d) IV, III, I, II
c) 5′ GAAHC 3′ 3′ CTTAAG 5′
c) Plasmid Boliver and Rodriguez
The correct answer is enzymes. Enzymes are used as biosensors in biotechnology applications. Biosensors are analytical devices that combine a biological component, such as an enzyme, antibody, or microorganism, with a physical or chemical transducer to detect and measure specific substances. Enzymes are particularly useful as biosensors because they exhibit high specificity for their substrates and catalyze specific biochemical reactions. When an enzyme biosensor encounters its target substrate, the enzymatic reaction produces a measurable signal such as a change in electrical potential, light emission, or heat. This signal is then detected and quantified by the transducer component of the biosensor. Enzyme biosensors are widely used in medical diagnostics, environmental monitoring, and food quality testing due to their sensitivity, specificity, and rapid response time.
b) 1-c, 2-d, 3-a, 4-b
d) Agrose Gel Electroporosis
e) Both assertion and reason are false
The correct answer is a) Ti plasmid causes the bunchy top disease. This statement is not correct. The Ti plasmid, or Tumor-inducing plasmid, is found in Agrobacterium tumefaciens and causes crown gall disease in plants, not bunchy top disease. The Ti plasmid is widely used as a vector in plant genetic engineering to introduce foreign genes into plant cells. Bunchy top disease is actually caused by a virus, not by the Ti plasmid. The other statements are all correct: the multiple cloning site is indeed known as a polylinker, which contains multiple restriction enzyme recognition sites; non-viral methods are used for transfection of nucleic acids into cells; and polyactic acid is a biodegradable and bioactive thermoplastic polymer used in various biotechnological applications.
d) Polymerase Chain Reaction
b) Transformed cells
d) High yield and resistance to bollworms
Biotechnology plays a pivotal role in modern practices across various industries, transforming product development and problem-solving. In the pharmaceutical sector, it is instrumental in the production of therapeutic proteins, vaccines, and diagnostic agents through recombinant DNA technology. The brewing industry utilizes microbial fermentation, a core biotechnological process, for the production of alcoholic beverages. Agro-industries benefit from genetically modified crops that exhibit enhanced yields, pest resistance, and nutritional value, alongside the development of biofertilizers and biopesticides. Modern biotechnology encompasses a broad spectrum of techniques, including not only recombinant DNA technology but also cell fusion, tissue culture, and genetic engineering, enabling precise manipulation of biological systems. These advanced tools allow for the development of novel solutions in medicine, agriculture, environmental management, and industrial processes, making biotechnology a significant economic driver worldwide.
Spirulina is a filamentous cyanobacterium that can be grown easily on various inexpensive and readily available materials, making it economically viable for large-scale cultivation. Spirulina can be cultured on waste water from potato processing plants, which contains residual starch that serves as a nutrient source. It can also be grown on straw, molasses, animal manure, and even sewage, all of which provide essential nutrients for its growth. These materials are often considered waste products from agricultural and industrial processes, so using them for Spirulina cultivation provides an economical and environmentally beneficial approach. The ability of Spirulina to grow on such diverse and inexpensive substrates makes it an excellent organism for producing large quantities of biomass rich in proteins, vitamins, and minerals. This characteristic has made Spirulina cultivation particularly attractive for food supplementation and nutritional programs in developing countries.
The exact kind of cleavage produced by a v restriction enzyme is important in the • design of a gene cloning experiment.
* Some cleave both strands of DNA through the centre resulting in blunt or flush end known as symmetric cuts.
* Some restriction enzymes cut the strand of DNA, a little away from the centre of palindrome sites, between the same two bases on the opposite strands, protruding and recessed ends known as sticky or cohesive end, cuts known as asymmetric cut or
staggered cuts.
* It is necessary that the vector and the source DNA are cut with the same restriction enzyme, so that the resultant DNA fragments have the same sticky ends facilitating the action of DNA ligase to join them.
Two types of restriction enzymes are used to cut DNA at different positions based on their specificity. Restriction exonucleases are enzymes that cut the phosphodiester bonds at the terminal ends of DNA molecules, removing nucleotides from the 5' or 3' ends. These enzymes work from the ends of the DNA strand inward. In contrast, restriction endonucleases cut the internal phosphodiester bonds within the DNA molecule, creating breaks at specific recognition sequences located in the interior of the DNA strand. Restriction endonucleases are more commonly used in molecular biology and genetic engineering because they produce predictable cuts at specific recognition sites, generating fragments with defined ends. These fragments can be used for cloning, mapping, and analyzing DNA. The cuts made by restriction endonucleases can produce sticky ends or blunt ends depending on the enzyme used, which is important for subsequent ligation and cloning procedures.
Gene transfer without the use of vectors is possible through chemical-mediated methods that facilitate the uptake of DNA into cells. Several chemicals are used to induce and enhance the uptake of foreign DNA into plant protoplasts and other cells. Polyethylene Glycol, commonly abbreviated as PEG, is one of the most widely used chemicals for this purpose. PEG acts by altering the permeability of the cell membrane, allowing DNA molecules to enter the protoplast more easily. Dextran Sulphate is another chemical used for similar purposes in gene transfer protocols. These chemicals work by creating temporary pores or disrupting the lipid bilayer of the cell membrane, facilitating the passage of DNA across the membrane barrier. Chemical-mediated gene transfer is particularly useful in plant biotechnology for creating transgenic plants without relying on biological vectors like Agrobacterium. This method is relatively simple, cost-effective, and can be applied to a wide range of plant species, making it a valuable alternative to vector-based gene transfer methods.
pBR332 – It is a reconstructed plasmid and most widely used as cloning vector.
* It contains 4361 base pairs.
* P denotes Plasmid.
* B&R – The names of Boliver and Rodriguez, the scientists developed this plasmid.
* 322 – The number of plasmid developed from their lab.
* It contains ampR & tet R – 2 different antibiotic resistant genes & the recognition sites for several restriction enzymes (Hindlll, ECoRI, Bam H-I, Sal I, Pvu II, Pst I, Cla I) ori & antibiotic resistance genes.
* Rop – Codes for the proteins involved in the replication of the plasmid.
