during sewage treatment. These bacteria are also
present in the rumen (a part of stomach) of cattle. A lot of cellulosic material present in the food of cattle
is also present in the rumen. In rumen, these bacteria help in the breakdown of cellulose and play an
important role in the nutrition of cattle. Thus, the excreta (dung) of cattle, commonly called gobar, is rich in
these bacteria. Dung can be used for generation of biogas, commonly called gobar gas.
A TYPICAL BIOGAS PLANT
The biogas plant consists of a concrete tank (10-15 feet deep) in which bio-wastes are collected and a
slurry of dung is fed. A floating cover is placed over the slurry, which keeps on rising as the gas is
produced in the tank due to the microbial activity. The biogas plant has an outlet, which is connected to a
pipe to supply biogas to nearby houses. The spent slurry is removed through another outlet and may be
used as fertiliser. Cattle dung is available in large quantities in rural areas where cattle are used for a
variety of purposes. So biogas plants are more after build in rural areas. The biogas thus produced is
used for cooking and lighting.
The technology of biogas production was developed in India mainly due to the efforts of Indian
Agricultural Research Institute (IARI) and Khadi and Village Industries Commission (KVIC).
MICROBES AS BIOCONTROL AGENTSBiocontrol refers to the use of biological methods for controlling plant diseases and pests. Use of
chemicals like insecticides and pesticides causes great harm.
These chemicals are toxic and extremely harmful, to human beings and animals alike, and have been
polluting our environment (soil, ground water), fruits, vegetables and crop plants. Our soil is also polluted
through use of weedicides to remove weeds.
Biological control of pests and diseases: It is a method of controlling pests that relies on natural predation
rather than introduced chemicals. A key belief of the organic farmer is that biodiversity furthers health.
The organic farmer works to create a system where the insects that are sometimes called pests are not
eradicated, but instead are kept at manageable levels by a complex system of checks and balances
within a living and vibrant ecosystem.
This is a holistic approach that seeks to develop an understanding of the webs of interaction between the
myriad of organisms that constitute the field fauna and flora.
The very familiar beetle with red and black markings – the Ladybird, and Dragonflies are useful to get rid
of aphids and mosquitoes, respectively.
An example of microbial biocontrol agents that can be introduced in order to control butterfly caterpillars is
the bacteria Bacillus thuringiensis (often written as Bt ). These are available in sachets as dried spores
which are mixed with water and sprayed onto vulnerable plants such as brassicas and fruit trees, where
these are eaten by the insect larvae. In the gut of the larvae, the toxin is released and the larvae get
killed.
A biological control being developed for use in the treatment of plant disease is the fungus Trichoderma.
Trichoderma species are free-living fungi that are very common in the root ecosystems.
Baculoviruses (genus Nucleopolyhedrovirus) are pathogens that attack insects and other arthropods.
These viruses are excellent candidates for species-specific, narrow spectrum insecticidal applications.
The beneficial insects are being conserved to aid in an overall integrated pest management (IPM)
programme, or when an ecologically sensitive area is being treated.
MICROBES AS BIOFERTILISERSBiofertilisers are organisms that enrich the nutrient quality of the soil. The main sources of biofertilisers
are bacteria, fungi and cyanobacteria.
The nodules on the roots of leguminous plants formed by the symbiotic association of Rhizobium. These
bacteria fix atmospheric nitrogen into organic forms, which is used by the plant as nutrient. Other bacteria
can fix atmospheric nitrogen while free-living in the soil (examples Azospirillum and Azotobacter), thus
enriching the nitrogen content of the soil.
Fungi are also known to form symbiotic associations with plants (mycorrhiza). Many members of the
genus Glomus form mycorrhiza. The fungal symbiont in these associations absorbs phosphorus from soil
and passes it to the plant. Plants having such associations show other benefits also, such as resistance
to root-borne pathogens, tolerance to salinity and drought, and an overall increase in plant growth and
development.
Cyanobacteria are autotrophic microbes widely distributed in aquatic and terrestrial environments many of
which can fix atmospheric nitrogen, e.g. Anabaena, Nostoc, Oscillatoria, etc. In paddy fields,
cyanobacteria serve as an important biofertiliser. Blue green algae also add organic matter to the soil and
increase its fertility. Currently, in our country, a number of biofertilisers are available commercially in the
market and farmers use these regularly in their fields to replenish soil nutrients and to reduce
dependence on chemical fertilisers.
HOTS
1 mark
1) Drinks like Whisky and Rum are more intoxicating than wine. Why?
2) Can we imagine a world without antibiotics? Explain briefly.
3) Bottled juices are clearer compared to homemade juice. Give reason.
4) Which one of these is a pertinacious infecting agent?
a) Viroids, b) prions, c) protern, d) wat.
