BIOTECHNOLOGY PRINCIPLES AND PROCESSES Class Notes PDF

# Prachand for NEET Aspirants: Zoology - Biotechnology: Principles and Processes (One Shot) ## By- Sujeet Tripathi Sir ### Topics to be covered 1. Introduction 2. Principles of Biotechnology 3. Tools of Recombinant DNA Technology 4. Processes of Recombinant DNA Technology ### Topic: Year Wise An...

# Prachand for NEET Aspirants: Zoology - Biotechnology: Principles and Processes (One Shot) ## By- Sujeet Tripathi Sir ### Topics to be covered 1. Introduction 2. Principles of Biotechnology 3. Tools of Recombinant DNA Technology 4. Processes of Recombinant DNA Technology ### Topic: Year Wise Analysis | Year | No. Of Questions | Marks | |---|---|---| | 2023 | 5 | 12-24 | | 2022 | 5 | 12-24 | | 2021 | 1 | 3-6 | | 2020 | 6 | 12-24 | | 2019 | 4 | 12-24 | | 2018 | 1 | 3-6 | | 2017 | 4 | 12-24 | | 2016 | 3-4 | 9-12 | | 2015 | 3 | 9-12 | | 2014 | 1 | 3-6 | | 2013 | 3 | 9-12 | ### Topic: Introduction * Biotechnology: Principles and Processes * Basic Theme: Industry * large-scale production * To provide benefit to humans * Biotechnology: Deals with the techniques: use of living organisms, enzymes produced by living organisms, products & processes, benefit of human beings. * Techniques (specific): Based upon the natural capability of living organisms, Formation of Products: Commercial benefit to mankind. * Biotechnology: Benefit to the mankind. ### Traditional/Olden View * Production of Cund, wine, bread etc. ### Modern Views (More Restricted Sense) 1. In-vitro fertilization (Test tube baby) 2. Production of Genes 3. Production of DNA vaccines 4. Gene Therapy #### Definition Given by EFB The European Federation of Biotechnology (EFB) has given a definition of biotechnology that encompasses both traditional view and modern molecular 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'. ### Topic: Principles of Biotechnology Among many, the two core techniques that enabled the birth of modern biotechnology are: 1. **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. **Bioprocess engineering**: 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. ### Modern Biotechnology (1970s) * 2 Core Techniques: 1. Genetic Engineering * Formation of r-DNA * Transfer into Host cell * Modify phenotype 2. Bio-process engineering (Chemical engineering) * Maintaining sterile environment * To prevent the growth of undesirable bacteria * Modify phenotype * Large scale production ### Idea of Biotechnological Growth? * Advantage of Sexual Reproduction over asexual reproduction: Variation, New combination * Traditional method of cattle/animal breeding and plant breeding: Drawback - undesired products, undesirable features * Body builder (morphological defect): सुन्दर स्त्री (IQ poon) ### Construction if 1st r-DNA * Usually E.coli does not show antibiotic resistance * Stanley cohen & Henbent Boyen: Isolated the antibiotic resistant gene from the DNA of Bacterium Salmonella typhimurium * Molecular Scissors: Desined DNA (Passenger DNA) * By the help of Chemical Glue (DNA Ligase): Bacterial cell E.coli * Vector DNA (Used as a vector) * r-DNA * Transfened into the target cell (Host cell) → Transformation * Fon cloning ### Topic: Principles of Biotechnology You can hence infer that there are three basic steps in genetically modifying an organism: 1. identification of DNA with desirable genes; 2. introduction of the identified DNA into the host; 3. maintenance of introduced DNA in the host and transfer of the DNA to its progeny. ### Topic: Tools of Recombinant DNA Technology * **Engineening**: Use of Machines * **Technological Processes**: 1. **Enzymes**: * **Restriction enzymes (Molecular scissors)** * **DNA Ligase (Chemical Glue)** * **Alkaline phosphatase**: Removal of Phosphate group * **DNA polymerase**: Polymerisation 2. **Vectors (Cloning vectors)** 3. **Competent Host** ### Topic: Restriction Enzymes * Anben, Nathans, H. Smith: Described about restriction enzymes, awarded Nobel prize. * Described about **Restriction Modification System**: In bacterial cells to restrict the growth of bacteriophages. * 2 types of enzymes: **Restriction