Biotechnology is one of the most important applied interdisciplinary sciences of the 21st century with tremendous promise for providing numerous benefits to humanity. The applications of biotechnology span multiple fields and sectors. In agriculture and environmental management, biotechnology is used to produce biofertilizers that enhance soil fertility through biological nitrogen fixation and nutrient cycling, and biopesticides that provide biological pest control without harmful chemical residues. Biotechnology enables the production of various enzymes used in industrial processes, food production, and medical applications. In the energy sector, biotechnology contributes to the production of biomass and biofuels as renewable energy sources, reducing dependence on fossil fuels. Environmental biotechnology applications include bioremediation and phytoremediation, which use microorganisms and plants respectively to clean up contaminated soil and water by breaking down or accumulating pollutants. In medicine and healthcare, biotechnology is used for the production of therapeutic proteins, vaccines, and diagnostic tools. Additionally, biotechnology plays a crucial role in food processing, textile production, and the synthesis of various biochemical compounds, making it an indispensable tool for sustainable development and improving quality of life.
Restriction enzymes are the enzymes of bacterial origin which cleaves DNA into fragments at or near specific recognition sites within DNA molecules. This principle is used in biotechnology to cut and insert the desired gene (gene of interest) thereby generating an rDNA with desirable characters.
a) Exonucleases – remove nucleotides one at a time from the end of DNA.
Eg: Bal 31, Exonuclease III
b) Endonucleases – break the internal phosphodiester bonds with in a DNA.
Eg: Hind II, EcoRI, Pvul, Bam HI, Taql.
Three classes of Restriction endonuclease
* Type 1, II & III – which differ slightly by their mode of action
* Type II – preferred in rDNA technology as they cut DNA with in a specific sequence consisting of 4 – 8 bp.
* Hind II – cut DNA at a point of specific sequence of 6 base pairs (recognition
sequence).
* From 200 strains 900 restriction enzymes isolated from over 230 strains of bacteria with different recognition sequences.
* Restriction endonucleases are named by a standard procedure.
* The first letter of the enzymes indicates the genus name, followed by the first two letters of the species, then comes the strain of the organism and finally a roman numeral indicating the order of discovery.
* For example ECORI is from Escherichia (E) coli (co) strain Ry 13 (R) and first endonuclease (I) to be discovered.
* It contains 2 different antibiotic resistance genes and recognition site for several restriction enzymes.
This sequence is referred to as a restriction site and is generally – palindromic which means that the sequence in both DNA strands at this site read same in 5′ – 3′ direction and in the 3′ – 5′ direction.
Yes, it is possible to transfer a suitable desired gene to a host plant using certain chemicals, microinjection method, electroporation or by biolistics.
a. Chemical mediated gene transfer:
Chemicals Poly Ethylene Glycol (PEG) & Dextran sulphate – induce DNA uptake into plant protoplasts.
b. Microinjection:
With a fine-tipped glass needle, DNA is directly injected into the nucleus.
The protoplasts are immobilized on solid support (agarose on a microscopic slide)
c. Electroporation method of gene transferJjjJU Protoplasts, cells or tissues subjected to a pulse of high voltage electric power to make transient pores in the plasma membrane, through which uptake of foreign DNA occurs.
d. Liposome – mediated methods of gene transfer
The gene or DNA is transferred in an encapsulated form from Liposome ( the artificial phospholipid vesicles) into the vacuole of plant cells.
e. Biolistics:
The DNA particle with gold or tungsten particle (1.3 gm) coating are bombarded into the target tissue by gene gun or microprojectile gun/shotgun The bombarded cells/tissues are cultured to regenerate plants from transformed cells.
Properties
Effect
Able to replicate automatically.
Multiple copies can be got along with insert in the host cell.
Small size, low molecular weight less than 10kbp
Entry into the host cell is easy.
Should contain ori
It can independently replicate within the host.
Contain suitable marker (Antibiotic resistance) etc.
It permit its detection in the transformed host cell.
Should have unique target sites for integration with DNA insert & should have ability to integrate with DNA insert.
So that it can be carried into the genome of the host cell.
Most of the cloning vectors have more than one restriction site (MCS) or polylinker.
Multiple cloning site (MCS) facilitates the use of restriction enzyme of choice.
Blotting techniques are molecular biology methods used to detect and analyze specific nucleic acids or proteins transferred from gels to membranes. Southern Blotting is used for the detection and analysis of DNA. In this technique, DNA is first separated by size using agarose gel electrophoresis, and then the DNA fragments are transferred from the agarose gel to a nitrocellulose or nylon membrane. The DNA is then detected using labeled probes that are complementary to the target DNA sequence. Northern Blotting is used for the detection and analysis of RNA. Similar to Southern Blotting, RNA is separated by electrophoresis in agarose gels and then transferred to a nitrocellulose membrane. The RNA is detected using labeled probes specific to the target RNA sequence. This technique is useful for studying gene expression patterns. Western Blotting is used for the detection and analysis of proteins. In this technique, proteins are first separated by size using polyacrylamide gel electrophoresis, and then transferred to a nitrocellulose or polyvinylidene fluoride membrane. Proteins are detected using specific antibodies that bind to the target protein, followed by detection of the antibody-protein complex. These three blotting techniques are fundamental tools in molecular biology research for analyzing the presence and quantity of specific biomolecules in samples.
Herbicide-tolerant crops offer multiple significant advantages in modern agriculture. First, they enable effective weed control, which directly improves crop yield and quality by reducing competition between crop plants and weeds, thereby assuring healthy plant growth. Second, the use of herbicide-tolerant crops reduces the overall spray and application of chemical herbicides, which decreases the economic effort required for weed management and simultaneously makes agriculture more environmentally friendly and less hazardous to human health. Third, herbicide-tolerant crops allow farmers to use low toxicity herbicide compounds that do not persist or remain active in the soil, thus preventing soil degradation and maintaining soil health for future crops. Fourth, by reducing the need for multiple herbicide applications and mechanical weeding, the cost of cultivation is potentially reduced, making farming more economically sustainable. These combined benefits make herbicide-tolerant crops an attractive option for sustainable and productive agriculture.