5) An organic farmer requires a bio control agent which is species specific and
has no negative impact on non-target organism. Suggest the name of such a bio
control agent.
6) Scientists have succeeded in introducing toxin gene of Bacillus thuringiensis
into plants like cotton. What purpose is achieved through this action? 1
7)A small amount of curd was added to cold milk for converting it into curd. But
milk did not get curdled. Why?
8) Consuming curd keeps the gastro-intestinal tract in tact. Give reason.
9) On fermentation the dough of idli and dosa puffs up. Which metabolic
pathway is responsible for this process?
10) How do lactic acid bacteria help in increasing the nutritional quality of curd?
11) The excreta of cattle do not contain any cellulose but human excreta may
contain cellulose. Why?
12) Antibiotics are always sold in combination with lactobacillus. Why?
13) Why are some microbes useful in detergent formulation?
14) Microbes release gases during metabolism. How will it help in the
production of energy?
15) Why does 'Swiss-cheese' contain large holes?.
16)In what way the relationship between BOD and organic matter in Sewage
will be useful in ecology?
17)Why do bottled fruit juices appear clearer than the home made ones?
18)What is the principle behind the conversion of milk into curd and partial
digestion of milk protein performed by Lactic Acid Bacteria?
19)Wine and beer are different from whisky and brandy though they are all
alcoholic beverages. What is the name of the process that brings out this
difference?
20)Name the group of bacteria that are capable of living at high temperature
above 1000C. How does it become possible by these bacteria?(1/2+1/2)
21)Why do doctors prescribe Streptokinases for brain hemorrhage patients?
Mention the source of industrial production of this biomolecule.(1/2 + ½)
22)How are the holes produced in bread and cheese?
23.) Three water samples namely river water, untreated sewage water and secondary
effluent discharge from a sewage treatment plan were subjected to BOD test. The
samples were labeled A, B and C but the lab attendant did not note which BOD is for
which water. The BOD values of three samples A, B and C were recorded as 20mg/L,
8mg/L and 400mg/L respectively. Which sample of the water is most polluted? Can
you assign the correct label to each, assuming that the river water is relatively clean.
24. What are prions?
25. What is an antibiotics?
26. Identify the microbe grown on nutritive media in the given figure.
27. Why
there are large holes in ‘Swiss cheese’? Name the
bacterium used for this.
28. Give reasons why Staphylococci could not grow in unwashed culture plates of Alexander Fleming
experiment?
29. Which bioactive molecule used as an immunosuppressive agent in organ-transplant patients?
30. What role do LAB play in our stomach?
31. What is toddy?
32. What are flocs?
33. Name the two plans initiated by Ministry of Environment and Forests to save major rivers of our
country from pollution.
34. Which antibiotic was extensively used to treat American soldiers wounded in World War II ?
35. Name the free-living fungi that are very common in the root ecosystems which are effective
biocontrol agents of several plant pathogens.
2 marks
1) A farmer was suggested to apply certain microbial culture in his field to
increase the yield. Suggest the types of microbes he could use in his paddy field
and how do they help in increasing the yield.
2) It was observed that certain plant roots are infected by fungus, in spite of this
infection the plant showed increased growth and development. Give reason.
3) Arrange the steps in sewage treatment in proper sequence.
a) Anaerobic sludge digestion
b) 'Floc' formation
c) Primary effluent in aeration tank
d) Formation of biogas
e) Constant agitation in presence of oxygen.
f)Digestion by anaerobic bacteria
4) Bacteria that convert effluent into activated sludge play one more beneficial
side.Mention the role
5) In the given figure label X & Y
and explain the functioning of the below plant.
6) How does Monascus help in lowering blood-cholesterol?
7) Complete the given table
Name of the organism
8.
9.
Product/Enzyme/Bioactive molecule
Aspergillus niger
(i)
(ii)
Ethanol
(iii)
(iv)
Cyclosporin-A
Acetic acid
Streptococcus
(v)
Monascus purpureus
(vi)
Name two alcoholic beverages produced in each of the following: (i) with distillation (ii)
without distillation.
Student tested the three water samples for BOD from different areas and he got following
results.
47
46.5
46
mg/L 45.5
45
44.5
44
Sample A
Answer the following based on above graph:-
Sample B
Sample C
(i)
Which water sample has more BOD?
(ii)
Which sample of the water is most polluted?
10. How Cyanobacteria increase fertility of soil?
11. What are the disadvantages of ‘conventional’ farming practices? How biocontrol helps in this
regard?
12. State the role of following enzymes where they are used;
(i) Lipases (ii) Streptokinase
13. Name the scientist who shared the nobel prize in 1945 for the discovery of antibiotic. From which
fungus that antibiotic was extracted?