enzyme**, **Modifien enzyme** * Restriction enzyme: Cleaves viral DNA into several fragments, restrict their multiplication, add methyl group. * **Methylase**: Protects bacterial own DNA from cleavage. * 1st restriction enzyme was: **Hind II** * **Restriction enzymes**: Site specific cleavage, Molecular scissors * Nucleases which cut/cleave the nucleotides (sugar-phosphate-sugar) * **Exonuclease**: Cut the DNA at ends (Terminal points) * **Endonuclease**: Inspect the entire length of DNA, binds with a specific site. * Cut at any intermediate position. * Fon Biotechnology **type II**, restriction endonucleases are used. ### Nomenclature of restriction endonucleases * 1st RE: Hind II: Isolated and described 5 years later. * Fon a restriction enzyme, nomenclature: * 4 points : 1. 1st letter of Genus of source bacterium 2. 1st,2 letters of species of source bacterium 3. May or may not be given: Type of strain (If known) 4. Roman numbers: Order of isolation from the same strain of bacteria. * 1st discovered RE * 2nd isolated from the same strain of bacteria: Hind II * Type of strain: influenzae * Order of isolation. * Haemophilus influenzae * Haemophilus * **Imp**: More than 900 RE have been isolated from over 230 strains of bacteria. * Anthrobacter luteus * A lu I : RY-13 (strain) * EcoRI: 1st isolated from strain of Eschenchia E. coli. ### 2 Types of cuts are obtained by using restriction enzymes * **Sticky cuts (Overhanging stretches)** * ECORI: 5' GAATTC 3' 3' CTTAAG 5'. * If RE cuts little away from the center of Pallinchomic sequence. * **Blunt/Flush ends**: 5' CCCGGG 3' 3' GGGCCC 5'. ### Blunt Ends - EcoRII : Escherichia coli - Hindll : Haemophilus influenzae - Smal : Serratia marcescens (CCCGGG) - Scal : Streptomyces caespitosus - EcoRV : Escherichia coli (GATATC) - Alul : Arthrobacter luteus - Haelll : Haemophilus aegypticus (GGCC) - Pvu II : Proteus vulgaris (CAGCTG ### Sticky Ends - BamHI: Bacillus amyloliquifaciens (GGATTC) - EcoRI: Escherichia coli (GAATTC) - HindIll: Haemophilus influenzae (AAGCTT) - Pst I : Providencia stuartii (CTGCAG) - Pvul: Proteus vulgaris (CGATCG) - Sall: Streptomyces albus - Xhol : Xanthomonas holcicola - Clal: Caryophanon latum ### Topic: Cloning Vectors * **Most common**: Plasmid & Bacteriophage * **Type of vector used in r-DNA technology**: Depends upon the size of desired DNA (Gene of interest), and the type of host cell (Prokaryotic/Eukaryotic). * **Plasmid**: Extrachromosomal hereditary material (Plant, Animal, Fungal cell). * Most prokaryotes & some eukaryotes * Circular, ds DNA, autonomous replication * Copy no. per cell: 1-on 2, sometimes 100 or more * Carries the insert length 0.5 to 8 Kb (smallest) ### Bacteriophages are also used as vectors * They have the ability to deliver its DNA into a bacterial cell. * Very high copy number. * Length of insert: 9 Kb -23 Kb. * Most commonly: M-13, lambda-phase vector. ### Cosmid * Plasmid + Cos sile (Cohesive ends): 45 Kb. ### BAC (Bacterial artificial chromosome) * 350-500 Kb. ### YAC (Yeast artificial chromosome) * 1000 Kb length of insert. ### Characteristics of a Cloning Vector * **Example**: pBR-322 (Artificially constructed vector) * **p**: Plasmid * **B**: Bolivar * **R**: Rodriguez. 1. **ORI site**: Controls the copy number, Alien DNA (Desired DNA) is inserted near ORI site. 2. **ROP site**: Repression of Plasmid. 3. **Selectable marker**: Gene segment used for identification of transformants, recombinants, origin of replication. 4. **Cloning sites**: For one restriction enzyme, there should be only one recognition site. * For 1 RE: Multiple site. * Unique site (only 1): For one RE * Different RE ### Selection of transformant & non-transformant & selection of recombinant & non-recombinant * E.coli (No antibiotic resistance) * **pBR-322**: Tranformed (transformant) antibiotic resistant gene * **Plasmid**: Non-transformant * **pBR-322**: Transformant * **Non-recombinant**: Neither (amp nor tet) * **Both (amp & tet) present**: Transformant * **Only ampicillin resistance**: Recombinant form * **pBR-322**: ampr tetr * **Alien DNA**: (Gene of interest) tetracycline resistance