Bt cotton, which contains genes from Bacillus thuringiensis, offers several important advantages and disadvantages. The major advantages include a significant increase in yield due to effective control of bollworms and other lepidopteran pests, a substantial reduction in the usage of chemical insecticides which decreases environmental pollution and health hazards, and a potentially reduced cost of cultivation due to lower pesticide expenses. However, Bt cotton has notable disadvantages as well. The cost of Bt cotton seeds is considerably higher than conventional cotton seeds, making initial investment expensive for farmers. The effectiveness of Bt toxin remains high only up to approximately 120 days after which the toxin expression decreases and effectiveness is reduced. Bt cotton is ineffective against sucking pests such as jassids, aphids, and whiteflies, which still require separate pesticide applications. Additionally, Bt cotton can negatively affect pollinating insects like bees, which may reduce pollination efficiency and ultimately decrease yield. These limitations necessitate integrated pest management approaches even with Bt cotton cultivation.
Bioremediation:
It is defined as the use of microorganisms or plants to clean up the environmental pollution. It is an approach used to treat wastes including wastewater, industrial waste, and solid waste. The bioremediation process is applied to the removal of oil, petrochemical residues, pesticides, or heavy metals from soil or groundwater.
In many cases, bioremediation is less expensive and more sustainable than other physical and chemical methods of remediation. The bioremediation process is a cheaper and eco-friendly approach and can deal with lower concentrations of contaminants more effectively. The strategies for bioremediation in soil and water can be as follows:
* Use of indigenous microbial population as indicator species for the bioremediation process.
* Bioremediation with the addition of adapted or designed microbial inoculants.
* Use of plants for bioremediation – green technology.
Genetically modified foods present both significant benefits and potential risks that require careful consideration. The major benefits include high crop yield without significant pest damage, reduction in the usage of chemical pesticides which decreases environmental pollution and health hazards, conservation of the microbial population of soil which maintains soil fertility, reduction in groundwater pollution from pesticide runoff, and reduction in air pollution from reduced chemical spray of pesticides. These environmental and agricultural benefits make GM crops attractive for sustainable food production. However, there are several believed and potential risks associated with genetically modified foods. Health hazards are a major concern, with some studies suggesting potential effects on liver and kidney function and possible links to cancer, though these remain controversial. Hormonal imbalance and various physical disorders have been reported in some cases. Anaphylactic shock and allergic reactions may occur in sensitive individuals due to introduction of novel proteins. Loss of seed viability, as seen in terminator seed technology, prevents farmers from saving and replanting seeds, creating dependency on seed companies. Additionally, genetically modified crops are not favored by some agriculturists and consumers due to concerns about long-term ecological and health impacts. These risks highlight the need for rigorous testing, regulation, and monitoring of GM crops before widespread adoption.
a) DMH -11 – Herbicide tolerant Pea
Biotechnology offers diverse applications for environmental remediation and resource recovery. Biopharming involves the cultivation of genetically engineered plants or animals to produce valuable pharmaceutical substances, such as vaccines or antibodies, in a cost-effective manner. Mycoremediation utilizes fungi to break down or absorb environmental pollutants, including hydrocarbons and heavy metals, thereby cleaning contaminated sites. Bioaugmentation enhances the natural degradation processes in the environment by introducing or stimulating specific microbial populations that are capable of breaking down persistent waste products more rapidly. Bioleaching employs microorganisms, particularly bacteria, to extract metals from low-grade ores or to remove metal pollutants from soil and water, offering an environmentally friendlier alternative to traditional mining and waste treatment methods.
d) Bacillus thuringiensis
d) Lactic acid
The incorrect pair is c) ROP – Protein involved in the replication of the plasmid. ROP (Repressor of Primer) is actually a protein that regulates and inhibits the replication of the plasmid rather than being involved in promoting its replication. The other pairs are correct: PBR 322 is indeed a cloning vector used in E. coli, EcoRI, ClaI, and HindIII are all restriction enzymes, and PCR is the technique in which multiple copies of a gene or DNA of interest are synthesized in vitro.
d) Toxic pigments
d) Karl Ereky
b) Kitchen Technology
a) Pharmaceuticals
d) All the above
b) ORI
a) Process Engineering
b) Edward Jenner
c) Invertase
a) H.G.Khorana
d) Both b & c
d) Vector DNA
d) Plasmid DNA of Ecoli
a) Palindromic region
c) EcoRI I & EcoRI II
d) E.Coli
c) Agrobacterium tumefaciens
b) rDNA
a) DNA fingerprinting
c. A Aequorea victoria – B Arabidopsis thaliana. Green Fluorescent Protein (GFP) is naturally isolated from Aequorea victoria, a bioluminescent jellyfish, and is spliced into Arabidopsis thaliana, a model plant organism, to create biosensors that can be used for tracking gene expression and protein localization in living cells and organisms.
b) Endonuclease
d) Restriction Endonuclease
d) Agrobacterium tumefaciens
d) All the above.
c. 5′ GAATTC 3′ 3’CTTAAG 5′
d) Transformation of plant cells
d) Gold or Tungsten
b) Production of CO 2
b) downstream process
d) All the above
a) Fermentation & its practical use
a) Genus
b) Biolistics or gene gun method
b) PHAs and PHB
d) Humulin
d) Live microbial food supplement
d. A Agrobacterium – B Lepidopteron
a) A common laboratory technique of making millions of copies of a particular region of DNA. PCR, or Polymerase Chain Reaction, is a fundamental molecular biology technique that amplifies specific DNA sequences through repeated cycles of denaturation, annealing, and extension, producing millions of identical copies of the target DNA region in vitro without requiring living cells.
c) Western blot and ELISA
iv) c & d. The characteristics of a molecular probe are that it is DNA or RNA in nature and is complementary to a part of the desired gene. A molecular probe does not need to be very long or necessarily double-stranded; rather, it must be complementary to the target sequence to enable specific hybridization and detection of the desired gene or DNA sequence.
d) both b and c
d) confer resistance to antibiotics
c) GAATTC
b) hybridization of DNA of one organism to that of the others. Genetic engineering is the process of deliberately introducing foreign DNA from one organism into the genome of another organism to create new genetic combinations and traits. This involves the isolation, modification, and insertion of specific genes into target organisms to produce desired characteristics, which is fundamentally different from making artificial genes, producing alcohol through fermentation, or creating artificial medical devices.