14.Fill in the blanks in the different columns of the table given below:
Organic Acid
Source Microbe
a
Lactobacillus
Acetic acid
b
c
Clostridium butylicum
Citric acid
d
15.What is the puffed up appearance of dosa due to? Which metabolic pathway involved in it?
16.What is the role of Baculoviruses in IPM? Name one of them used in this programme.
17.List the common uses of microbes in human welfare.
3 marks
1) For what significant properties the Baculoviruses are considered as best Biological
control agent?. Mention its importance in organic farming.
2). Give one example and one use of the following:i) Free living fungi
ii) Symbiotic fungi
iii) Free living bacteria
3. How do biofertilizers enrich the fertility of the soil?
4. What is biological treatment? How it helps in treatment of effluents?
5. What are statins? Where are they produced from? How are they useful to man?
6. (i) Why is cow dung used in the generation of biogas?
(ii) What are the major components of biogas?
(iii) Name the common bacterium involve in biogas formation
5 Marks
1. Why is organic farming favoured these days? Describe the methods employed in the
process.
2. Describe the treatment of sewage before it is discharged into a water body like a river.
3. (i) Why crystals of Bt not kill bacteria it self? How they kill insects?
(ii) Do you think microbes can also be used as source of energy? If yes, how?
4. With the help of labeled diagram explain the production of biogas in your locality.
Questions with hints of model answer
Section - A: 1 Mark Questions [To be answered in one word or in one sentence]
1. Expand the following (each of 1 mark)
i. STP
Answer: Sewage Treatment Plant
ii)
BOD
Answer: Biochemical Oxygen Demand.
iii) IPM
Answer: Integrated Pest Management
iv. IARI
Answer: Indian Agricultural Research Institute.
v. LAB
Answer: Lactic Acid Bacteria
xiv) IVRI
Answer: Indian Veterinary Research Institute
2. Why there is a big hole in Swiss cheese?
Answer: The large holes in “Swiss cheese” are due to production of a large amount of CO 2 by a
bacterium.
ii) Ratna
3. Write the name of micro-organism responsible for production of citric Acid and Butyric Acid.
Answer: i) Aspergillus niger (Fungus) – Citric Acid.
ii) Clostridium butylicum (a bacterium) – butyric Acid
4. Name the fungus which is very commonly found in the root ecosystem used in treatment of plant
diseases.
Answer: Trichoderma.
5. Which fungus is effective bio control agents of several Plants Pathogens?.
Answer: Trichoderma
Section – B: 2 Marks Questions [To be answered in 20-30 words approximately]
1. Define “bioactive molecules”. Name two such molecules and give their sources.
Answer: Bioactive molecules are those organic compounds which are produced on commercial
scale by the microbes and are useful in the human welfare.
Cyclosporin A - produced by fungus
Statins
- produced by yeast.
2. What is activated sludge?
Answer: It is a measure for secondary treatment of sewage water. In this measure sludge is used
which contain bacteria & algae.
7. Name a bioactive molecule used as an immunosuppressive agent in organ transplant patients
also mention its source.
Answer: Bioactive molecule – Cyclosporin A
Source – Trichoderma Polysporum(Fungus)
10. What is mycorrhiza? How is it useful to some plants?
Answer: Fungal association with the roots of some higher plants are known as mycorrhiza.
It increase phosphorous absorption from the soil.
Section – C: 3 Marks Questions [To be answered in 30-50 words approximately]
1. What are antibiotics? Give two examples. What is their significance?
Answer:
Antibiotics : - are the chemical substances produced by certain microbes, that can kill or
retard the growth of other disease causing microbes.
e.g – Penicillin, Streptomycin
Significance: Antibiotics are used in the treatment of many human, animal and plant diseases.
2. Give the name of the microbes from which cyclosporine A and statins are obtained.
cyclosporine A and status are useful to man?
How
Answer:
Cyclosporin A is produced by the fungus
Trichoderma polysporum
Statins are produced by a yeast, Monascus purpureus.
Uses – Cyclosporin A is used as an immuno suppressive agent during organ
transplantation. Statins are used for lowering the levels of blood cholesterols, as it acts as a
competitive inhibitor of the enzyme involved in cholesterol synthesis.
3. Name any three organic acids and the bacteria that produce them.
Answer:
Organic Acids
i) Acetic Acid
ii) Butyric acid
iii) Lactic acid
Bacteria
Acetobacter aceti
Clostridium butylicum
Lactobacillus delbrueckii
Section – D: 5 Marks Questions [To be answered in 80 -120 words approximately]
1. How do biofertilisers enrich the fertility of the soil?
Answer:
Biofertilizers are the organisms that enrich the nutrient quality of soil.