loss: recombinant * **recombinant DNA**: DNA * **Masten plate**: 1,2,5 → Transformant antibiotic resistance * **Non-transformant**: 3,4 → not survived * **Transferned into tetracycline containing medium**: * **Recombinants**: with only ampr but Tetr is lost due to insertion of Foreign DNA (Insertional inactivation) * **Only colony grows**: Shows resistance against tetracycline. * **By using pBR-322**: Simultaneous plating is required in 2 different medium (amp medium, tet medium) * Cumbersome process, difficult task * **To over come this, we have designed some other vectors, with more high numbers**: eg: puc 8 (puc-19, 21) * **Selective marker gene**: Lac-Z gene (Codes for enzyme β-galactosidase) * **In a single plating by Blue-White selection, we can identify the recombinant/Non-recombinant**: Transformant and non-transformant ### pBR-322 * **Blue**: Non-recombinant (transformant) * **White**: Recombinant (transformant). * **Only pBR-322 without GOI**: Non-recombinant * **pBR-322 with GOI**: Recombinant * **2 plating**: Amp medium/Tet medium * **β -galactosidase**: x-gal (colured product): * Ampicillin sensitive: Non-transformant, Colured (Blue): Lac-Z present * No colour, white: Recombinant, Lac-Z absent. ### puc-8 * **lac Z** * **Gene of interest**: Recombinant * **B+C survived**: Transformants * **Ampillin medium**: Chemical (Chromogenic substrate) * **x-gal** ### Vectors for plants * **Ti plasmid used**, obtained from a bacterium **Agrobacterium tumefaciens**. * This bacterium targets only most **dicots (Broad leaf plants)** * Enters in their body through wounds and cause **crown gall disease** * **Ti- plasmid acts like a natural genetic engineer** * **Cause of cancerous growth in plants**: * **Shuttle vector**: Multiply in both **prokaryote & eukaryotes** ### Animals * **Retrovirus/Adenoviruses**: These viruses have the ability to multiply into animals, can cause cancerous growth. ### These vectors are disarmed * Their disease-causing ability is removed/lost, and then transferred into host * No more pathogenic ### Topic: Competent Host * Tanget cell/Host: should be made **competent** to allow the entry of DNA * DNA: Hydrophilic molecules * **Heat Shock Method**: * ICE * DNA * Heat (42°c, short term * ICE * Host cell * **Ca++ (Divalent cation)/CaCl₂ sol**: Adherence of DNA at the cell surface, make the entry easy into Host cell * **Heat shock**: Pones (minute) created through which DNA mol enter easily. ### Some More Methods 1. **Gene Gun**: In Plants: DNA coated with Tungsten/Gold particles, directly bombarded into target cell (high velocity) 2. **Electroporation**: Weak electric pulses, create minute pores. 3. **Micro injection**: For animal cell (ICSI) ### Topic: Processes of Recombinant DNA Technology * **Upstream processing**: * **Isolation of the genetic material** (Plant/Bacteria/Fungal/Animal) * **Cutting of DNA at specific location** (RE) * **Amplification of gene of interest using PCR** * **Insertion of Recombinant DNA into the host cell/organism** * **Downstream processing**: * **Obtaining the foreign gene product** (last step) * **Product Separated/Purified** * **r-DNA** * **PCR**: Photocopy/Xenox, billions copies (few hours) * **Competent Host**: Industrial scale * **Product**: Ready for sale, separate/purification ### Isolation of DNA from a source cell - Plant - Cellulase - Animal - Lipase: - Fungal - Chitinase - Bacterial - Lysozyme - Protein - Protease - RNA- RNase - Treated by chilled Ethanol - DNA is precipitated - Spooling (Taking out of ppt-DNA) ### Treatment of isolated DNA by restriction endonuclease * Desined DNA * Gene of interest ### Gel Electrophoresis * In r-DNA technology the cleaved DNA fragments are separate on the basis of size only (since all are negative charge) * Agonose gel medium is used, extracted from Seaweeds (Gelidium, Gracilaria), Red alga * Well: For DNA input * -ve electrode (cathode) * +ve electrode (anode): DNA molecules move through agarose gel, seiving effect, DNA mol are not visible. * ETBR: (Ethidium Bromide): Followed by U-V Rays exposure (bright orange) * Elution: All DNA fragments are -ve charged: Visible DNA is now cut from agarose get & extracted ### 1 PCR = 2 Copies * 30 cycles repeated - Billions copies * **Desined DNA (GOI):** Is now linked with a suitable vector * **Gene amplification** - PCR technique (polymerase chain reaction) * by the help of **thermostable DNA Polymerase** (isolated from a bacterium: **Thenmus aquatic**) (tolenerate very high temp) - Billions copies * **Main steps**: 1. **Denaturation**: At high temp (94°C), ds DNA → ss DNA 2. **Annealing**: Addition of primer (DNA): small fragment of oligonucleotide, added (at 3'OH) 3. **Chain Extension**: polymerization DNA (Taq polymerase at 72°C temp, Mg++) ### Transfer of DNA into a competent Host * Heat shock method ### Obtaining foreign gene product * Recombinant protein: Desired product * Heterogeneous Host: Consists GOI ### Bioreactors * **Laboratory scale**: (Depending upon requirement of product) * **Industrial scale**: Big sized containers (100L-1000L) * To get desired protein, bioneactors * **pH control, Temp monitoring & O2 transport controlling system**: waste removal ### Simple stirred tank bioreactor * Common * (Bacterial transfer of host) **Nutrient**: * **Temp. sensor** * **pH monitor** * **Foam breaker** * **Flat blade impellera** * Product line * Sterile air ### Sparged Tank * Spangen: Tube with many pores * To provide extra oxygen ### Downstream Processing * **Last step**: isolation of the product, purification, added with preservatives, Ready for commercial use ### Question: During the purification process for recombinant DNA technology, addition of chilled ethanol precipitates out: (2023) - **DNA** ### Question: Gene gun method used to introduce alien DNA into host cells, microparticles of **Tungsten/gold** metal are used. (2023) ### Question: Which of the following is not a cloning vector (2023) - **Probe** ### Question: Upon exposure to UV radiation, DNA stained with ethidium bromide will show **bright orange colour** (2023) ### Question: Main steps in the formation of recombinant DNA are given below. Arrange these steps in a correct sequence: A. Insertion of recombinant DNA into the host cell. B. Cutting of DNA at specific location by restriction enzyme. C. Isolation of desired DNA fragments. D. Amplification of gene of interest using PCR. (2023) - **B,C,D,A** ### Question: Which one of the following statement is not true regarding gel electrophoresis technique? (2022) - **Bright orange colored bands of DNA can be observed in the gel when exposed to UV light.** ### Question: Given below are two statements; one is labelled as Assertion (A) and the other is labelled as Reason (R). **Assertion (A):** Polymerase chain reaction is used in DNA amplification. **Reason (R):** The ampicillin resistant gene is used as a selectable marker to check transformation. (2022) - **Both (A) and (R) are correct but (R) is not the correct explanation of (A).** ### Question: In the following palindromic base sequences of DNA, which one can be cut easily by a particular restriction enzyme? (2022) - **5'GAATTC 3'; 3' CTTAAG 5'** ### Question: Given below are two statements: **Statement I:** Restriction endonucleases recognise specific sequence to cut DNA known as palindromic nucleotide sequence. **Statement II:** Restriction endonucleases cut the DNA strand a little away from the centre of the palindromic site. (2022) - **Both Statement I and Statement II are correct.** ### Question: Which of the following is not a desirable feature of a cloning vector? (2022) - **Presence of two or more recognition sites.** ### Question: When gene targeting involving gene amplification is attempted in an individual’s tissue to treat disease, it is known as **gene therapy**. (2021) ### Question: Match the organism with its use in biotechnology. (1). _Salmonella typhimurium_ (2). _Agrobacterium tumefaciens_ (3). _Bacillus thuringiensis_ (4). _Thermus aquaticus_ (2020) - (iv) (iii) (i) (ii) ### Question: In gel electrophoresis, separated DNA fragments can be visualized with the help of **Ethidium bromide in UV radiation**. (2020) ### Question: The sequence controls the the copy number of the linked DNA in the vector is termed **Ori site**. (2020) ### Question: The specific palindromic sequence which is recognized by EcoRI is **5'-GAATTC-3'; 3'-CTTAAG-5'** (2020) ### Question: Identify the wrong statement with regard to restriction enzymes. (2020) - **Each restriction enzyme functions by inspecting the length of a DNA sequence.