d) I, II & III
a) If both ‘A’ and ‘R’ are true and ‘R’ is the correct explanation of A
b) It both ‘A’ and ‘R’ are true but ‘R’ is not the correct explanation of A
d) If both A & R are false
b) It both ‘A’ and ‘R’ are true but ‘R’ is not the correct explanation of A
a) It both ‘A’ and ‘R’ are true but ‘R’ is not the correct explanation of A
a) It both ‘A’ and ‘R’ are true but ‘R’ is not the correct explanation of A
a) It both ‘A’ and ‘R’ are true but ‘R’ is not the correct explanation of A
III. Choose the correct statement
Plasmid DNA and chromosomal DNA differ significantly in their structure, location, and function within a cell. Plasmid DNA is typically found as extra-chromosomal DNA, meaning it exists independently of the main bacterial chromosome. It is usually in a circular, double-stranded form and does not associate with histone proteins. Plasmids possess their own origin of replication (ori), allowing them to replicate autonomously within a suitable host cell, independent of the chromosomal DNA replication. They often carry genes that confer advantageous traits, such as antibiotic resistance, but they do not generally act as the primary genetic factor for essential cellular functions. Plasmids typically lack introns. In contrast, chromosomal DNA constitutes the main genetic material of the organism, organized into chromosomes. This DNA is tightly associated with histone proteins to form chromatin. Chromosomal DNA replicates along with the entire genome during cell division. It can exist in linear or circular forms and can be single or double-stranded. Chromosomal DNA carries the essential genes required for the organism's survival and reproduction, thus acting as the primary genetic factor. Eukaryotic chromosomal DNA contains introns, which are non-coding sequences interspersed within the coding sequences (exons).
- E.coli genetic makeup has been extensively studied
- It is easy to handle & grow in short time
- It can accept a range of vectors & also been studied for safety
- Under optimal growing conditions the cells divide every 20 minutes
- It is a method of transfecting cells by bombarding them with microprojectiles coated with DNA
- It is most useful for inserting genes(such as pesticide/ herbicide resistance genes) into plant cells
- The bombarded cells or tissues are cultured on selected medium to regenerate plants from the transformed cells
Agrobacterium tumefaciens is appropriately referred to as a natural genetic engineer of plants because of its remarkable natural ability to transfer genetic material into plant cells. This bacterium possesses a large plasmid called the Ti plasmid, which stands for Tumor-inducing plasmid. A specific portion of this Ti plasmid is designated as T-DNA, or transfer DNA. When Agrobacterium tumefaciens infects plant cells, particularly at wound sites, it has the natural capacity to transfer the T-DNA region of its plasmid directly into the plant cell's genome, integrating foreign genetic material into the plant's chromosomes. This natural genetic transfer mechanism was one of the first discovered examples of horizontal gene transfer in nature and has been extensively exploited by biotechnologists to create transgenic plants by inserting desired genes into the T-DNA region before infection. This inherent ability to naturally engineer plant genomes makes Agrobacterium tumefaciens a valuable tool in plant genetic engineering and justifies its designation as a natural genetic engineer of plants.
- In gene targeting experiments the nuclei has been targeted. This is known as gene knock out
- Two types of vectors are used for it. They are insertion vectors & the replacement vectors
The Genome Project is a comprehensive research initiative in which the entire genome of an organism, particularly plants, is systematically analyzed using advanced sequence analysis techniques and sequence homology comparisons with genomes of other organisms. This project involves determining the complete DNA sequence of an organism's genome and identifying all genes and their functions. Examples of plant genome projects include the analysis of Chlamydomonas, an algae, Arabidopsis thaliana, a model dicot plant, and important crop plants such as rice and maize. These genome projects provide fundamental information about gene organization, function, and evolution, serving as reference databases for understanding plant biology and improving crop varieties through biotechnology.
- A process in biotechnology by which the nuitritive quantity of food material is increased by gene transfer technology.It is also known as Biofortification
- The nutritive protein, carbohydrate, Vitamins can be enriched by this process.
- Eg: Golden rice with vitamin A
- Weed control, improves higher crop yields
- Reduces usage of herbicides
- Reduces competition between crop & weed
- Use of low toxicity compounds ( not remain active in soil)
- Conservation of soil structure and soil microbes
Bacillus thuringiensis is protected from its own Bt toxin because the toxin is produced and stored in an inactive form called protoxin within the bacterial cells. The protoxin is harmless to the bacterium itself and remains inactive under the acidic conditions present within the bacterial cytoplasm. However, when an insect ingests the bacterium or Bt toxin, the alkaline pH of the insect's alimentary canal activates the protoxin, converting it into its active toxic form. Once activated, the Bt toxin binds to specific receptor proteins on the epithelial cells of the insect's midgut, forming pores in the cell membrane. This pore formation causes the epithelial cells to swell and undergo lysis, leading to the rupture and destruction of the midgut cells. This cellular damage disrupts the insect's digestive system and causes death. This selective activation mechanism ensures that the Bt toxin is only dangerous to susceptible insects with alkaline digestive systems while remaining harmless to the bacterium that produces it and to organisms with acidic digestive systems.
- Gene for BT.toxin is written as cry and the prote in as cry III At
- The first letter of protein symbol is always written capital form and written as cry III Ab.
Bioremediation is an environmental biotechnology approach in which genetically engineered microorganisms (GEMs) or green plants are used to treat nonbiodegradable and toxic wastes such as oil, petrochemical residues, pesticides, and heavy metals. These organisms can be deployed in contaminated soil, groundwater, and marine environments to break down or neutralize pollutants, thereby making the environment more sustainable and safe. The principle behind bioremediation is that certain microorganisms possess metabolic pathways capable of degrading toxic compounds into harmless substances, while plants can accumulate heavy metals in their tissues through phytoremediation. This approach is cost-effective, environmentally friendly, and represents a significant application of biotechnology in environmental management and restoration of polluted ecosystems.
- Only biodegradable contaminants can be degraded
- The process must be specific to the contaminated site
- Small scale tests to be conducted before carrying out on a pilot scale
- It is a costly affair also need more research in these areas.
- The use of Algae as a source of energy
- It is an alternative to i) Fossil fuels, ii) Fuel from corn, iii) Sugar cane
- It is also used for making bio-fuel or bio-iesal
- Land unsuitable for Agriculture can be utilised for (farming algae) algal culture. Eg.Botryococcus braunii
- The Technology is photo biological water splitting
- When thenormal condition of photosynthesis was altered, or when it is deprived of sulfur it switches to the production of Hydrogen and the electrons are transported to ferredoxins
- [Fe]-hydrogenase enzymes combine them into the production of Hydrogen gas, an alternative fuel for the next generation
- Electrophoresis is a separating technique used to separate different biomolecules with positive and negative charges.