Bacteria, cyanobacteria and fungi are the three groups of organisms used as biofertilisers.
i)
Bacteria
a) symbiotic bacteria Rhizobium.
b) Free living bacteria Azospirillum & Azotobactor.
c) They fix the atmospheric nitrogen and enrich soil nutrients.
ii)
Cyanobacteria
Cyanobacteria are autotrophic microbes widely distributed in aquatic and
terrestrial environment. Many of them can fix atmospheric nitrogen and increase the organic
matter of the soil through their photosynthetic activity.
In paddy field cyanobacteria serves as important biofertilizer. Commercially fermers use
these cyanobacteria in their fields to replenish soil nutrients and to reduce dependency on
chemical fertilizers.
Important examples are Anabaena, Nostoc and Oscillatoria.
iii)
Fungi/ Micorrhiza
Fungi are also known to develop symbiotic relationship with plant roots. Such a
relationship is called micorrhiza. Many members of the genus Glomus form micorrhizae.
The fungus absorbs phosphorus from the soil and passes it to the plant.
Plants having such association show other benefits alsoa) Resistance to root borne pathogens
b) Tolerance to salinity.
c) Tolerance to drought
d) Overall increase in the plant growth and development.
Ch.11 BIOTECHNOLOGY-PRINCIPLES & PROCESSES
IMPORTANT CONCEPTS AND DEFINNITIONS1. Biotechnology deals with techniques of using live organisms or enzymes from organisms to
produce products and processes useful to humans.
2. Biotechnology - The definition given by EFB is as follows:
‘The integration of natural science and organisms, cells, parts thereof, and molecular analogues
for products and services’.
3.
Genetic engineering : 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.
4.
Restriction enzymes: Enzymes that are used to cut DNA segment at a specific site are called
restriction enzymes.
5. Exonucleases remove nucleotides from the ends of the DNA molecule.
6. Endonucleases make cuts at specific positions within the DNA molecule.
7.
Plasmid - autonomously replicating circular extra-chromosomal DNA of any bacteria.
8.
Origin of replication - a specific DNA sequence which is responsible for initiating replication is
called origin of replication
9.
Vectors - These are plasmid DNA or viruses act as vehicle to transfer the piece of DNA attached
to it.
10. Palindromic Nucleotide Sequences - 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.
11. Gel electrophoresis - a technique which is used to separate the fragments of DNA is known as
gel electrophoresis.
12. Transformation is a procedure through which a piece of DNA is introduced in a host bacterium
13. Insertional inactivation – The procedure of inserting a recombinant DNA within coding sequence of a
functional gene which makes that gene inactive (unable to express) is called insertional inactivation.
14. Selectable marker- the gene encoding desirable information useful in identifying and eliminating
nontransformants and selectively permitting the growth of the transformants is called selectable
marker.
15. Micro-injection- a technique in which recombinant DNA is directly injected into the nucleus of an
animal cell.
16. Biolistics or Gene gun - plant cells are bombarded with high velocity micro-particles of gold or tungsten coated
with DNA in a method known as biolistics or gene gun.
17. Bioreactors - bioreactors can be thought of as vessels in which raw materials are biologically
converted into specific products, individual enzymes, etc., using microbial plant, animal or human cells.
IDENTICAL TERMS1. Endonucleases / Exonucleases
2. Vectors / Plasmids
3. Genetic Engineering / Recombinant DNA Technology
4. Recombinant DNA / Recombinant Protein
5. Simple Stirred-tank Bioreactor / Sparged Stirred-tank Bioreactor
IMPORTANT POINTS, PRINCIPLES AND THEORY1. Genetic engineering : 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.
2. 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.
3. Three basic steps in genetically modifying an organism —
(i) identification of DNA with desirable genes;
(ii) introduction of the identified DNA into the host;
(iii) maintenance of introduced DNA in the host and transfer of the DNA
to its progeny.
TOOLS OF RECOMBINANT DNA TECHNOLOGY--1. DNA technology can be accomplished only if we have the key tools, i.e., restriction enzymes,
polymerase enzymes, ligases, vectors and the host organism.
2. The first restriction endonuclease–Hind II
3.
Hind II always cut DNA molecules at a particular point by recognising a specific sequence of
six base pairs. This specific base sequence is known as the recognition sequence for Hind II.
4. The convention for naming these enzymes is the first letter of the name comes from the genes
and the second two letters come from the species of the prokaryotic cell from which they were
isolated, e.g., EcoRI comes from Escherichia coli RY 13.
5.
In EcoRI, the letter ‘R’ is derived from the name of strain. Roman numbers following the names
indicate the order in which the enzymes were isolated from that strain of bacteria.
6.
Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the
DNA.
7.
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. For example, the following sequences reads the same on
the two strands in 5' 
This is also true if read in the 3'  ' direction.