** ### Question: First discovered restriction endonuclease that always cuts DNA molecule at a particular point by recognising a specific sequence of six base pairs is **Hind II**. (2020) ### Question: In a mixture, DNA fragments are separated by **Electrophoresis.** (2020) ### Question: Match the following techniques or instruments with their usage. 1. Electrophoresis 2. Bioreactor 3. ELISA 4. PCR (2020) - (ii) (i) (iv) (iii) ### Question: Spooling is **Collection of isolated DNA** (2020) ### Question: Select the correct statement. (2020) - **Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzymes of optimum conditions.** ### Question: Which one of the following equipment is essentially required for growing microbes on a large scale for industrial production of enzymes? (2019) - **Bioreactor** ### Question: DNA precipitation out of a mixture of biomolecules can be achieved by treatment with **chilled ethanol**. (2019) ### Question: Following statements describe the characteristics of the enzyme restriction endonuclease. Identify the incorrect statement. (2019) - **The enzyme binds DNA at specific sites and cuts only one of the two strands.** ### Question: The correct order of steps in Polymerase Chain Reaction (PCR) is **Denaturation, Annealing, Extension.** (2018) ### Question: What is the criterion for DNA fragments movement on agarose gel during gel electrophoresis? (2017) - **The smaller the fragment size, the farther it moves** ### Question: The process of separation and purification of expressed protein before marketing is called **Downstream processing**. (2017) ### Question: A gene whose expression helps to identify transformed cell is known as **selectable marker**. (2017) ### Question: The DNA fragments separated on an agarose gel can be visualised after staining with **Ethidium bromide**. (2017) ### Question: Restriction endonucleases are **Synthesised by bacteria as part of their defense mechanism**. (2017) ### Question: Which of the following restriction enzymes produces blunt ends? (2016-II) - **Eco RV** ### Question: Which of the following is not the product of downstream processing? (2016-II) - **Expression** ### Question: A foreign DNA and plasmid cut by the same restriction endonuclease can be joined to form a recombinant plasmid using **Ligase**. (2016-II) ### Question: Stirred-tank bioreactors have been designed for **Availability of oxygen throughout the process**. (2016-II) ### Question: Which of the following is a restriction endonuclease? (2016-1) - **Hind II** ### Question: Taq polymerase enzyme is obtained from **Thermus aquaticus**. (2016-1) ### Question: Which of the following is not a feature of the plasmids? (2016-1) - **Single-stranded** ### Question: The DNA molecule to which the gene of interest is integrated for cloning is called **Vector.** (2015 Re) ### Question: The cutting of DNA at specific locations became possible with the discovery of **Restriction enzymes.** (2015 Re) ### Question: The introduction of T-DNA into plants involves **Infection of the plant by Agrobacterium tumefaciens**. (2015 Re) ### Question: Which vector can clone only a small fragment of DNA? (2014) - **Plasmid** ### Question: DNA fragments generated by the restriction endonuclease in a chemical reaction can be separated by **Electrophoresis**. (2013) ### Question: The colonies of recombinant bacteria appear white in contrast to blue colonies of non-recombinant bacteria because of **Non-recombinant bacteria containing β-galactosidase.** (2013) ### Question: Which of the following is not correctly matched for the organism and its cell wall degrading enzyme? (2013) - **Algae - Methylase** ### Revision * **NCERT: Read** * **Biotech: Application (Example based)** ### Homework * **Module Question** * **NCERT ही पार लगाएगी** # Thank You