- By applying electricity (DC) the molecules migrate according to the type of charges they have.
- The electrical charges on different molecules are variable.
- +ve charged cation will move towards -ve cathod.
- -ve charged anions will move towards +ve anode.
- After the introduction of r-DNA into a suitable host cell
- It is essential to identify those cells which have received the r-DNA molecule.
- This process is called screening
- Direct or vector less gene transfer
- Indirect or vector mediated gene transfer
Zymology is the scientific study of fermentation and its practical applications. It encompasses the investigation of fermentation processes, the microorganisms involved in fermentation, and the various industrial and biotechnological uses of fermentation. Zymology is particularly important in industries such as brewing, winemaking, bread production, and the manufacture of various fermented foods and beverages, as well as in the production of antibiotics and other pharmaceutical compounds through microbial fermentation.
b. Aeration, agitation, temperature and pH
a) The discoverer has the full rights on his/her property. Intellectual Property Rights (IPR) protect the intellectual creations and innovations of individuals or organizations. IPR grants the discoverer or inventor exclusive rights to their discovery, invention, or creation, allowing them to control its use, reproduction, and commercialization. This protection is enforced by laws framed by individual countries and international agreements, ensuring that the creator can benefit from their work and prevent unauthorized use by others.
d) Goldenrice has been genetically altered so that the endosperm now accumulates Beta-Carotene. Golden rice is a genetically modified variety of rice developed to address vitamin A deficiency in populations that rely heavily on rice as a staple food. The endosperm of golden rice has been engineered to produce and accumulate beta-carotene, a precursor to vitamin A, giving the rice its characteristic golden color. This represents a significant advancement in agricultural biotechnology aimed at improving human nutrition and health.
a) Cosmids are hybrid vectors derived from plasmids. Cosmids are indeed hybrid vectors that combine features of both plasmids and bacteriophage lambda, allowing them to be used effectively in recombinant DNA technology. YAC (Yeast Artificial Chromosome) vectors do behave like yeast chromosomes, possessing centromeres and telomeres. BAC (Bacterial Artificial Chromosome) vectors are extensively used in rDNA technology for cloning large DNA fragments. Shuttle vectors are plasmids capable of replicating in more than one type of organism, not just prokaryotes.
c) Eugenics
c) Tissue culture
b) transposon
b) Nematodes
c) Golden rice
d) Insertional Inactivation (Blue white colony selection method). LacZ is a reporter gene that encodes beta-galactosidase enzyme and is used in the blue-white colony selection method for insertional inactivation. When foreign DNA is inserted into the LacZ gene within a vector, the gene is disrupted and becomes nonfunctional. Colonies containing recombinant plasmids with inserted DNA produce white colonies, while colonies with non-recombinant plasmids produce blue colonies when grown on X-gal substrate, allowing researchers to easily identify and select recombinant clones.
c) GFP
b) Transfection
a) Bakery and Brewery
b) Alkaline Phosphatase
a) Arabidopsis thaliana & Escherichia coli
d. none of the above
a) Medicago sativa
a) 200
b) Bacillus thuringiensis – cotton bollworm
b) Rice
d) Natural selection
b) Round up Enzyme
| Plasmid | ds-circular gene |
| Ti plasmid | Has one, ori & inc genes |
| pBR322 plasmid | most widely used as cloning vector |
| Transposable elements | Jumping gene |
a) i-III, ii) I, iii) IV, iv) II. The correct matching is: Fermentation is associated with Louis Pasteur (iii), Monoclonal antibodies with Kohler (i), Viral Vaccine with Edward Jenner (iv), and Double helix structure of DNA with Francis Crick (ii). These scientists made fundamental contributions to their respective fields in biology and biotechnology.
d. Molecular pharming to produce transgenic organisms.
d) The presence of Mycobacterium tuberculosis is also traced by ELISA test. ELISA (Enzyme-Linked Immunosorbent Assay) is a diagnostic tool used to identify pathogens by detecting specific antigens or antibodies using enzyme-linked antibodies. In plant pathology, ELISA is employed to identify and eliminate virus-infected plants. ELISA is also used in the diagnosis of AIDS by detecting HIV antibodies. Additionally, ELISA can be used to detect Mycobacterium tuberculosis antigens or antibodies in clinical samples, making it a versatile diagnostic technique in both medical and agricultural microbiology.
b) RNA probes are used in the identification of bacteria as pathogens
IV. Fill in the blanks Answer
1. The method that involved the growth of tissues & cells in a suitable new medium and away from the parent plant is known as…………………….
Tissue culture
2. The range of insects killed by Bt. Toxins are…………………….
Lepitopteron
3. The genes that code for Bt toxins are commercially called…………………….
Cry genes
4. The first company to produce insulin by rDNA technology is…………………….
Eli Lilly
5. The Indian scientist who was the innovator of ELISA in India is…………………….
Usha M.Joshi
6. PCR is usually used to detect the……………………. in a suspected …………………….patient.
HIV & AIDS
7. Are present in increased quantities in glutelin is…………………….
rice
8. Protein encoded by cry Ab control…………………….
Cotton borer
9. Use of microorganism in solution to recover toxic metal pollutants from contaminated sites is…………………….
Bioleaching
10. The endosperm of normal rice doesnot contain…………………….
Beta carotene
V. Two Marks
- Bio-pesticide: pesticide derived from plants bacteria, animals, etc.,
- Bio-fertilizer: all nutrient outputs of biological origin include plants, animals & microbes
- Bio venting: The process that increases oxygen to accelerate the degradation of environmental pollutants
- Bio leaching: Microbes in solution, used to recover poisonous metal pollutants in the soil
- Bioprospecting: The process of commercialization of new products of based on biological resources
- Bio – pharming: use of genetically engineered plants/microbes to produce molecular pharming: pharmaceutical products
- BioFuel: plant/microbes/algae used as an alternative fuel source
- Biofortification: Breeding crops to enrich the nutritional value either by conventional or gene by genetic engineering
- Bioremediation: use plants/microbes to clean up environmental pollutants
- Biopiracy: exploiting the traditional knowledge/invention of poor countries by MNC or developed countries without approval or proper compensation
- Bio patency: The legal exclusive right for the inventor and thereby excluding others from exploiting the knowledge/invention.