5' —— GAATTC —— 3'
3' —— CTTAAG —— 5'
8. Restriction endonucleases are used in genetic engineering to form ‘recombinant’ molecules of DNA,
which are composed of DNA from different sources/genomes.
9. When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of ‘stickyends’ and, these can be joined together (end-to-end) using DNA ligases
Diagrammatic representation of recombinant DNA technology
(A) Separation and isolation of DNA fragments : The cutting of DNA by restriction endonucleases
results in the fragmentes of DNA. These fragments can be separated by a technique known as gel
electrophoresis. Since DNA fragments are negatively charged molecules they can be separated by
forcing them to move towards the anode under an electric field through a medium/matrix. Nowadays the
most commonly used matrix is agarose which is a natural polymer extracted from sea weeds. The DNA
fragments separate (resolve) according to their size through sieving effect provided by the agarose gel.
Hence, the smaller the fragment size, the farther it moves.
The separated DNA fragments can be visualised only after staining the DNA with a compound known as
ethidium bromide followed by exposure to UV radiation (you cannot see pure DNA fragments in the
visible light and without staining). You can see bright orange coloured bands of DNA in a ethidium
bromide stained gel exposed to UV light. The separated bands of DNA are cut out from the agarose gel
and extracted from the gel piece. This step is known as elution. The DNA fragments purified in this way
are used in constructing recombinant DNA by joining them with cloning vectors.
A typical agarose gel electrophoresis showing migration of undigested (lane 1) and digested set of DNA fragments
(lane 2 to 4)
(B) Cloning Vectors: Making many copies of rDNA is possible through multiplying the vector to which it
has aligned. We are able to link an alien piece of DNA with bacteriophage or plasmid DNA, we can
multiply its numbers equal to the copy number of the plasmid or bacteriophage.
(C) The following are the features that are required to facilitate cloning into a vector.
(i) Origin of replication (ori) : This is a sequence from where replication starts and any piece of DNA
when linked to this sequence can be made to replicate within the host cells.
(ii) Selectable marker : In addition to ‘ori’, the vector requires a selectable marker, which helps in
identifying and eliminating nontransformants and selectively permitting the growth of the transformants.
Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or
kanamycin, etc., are considered useful selectable markers for E. coli. The normal E. coli cells do not carry
resistance against any of these antibiotics.
(iii) Cloning sites: In order to link the alien DNA, the vector needs to have very few, preferably single,
recognition sites for the commonly
used restriction enzymes. Presence
of more than one recognition sites
within the vector will generate
several fragments, which will
complicate the gene cloning.
E. coli cloning vector pBR322 showing restriction sites (Hind III, EcoR I, BamH I, Sal I, Pvu II, Pst I, Cla I), ori
and antibiotic resistance genes (ampR and tetR). Rop codes for the proteins involved in the replication of the plasmid.
Alternative selectable markers have been developed which differentiate recombinants from nonrecombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. In
this, a recombinant DNA is inserted within the coding sequence of an enzyme, â-galactosidase (gene
gets ‘inactivated due to insertion’ of alien DNA). This results into inactivation of the enzyme, which is
referred to as insertional inactivation. The presence of a chromogenic substrate gives blue coloured
colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional
inactivation of the â-galactosidase and the colonies do not produce any colour, these are identified as
recombinant colonies.
(iv) Vectors for cloning genes in plants and animals: The tumor inducing (Ti) plasmid of
Agrobacterium tumifaciens has now been modified into a cloning vector which is no more pathogenic to
the plants but is still able to use the mechanisms to deliver genes of our interest into a variety of plants.
Similarly, retroviruses have also been disarmed and are now used to deliver desirable genes into animal
cells. So, once a gene or a DNA fragment has been ligated into a suitable vector it is transferred into a
bacterial, plant or animal host (where it multiplies).
(D) Competent Host (For Transformation with Recombinant DNA)
In order to force bacteria to take up the plasmid, the bacterial cells must first be made ‘competent’ to
take up DNA. This is done by treating them with a specific concentration of a divalent cation, such as
calcium, 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 briefly at 420C (heat shock), and then putting them back on ice. This
enables the bacteria to take up the recombinant DNA.
Other Methods:1. In a method known as micro-injection, recombinant DNA is directly injected into the nucleus of an
animal cell.
2. In another method, suitable for plants, cells are bombarded with high velocity micro-particles of gold or
tungsten coated with DNA in a method known as biolistics or gene gun.
3. Another method uses ‘disarmed pathogen’ vectors, which when allowed to infect the cell, transfer the
recombinant DNA into the host
(E) Isolation and purification of the Genetic Material (DNA)Since the DNA is enclosed within the membranes, we have to break the cell open to release DNA
along with other macromolecules such as RNA, proteins, polysaccharides and also lipids. This can be
achieved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacteria),
cellulase (plant cells), chitinase (fungus).