- Bio chip: microchip designed intended to function in a biological environment or inside the body of an organism
- Enzymes (1) restriction endonuclease (2) DNA ligase
- Vectors
- Host organisms
PCR (Polymerase Chain Reaction) is a common laboratory technique used to amplify and make millions of copies of a particular region of DNA. This technique is based on repeated cycles of heating and cooling to denature DNA, allow primers to anneal to target sequences, and enable DNA polymerase to synthesize new DNA strands. PCR is widely used in molecular biology for DNA analysis, genetic testing, cloning, and various diagnostic applications.
- They exist in many bacteria, where they function as a part of their defence mechanism called restriction-modification system
- It helps the bacteria to cut the genetic material of the virus that attack it and render them harmless. EgiE.coli
DNA ligase joins DNA fragments by catalyzing the formation of phosphodiester bonds between the sugar and phosphate molecules of double-stranded DNA. Specifically, DNA ligase seals the nick between the 5' phosphate group of one DNA strand and the 3' hydroxyl group of an adjacent strand in an ATP-dependent reaction. This enzyme is essential in recombinant DNA technology for joining DNA fragments together and in DNA replication and repair processes within cells.
The two main types of vectors used in recombinant DNA technology are cloning vectors and expression vectors. Cloning vectors are designed primarily for the insertion and replication of foreign DNA within a suitable host cell, allowing for the amplification and maintenance of the cloned DNA sequences. Expression vectors, on the other hand, are engineered not only to carry the foreign DNA but also to facilitate its transcription and translation, enabling the host cell to produce the specific protein encoded by the inserted DNA. Expression vectors contain additional regulatory elements such as promoters and terminators that are necessary for gene expression in the host organism.
The origin of replication, often abbreviated as 'ori', is a specific nucleotide sequence on a DNA molecule where DNA replication begins. This sequence serves as the recognition site for initiator proteins that bind to the DNA and unwind it, allowing the replication machinery to assemble and start synthesizing new DNA strands. In the context of genetic engineering and molecular cloning, a DNA fragment of interest can be inserted into a cloning vector, such as a plasmid. For this recombinant DNA molecule to be replicated within a host cell, it must contain an 'ori' sequence recognized by the host's replication machinery. When a piece of DNA is ligated to a vector containing a functional 'ori', it can be replicated along with the vector, leading to the amplification of the inserted gene, a process crucial for gene cloning and expression studies.
- Selectable marker, which helps in identifying and eliminating nontransformants
- It will selectively permit the growth to the transformants
Walking genes, jumping genes, or transposons are DNA sequences that possess the remarkable ability to move or insert themselves at new locations within the genome without requiring any sequence homology or relationship with the target locus. These mobile genetic elements can change their position within the DNA, moving from one location to another, which is why they are termed 'walking' or 'jumping' genes. Transposons were first discovered by Barbara McClintock in maize and have since been found in various organisms including bacteria, plants, and animals. They can cause mutations when they insert into genes, potentially disrupting normal gene function. Some transposons carry additional genes such as antibiotic resistance genes, which can be transferred between organisms. The movement of transposons is regulated by specific enzymes, particularly transposase, which catalyzes the excision and insertion of these elements. Understanding transposons has been crucial in molecular biology and genetic engineering, as they provide a natural mechanism for genetic variation and have been utilized in creating transgenic organisms.
- Ability to change the -genetic material for getting new products according to the requirement through r DNA technology
- Ownership of the newly developed technology and its social impact
A bioreactor is a specially designed vessel or container engineered to provide an optimum environment in which microorganisms or their enzymes interact with a substrate to produce desired products on a large scale. The primary function of a bioreactor is to maintain controlled conditions that are essential for efficient microbial growth and product formation. These controlled parameters include aeration, which supplies oxygen to aerobic microorganisms; agitation, which ensures uniform mixing of the culture medium and nutrients; temperature, which is maintained at the optimal level for enzyme and microbial activity; and pH, which is kept within the range suitable for the specific microorganism and product. Bioreactors are essential in biotechnology and pharmaceutical industries for the mass production of antibiotics, vaccines, enzymes, and other biological products. The bioreactor process involves two main stages: upstream processing, which includes the preparation of the culture medium, sterilization, inoculation, and fermentation; and downstream processing, which involves the extraction, purification, and formulation of the final product. Modern bioreactors range from small laboratory-scale vessels to large industrial fermenters with capacities of thousands of liters.
Upstream and downstream processes are two distinct phases in industrial biotechnology operations, particularly in fermentation. The upstream process encompasses all activities that occur before the main bioprocess, such as fermentation, begins. This phase is critical for ensuring optimal conditions for microbial growth and product formation. It includes the meticulous sterilization of the bioreactor to eliminate any contaminating microorganisms, the preparation and sterilization of the culture medium containing all necessary nutrients, and the cultivation of a suitable inoculum of the desired microorganism. The downstream process, on the other hand, follows the fermentation stage and involves all operations required to recover, purify, and formulate the desired product from the fermentation broth. This phase typically includes techniques like distillation, centrifugation, filtration, chromatography, and solvent extraction, all aimed at isolating the target product with high purity and yield, and preparing it for its intended use.
Agrobacterium tumefaciens, a soil bacterium, serves as a highly effective natural vector for gene transfer into plant cells due to its unique Ti (tumor-inducing) plasmid. This plasmid contains a specific segment of DNA known as the T-DNA (transfer DNA), which is capable of being transferred from the bacterium into the plant genome. The Ti plasmid harbors genes, including transfer (tra) genes, that facilitate this DNA transfer process. It also contains genes responsible for tumor formation (oncogenes) and replication (ori gene) and regulates its compatibility with other plasmids (inc gene). In genetic engineering, the disease-causing genes on the T-DNA are often removed and replaced with a gene of interest. When Agrobacterium is co-cultivated with plant cells or tissues, the modified T-DNA, carrying the desired gene, is integrated into the plant's chromosomal DNA. This stable integration allows the plant cells to express the introduced gene, leading to the development of transgenic plants with new, desirable traits.
- Protein supplement
- Cosmetic product for healthy hair & skin
- Poultry industry as excellent source of proteinacious food.
- In food industry – canbe carrier in production of aroma, tic compounds vitamin, emulsifying agent improve the nutritive value of baked products & ready to serve meals.
- In the processing of paper & leather as foam stabilizers.
- It is genetic form refer to the identify of the taxon based on its genetic makeup.
- It is an optical machine readable representation of data which describes about A the characters of any plants / objects.
- A group of technologies that has the ability to change an organism’s DNA.
- Genetic material can be added, removed or altered at particular locations in the genome – known as genome or gene editing.
- Eg. GRISPR – mediated gene replacement – Rice can be switched from sexual to an asexual mode.
- Affect Liver, Kidney functioning
- Carcinogenic (cause cancer)
- Hormonal imbalance & Physical disorder
- Anaphylactic shocks (sudden hypersensitive reaction) & Allergies
- Adverse effects on immune system – due to interference of bacterial protein
- Loss of viability of seeds, (shown in terminator seed technology of GM crops).
Northern blotting is a molecular biology technique, devised by Alwin et al. in 1979, used to detect specific RNA molecules within a sample. The process involves separating RNA fragments by gel electrophoresis, followed by transferring these separated bands onto a solid support membrane, typically Amino Benzyloxymethyl (ABM) paper, which can be prepared from Whatman 540 paper. The transferred RNA is then hybridized with a labeled probe that is complementary to the target RNA sequence, allowing for its detection and quantification. Western blotting, in contrast, is a technique used to detect specific proteins. It involves separating proteins by gel electrophoresis and then transferring them to a blotting paper, usually nitrocellulose. The specific protein of interest is then identified by probing the blot with a radio-labeled antibody that specifically binds to that protein. Thus, Northern blotting targets RNA, while Western blotting targets proteins.
- The native genes in Tomato produce enzyme Polygalacturonase and this leads to ripening follow by senescence & fruits get spoit.
- When Anitsense RNA genes inserted into Tomato plant via Agrobacterium mediated gene transfer the gene interfere with the production of Polygalacturonase, there by delay ripening, softening and further spoiling (shelf life of fruits increased).
- Transgenic tomatos can be transported to long distance with out getting spoilt.
Both ELISA and Western Blotting are indirect tests – to measure he immune system’s response to an infections agent rather than looking for the components of the agent itself.
ELISA Test
Western Blot Test
It detects the antibodies which the
body starts to produce between 2-12 weeks after being infected,
It is a confirmative test. It is less likely to have false positive results – as it can effectively distinguish between the anti bodies of the particular disease from other antibodies
It is a qualitative, sensitive test – but not a confirmative test. Eg.: HIV – AIDS
It is a clear confirmative test.
Eg.: HIV – AIDS
VII. Five marks
The production of a desired product using biotechnology typically involves three sequential steps: upstream processing, fermentation, and downstream processing. The upstream process is the preparatory phase, which includes essential tasks like the thorough sterilization of the fermenter vessel to prevent contamination, the preparation and sterilization of the culture medium providing all necessary nutrients for microbial growth, and the cultivation of a suitable inoculum, which is a concentrated starter culture of the microorganism. The second step is the fermentation process itself, where the inoculated medium in the sterilized fermenter is incubated under controlled conditions to allow the microorganisms to grow and produce the target product. Finally, the downstream process involves the recovery and purification of the desired product from the fermentation broth. This phase includes various separation and purification techniques such as distillation, centrifugation, filtration, and solvent extraction, ultimately yielding the purified product. The success of the fermentation process is highly dependent on the meticulous execution of both the upstream and downstream steps; for instance, inadequate sterilization during upstream processing can lead to contamination and spoilage, while inefficient downstream processing can result in low yields or purity of the final product.
- Antibiotic Resistant Marker (ARM) is – a gene when introduced into bacterial cells – (Recombinant) produce – a protein that provide resistance to antibiotics.
- Recombinants (A) may grow well in a medium with antibiotics (such as ampicillin, chioramphenicol, teiracycline or kanamycinetc)
- Non recombinants (B) may not be able to grow in these media with these antibiotics.
- Thus Antibiotic resistant marker is a useful selectable marker in distinguishing the two.
The replica plating technique is a method used to select transformed cells, particularly in bacterial cultures, by transferring colonies from an initial growth plate to new plates under different conditions. The procedure begins with a master culture plate (A) containing bacterial colonies that have been grown on a nutrient medium. A sterile filter paper or velveteen cloth is then pressed firmly onto the surface of the master plate (A) to pick up an imprint of the colony pattern. This filter is subsequently pressed onto a new, sterile culture plate (B), transferring the colonies to the new medium in the same relative positions. This process can be repeated to inoculate multiple plates. To select for transformed cells, one of the replica plates (e.g., plate C) can be supplemented with a selective agent, such as an antibiotic, to which only transformed cells are resistant. Colonies that grow on plate C but not on a non-selective plate, or that show differential growth patterns, can be identified as transformed cells. Similarly, replica plating can be used to test for auxotrophic mutants by comparing growth on media with and without specific growth factors.
- Agarose GEL Electrophoresis is a,medium used to separate DNA fragements of larger sizes (few 100S to 20,000 bp)
- Polycrylamide is a medium used to separate DNA fragments of smaller sizes.
- Agarose GEL provides – a three dimensional matrix & DNA molecules migrates through the – gel and DNA bands can be readily detected at highter sensitivity.
- Energy – The electric field provide energy
- Technique – DNA are negatively charged and migrate towards the positive pole (anode)
- (The marker DNA fragments of known size which allow accurate size determination of an unknown DNA molecule by interpolation)
- The bands of DNA can be stained by a dye Ethium bromide and can be detected as visible orange fluorescence under UV light and can also be photographed.
RNA interference, also known as RNAi or RNA-mediated gene silencing, is a biological phenomenon in which double-stranded RNA molecules are used to selectively target and silence specific messenger RNA molecules, thereby inhibiting or neutralizing their expression into proteins during translation. When double-stranded RNA is introduced into a cell, it is recognized by an enzyme called Dicer, which cleaves it into small interfering RNAs of approximately 21 to 23 nucleotides in length. These small interfering RNAs are then incorporated into a protein complex called the RNA-induced silencing complex (RISC). The RISC complex uses these small RNAs as guides to locate and bind to complementary mRNA sequences. Once the target mRNA is bound, it is degraded or its translation is blocked, effectively silencing the expression of that particular gene. RNAi has become a powerful tool in molecular biology for studying gene function, as it allows researchers to selectively knock down the expression of specific genes without permanently altering the genome. This technique has also shown promise in therapeutic applications for treating genetic disorders, viral infections, and certain cancers by silencing disease-causing genes or viral genes.