Genes are located on long molecules of DNA interwined with proteins such as histones. 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 and purified DNA ultimately precipitates out
after the addition of chilled ethanol. This can be seen as collection of fine threads in the suspension by
spooling.
(F) Amplification of Gene of Interest using PCRPCR stands for Polymerase Chain Reaction. In this reaction, multiple copies of the gene (or DNA) of
interest is synthesised in vitro using two sets of primers (small chemically synthesised oligonucleotides
that are complementary to the regions of DNA) and the enzyme DNA polymerase. The enzyme extends
the primers using the nucleotides provided in the reaction and the genomic DNA as template. If the
process of replication of DNA is repeated many times, the segment of DNA can be amplified to
approximately billion times, i.e., 1 billion copies are made. Such repeated amplification is achieved by the
use of a thermostable DNA polymerase (isolated from a bacterium, Thermus aquaticus), which remain
active during the high temperature induced denaturation of double stranded DNA. The amplified fragment
if desired can now be used to ligate with a vector for further cloning.
Polymerase chain reaction (PCR) : Each cycle has three steps: (i) Denaturation; (ii) Primer annealing; and (iii) Extension of
primers
(G) Obtaining the Foreign Gene ProductAfter having cloned the gene of interest and having optimised the conditions to induce the expression of
the target protein, one has to consider producing it on a large scale. If any protein encoding gene is
expressed in a heterologous host, is called a recombinant protein.
The cells harbouring 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.
Small volume cultures cannot yield appreciable quantities of products. To produce in large quantities, the
development of bioreactors, where large volumes (100-1000 litres) of culture can be processed, was
required.
Bioreactors can be thought of as vessels in which raw materials are biologically converted into specific
products, individual enzymes, etc., using microbial plant, animal or human cells. A bioreactor provides the
optimal conditions for achieving the desired product by providing optimum growth conditions
(temperature, pH, substrate, salts, vitamins, oxygen).
(a) Simple stirred-tank bioreactor; (b) Sparged stirred-tank bioreactor through which
sterile air bubbles are sparged
A 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. Alternatively
air can be bubbled through the reactor. The bioreactor has an agitator system, an oxygen delivery
system and a foam control system, a temperature control system, pH control system and sampling ports
so that small volumes of the culture can be withdrawn periodically.
(H) Downstream ProcessingAfter 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,
which are collectively referred to 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.
HOTS
1 marks
1 Restriction Enzymes are called “Molecular scissors”. In what context they are
referred so?
2 In what way the following two plasmids differ? 1
3. What is the source of thermostable DNA polymerase and Name that polymerase. Why
thermostable DNA polymerase is essential in PCR? 1
4 “The prophase I of meiosis plays a vital role in r-DNA formation” Justify the statement.1
5 Eukaryotes do not have restriction endonuclease, then how they manage with normal
endonuclease enzyme?1
6What special feature do prokaryotes have to defend themselves from bacteriophages?1
7_____= Denaturation +______+Extenton.1
8.Complete the following palindrome sequence and name the restriction endonuclese that
recognises this.
5’
G ? A ? T C
3’
3’ C ? T ? A G
5’
9. Which is not a tool of Recombinant DNA technology.
a) Restriction enzyme
b) Vector
c)Bioreactor
10. In what stage of meiosis recombination of DNA takes place?
11. How has EFB defined biotechnology?
12. Which group of enzymes known as molecular scissors?
13. From which organism restriction endonuclease was first extracted?
14. How is a DNA fragment amplified?
15. Name the common substance used as matrix for gel electrophoresis.
16. Which of the following DNA sequences would a Restriction enzyme recognize and cut?
(a) ATGCAC
(b) GATATC
(c) TAGATA
(d) AATATA
TACGTG
CTATAG
ATCTAT
TTATAT
17. Who isolated the antibiotic resistance genes from bacteria?
18. What are recognition sequences?
19. What is elution in the process of separation of DNA fragments?
20. Name the chemical used to stain DNA in gel to view them.
21. What is Rop in cloning vector pBR322?
22. Which enzyme is used to treat the fungal cells during isolation of DNA?
23. Identify the type of bioreactor given in below figure:-
24. Why does DNA move towards
25. What is gene gun?
the anode in gel electrophoresis?
2marks
1. Is advisable to use different restriction endonucleases to cut the vector DNA
and source DNA? Why?
2. What will happen if more than one recognition sites are present in a vector?
3.Uncontrolled recombinant DNA technology experiments is dangerous to
mankind. Comment on it.
4.
+
=
--------------------------------
Foreign DNA
+
plasmid
= …………??…………
Complete the above sequence of diagrammatic representation and name it.
5. (a) Which is the most commonly used matrix in gel electrophoresis ?
(b) What is the source of it?
6 Find the ‘odd one out and write why that is ‘odd’
(a) Sal I, Pst I, Cla I, BamH I, pBR 322
(b) Bacteria, Virus, Gene-gun, Fungi
7 Detect the mismatch from the following and replace the
wrong match with a right one
(a) ECOR I
–
Gel electrophoresis
(b) Ethidium Bromidqe Bacteria
(c) Lysozyme
Restriction enzyme
(d) Palindrome sequence Fungi
8.Write the use of the following in Biotechnology.
(a) Chilled ethanol (b) Microinjection
(c) Bioreactor (d) plasmid
9. Is there any difference between recombinant DNA and
recombinant protein? Support your answer.
10. What are selectable markers? Give two examples.
11. What are the optimal conditions bioreactor provides for the achieving the desired product of
gene?
12. Few gaps have been left in the following table showing certain terms and their meanings.
Sl.No
Terms
Meanings
1.
---------------any protein encoding gene is expressed in a
heterologous host
2.
lysozyme
------------------------------------------------3.
---------------In this recombinant DNA is directly injected
into the nucleus of an animal cell.
4.
Ti
---------------------------13. How you overcome inclusion and multiplication of undesirable genes along with the desired
genes by hybridisation procedures used in plants? What techniques are included in this process?
14. A scientis cultured the recombinant DNA bearing gene for resistance to an antibiotic ampicillin is
transferred into bacterial cells on agar plates containing ampicillin.Which one will grow on this
medium? What you call such type genes in bacterial cells?
15. How you Isolate the Genetic Material (DNA) from plant cells?
16. Why the bacterial cells must first be made ‘competent’ to take up DNA? List the methods used to
achieve this Process.
17. How has Agrobacterium tumifaciens been suitably modified to act as a cloning vector?
18. Name the 1,2,3 And Enzyme 4 in given below diagram.
19. What are three basic steps in genetically modifying an organism?
3 MARKS
1. Give the correct term for the following; (a) Replacing a defective mutant allele with a normal functional allele /gene.
(b) Increasing the copies (content) of a gene
(c) Preventing m-RNA translation
(d) Specific pattern of base pairs that are recognized by restriction enzymes.
(e) Mobile genetic element
(f) One DNA copies itself and produces two
2. Name the enzyme involved in the following process:
(a) Repeated amplification of DNA fragments.
(b) Formation of short piece of RNA strand for annealing.
(c) Breaking of bacterial cell to release DNA and other macromolecules.
(d) Cutting and rejoining DNA fragments.
(e) Formation of m-RNA.
(f) Joining of foreign DNA fragments with plasmid.
3. Explain the following, emphasizing their formation.
(a) rDNA
(b) cDNA
(c) dsRNA
4. Name the diagram and label the parts ‘a’ to ‘e’.
5. (i) Label the following diagram & Identify the selectable markers (2)
(ii) What is the role of polymerase? (1)
6. Two of the steps involved in producing rDNA are mentioned below. Write the missing steps in its
proper sequence?
a. Isolation of DNA,
b.---------------------------------c.---------------------------------d.--------------------------------e. ligation of the DNA fragment into a vector,
f.--------------------------------------------------g.-------------------------------------------------.
7. Describe the naming of restriction enzymes with an example.
8. What is insertional inactivation? Which enzyme is used in this process? What type of colour it
gives in its reaction?
9. What Polymerase Chain Reaction? Name the steps involved in this process? Name the bacteria
used to isolate thermostable polymerase enzyme.
5 MARKS
1. (a) Explain how recombinants and non- recombinants are differentiated on the
basis of colour production in the presence of a chremogenic substrate. Name that
procedure.
(b) Describe the temperature treatment (-3 step-) that enhances the bacteria to
take up the rDNA.
2. What features are required to facilitate as cloning vector? Describe in brief.
3. Diagrammatically represent the experimental set up in cloning and expressing a human genome like
growth hormone in to a bacterium E.coli
Questions with hints of model answers
Section - A: 1 Mark Questions [To be answered in one word in one sentence]
1. What is plasmid?
Answer: Plasmid is autonomously replicating circular extra chromosomal DNA.
2. Write any two ways of introducing foreign DNA into host cell.
Answer:
i) Micro-injection
ii) biolistics/ gene gun.
3. Which compound is used for staining DNA in gel?
Answer: Ethidium Bromide.
4. Name the enzyme that digest bacterial cell wall in genetic engineering.
Answer: Lysozyme.
Section – B: 2 Marks Questions [To be answered in 20 to 30 words approximately]
1. A farmer found that agrochemicals cause pollution of soil and water and are too expensive. What
alternative should be use?
Answer: Farmer should use genetically modified crops and sould have organic farming as a
possible solution for given problems.