Herbicide-tolerant plants, particularly those resistant to Basta and related herbicides, have been developed through genetic engineering to survive herbicide application. Basta is a non-selective herbicide containing the chemical compound phosphinothricin, which functions by inhibiting the enzyme glutamine synthetase, a key enzyme involved in ammonia assimilation and nitrogen metabolism in plants. When phosphinothricin is applied to non-transgenic plants, it blocks glutamine synthetase activity, leading to accumulation of toxic ammonia levels and ultimately plant death. To create herbicide-resistant transgenic plants, the PAT gene (phosphinothricin acetyltransferase) or the bar gene (bialaphos resistance gene) is introduced into the plant genome. These genes encode enzymes that can inactivate or detoxify phosphinothricin before it can inhibit glutamine synthetase. The PAT enzyme acetylates phosphinothricin, converting it into an inactive form that cannot inhibit the target enzyme. Similarly, the bar gene product can also detoxify the herbicide. Transgenic plants carrying these genes can therefore tolerate application of Basta and related herbicides, allowing farmers to control weeds effectively while protecting the crop. This approach has been successfully applied to develop herbicide-resistant varieties of crops such as mustard, cotton, and other important agricultural plants, providing an efficient weed management strategy.
Transgenic plants are organisms that have had foreign genes deliberately introduced into their genome to confer desirable traits. Bt cotton is a transgenic plant containing genes from Bacillus thuringiensis that produce Cry toxins, enabling it to resist bollworms and other lepidopteran insects, thereby increasing yield and significantly reducing the need for chemical insecticides. Bt brinjal is another transgenic variety engineered to resist lepidopteran insects such as the fruit and shoot borer (Leucinodes orbonalis), protecting the crop from serious pest damage. Golden rice is a biofortified transgenic rice variety that has been modified to produce beta-carotene, a precursor to vitamin A, addressing nutritional deficiencies in populations that rely heavily on rice as a staple food. Dhara mustard hybrid (DMH-11) is a transgenic mustard variety that is resistant to the herbicide Basta, allowing for effective weed control while increasing overall yield through improved agricultural management. Flavr Savr tomato is a transgenic tomato variety engineered to delay ripening while retaining color and flavor, allowing the fruit to be transported over long distances without spoilage, thereby improving shelf life and market quality. These transgenic plants demonstrate the diverse applications of biotechnology in agriculture, ranging from pest resistance and herbicide tolerance to nutritional enhancement and improved post-harvest characteristics, contributing significantly to food security and agricultural sustainability.
The development of herbicide glyphosate-tolerant transgenic plants involves the introduction of the EPSPS gene (5-enolpyruvylshikimate-3-phosphate synthase) or the bar gene into the plant genome. The EPSPS gene encodes an enzyme that is insensitive to glyphosate inhibition, allowing the plant to continue normal metabolic processes even in the presence of the herbicide. Glyphosate functions by inhibiting the shikimate pathway, which is essential for the synthesis of aromatic amino acids in plants. By introducing a glyphosate-insensitive version of EPSPS, transgenic plants can tolerate glyphosate application. The protocol involves selecting an appropriate explant, typically leaf tissue or callus, which is then transformed using Agrobacterium tumefaciens carrying the desired gene construct. The transformed cells are cultured on selection medium containing glyphosate to identify successfully transformed plants. Regenerated plantlets are then subjected to a hardening process in a greenhouse or hardening chamber before being transferred to field conditions. This approach has been successfully applied to develop glyphosate-tolerant potato varieties and other crops, providing farmers with an effective herbicide for weed management.
Bt cotton is a transgenic plant that has been engineered to resist pests through the incorporation of genes from the bacterium Bacillus thuringiensis. Bacillus thuringiensis produces more than 200 different Bt toxins, with most of these toxins being particularly effective against lepidopteran insects including moths, butterflies, beetles, cotton bollworms, and other gatflies that are major agricultural pests. The Cry genes present in Bt cotton encode crystalline proteins known as Cry toxins or delta-endotoxins. When an insect larva ingests plant tissue containing these Cry toxins, the toxins are dissolved in the alkaline pH environment of the insect's gut. Once activated in this alkaline environment, the Cry toxins undergo conformational changes and insert themselves into the epithelial cell membranes of the insect's digestive tract, forming pores or channels. The formation of these pores disrupts the normal ion balance within the epithelial cells, leading to an uncontrolled influx of ions, particularly potassium ions, and water into the cells. This osmotic imbalance causes cell lysis and destruction of the epithelial cell layer. The breakdown of the gut epithelium results in leakage of gut contents into the insect's body cavity, leading to septicemia and ultimately death of the insect larva. This mechanism of pest resistance has made Bt cotton highly effective in controlling major cotton pests while reducing the need for chemical insecticides, thereby providing both economic and environmental benefits to farmers.
GFP stands for Green Fluorescent Protein. It is a protein, with a molecular weight of approximately 26.9 kilodaltons, that exhibits bright green fluorescence when exposed to blue or ultraviolet light, typically in the range of 395 nm. The protein was first isolated from the jellyfish Aequorea victoria. A key property of GFP is its ability to form an internal chromophore, the part of the molecule responsible for light absorption and emission, without requiring any cofactors other than molecular oxygen. This self-sufficient fluorescence makes it exceptionally useful in biological research. GFP is widely employed as a reporter molecule to monitor gene expression levels or protein localization within cells or organisms. Modified forms of GFP have also been developed to serve as biosensors, allowing researchers to detect specific cellular events or the presence of particular molecules by changes in fluorescence.
It has wide applications in various sectors
I. Agriculture – Transgenic plants
Bt.cotton, Bt.brinjal, Golden rice, Flavr Savr tomato, Cauliflower, Potato, and Banana – are
the outcome of Biotechnology Resistant varieties They are Resistant to pest, stress, disease, etc.,
II. Medicine:
* Insulin – is produced by r DNA technology is a breakthrough in medicine
* Vaccines, enzymes, antibiotics, dairy products & beverages are also products of biotechnology
III. Biochip:
Bio chip-based biological computer
IV. Genetic engineering:
It involves
* gene manipulation
* Tissue culture
* Single-cell protein (food industry) SCP
* secondary metabolites & etc.,
* biofertilizers – biopesticides etc.,
V. Environmental aspects Include
* Bio mass-energy
* Biofuel
* Bio & phytoremediation
* Environmental biotechnology etc.,