2. What are the key tools needed for the recombinant technology?
Answer: The key tools needed for the recombinant technology to be accomplished are:i)
cell culture with desired DNA
ii)
restriction enzymes
iii)
DNA polymerase
iv)
Ligases
v)
Vector
vi)
Host organism/ Cell
3. To which class of enzyme restriction enzyme belong and write its types.
Answer: Restriction enzyme belong to a class of enzyme, called nuclease. There are following
two kinds of restriction enzymes:a) Exonucleases, which remove nucleotides from two ends of DNA.
b) Endonucleases, which cut the DNA at specific position anywhere in its length.
8. What is palindrome nucleotide sequence?
Answer: The recognition sequence is a palindrome, where the sequence of base pairs reads the
same on both the DNA strands.
Example5’- GAATTC- 3’
3’- CTTAAG- 5’
4. What is vector in genetic engineering? Write its one property.
Answer: Plasmid and bacteriophages are the commonly used vectors, the vector should have a
few (or) at least one unique recognition site, to link the foreign/ alien DNA.
5. What is marker?
Answer: A marker is a gene, which helps in selecting those host cells which contain the vector
(transformant) and eliminating the non-transformants.
Section – C: 3 Marks Questions [To be answered in 30 to 50 words approximately]
1. Why are cloning vectors necessary in cloning? Name any two such vectors that are used in
experiments with Escherichia coli.
Answer: Cloning vectors are necessary in cloning because these are the vehicle for cloning. The
vector carries a foreign DNA sequence into a given host cell. Bacterial plasmids and
bacteriophages are considerd the most useful.
Name of two vectors are:i)
PBR 322 and
ii)
PUC (named after University of California) is reconstructed plasmid vector.
2. What is genetic engineering? List the steps involved in rDNA technology?
Answer: Genetic engineering is a technique of molecular biology which includes deliberate
manipulation of genetic make up of an organism, synthesis of new DNA, repair of DNA etc.
Recombinant DNA (rDNA) technology involves the following steps:i)
Isolation of DNA.
ii)
Fragmentation of DNA by restriction endonuclease
iii)
Isolation of the desired DNA fragment.
iv)
Amplification of the gene of interest.
v)
Ligation of two DNA fragment into a vector using DNA ligase.
4. Which convention do you use to write name of restriction enzymes?
Answer: first of all scientific name, strain and then order of enzyme isolation is written in roman.
Example- ECoRI where,
E- Escherichia (Genus)
Co- coli (species)
R- Strain of organism
I- order of enzyme isolation.
5. What is palindrome in DNA? Give one example.
Answer: Palindrome in DNA is a sequence of base pairs that pairs that reads same in two strands
when orientation of reading is kept the same.
5’- GAATTC- 3’
3’- CTTAAG- 5’
These two strands read same in 5’-3’ direction as well as in 3’-5’ direction.
6. Define the terms:
i) Recombinant DNA
iii) Plasmid
v) Transgenic
ii) sticky ends
iv) gene gun
vi) Bioconversion
Answer:
i)
Recombinant DNA:
It is the DNA formed by combining DNA from two different
organisms.
ii)
Sticky ends:
refers to the single stranded proteins at the ends of DNA when cut by a
restriction enzyme.
iii)
Plasmid:
extra chromosomal fragments of DNA present in bacteria which is
circular.
iv)
Gene Gun:
bombarding micro projectiles coated with foreign DNA with great velocity
into the target cell.
v)
Transgenic:
genetically modified organisms that contain a foreign gene.
vi)
Bio conversion: raw materials are biologically converted into specific products using
microbial, plant or animal cells.
Section – D: 5 Marks Questions [To be answered in 80 to 120 words approximately]
1. What are Bioreactors? Write its types and describe them.
Answer: Bioreactors: These bio appliances could be considered as vessels in which certain raw
materials are biologically converted into specific products by microbes (or) plants/ animals cells and/or
their enzymes.
Two common types of bioreactors are the following:a) Simple stirred tank bioreactor
b) Sparged stirred tank bioreactor
Stirred tank bioreactors are the most commonly used bioreactors. In a simple stirred tank
bioreactor, there is usually a cylindrical vessel (or) a vessel with a curved base to facilitate mixing
of the content.
But in the sparged stirred tank bioreactor sterile air bubble are sparged.
2. What is rDNA? Explain the technique of cloning DNA.
Answer: To alter the chemistry of genetic material by gene cloning and gene transfer.
Techniques involved in Recombinant DNA:i)
Isolation of DNA.
ii)
Fragments of DNA by restriction Endonucleases.
iii)
Isolation of desired DNA fragments.
iv)
Amplification of the gene.
v)
Ligation of the DNA fragments.
vi)
Transfer of recombinant DNA into the host.
vii)
Culturing the host cells.
viii)
Extraction of desired product.