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Biotechnology: Principles and Processes Class XII Chapter 11.pptx

The document elaborates on biotechnology, emphasizing the use of microorganisms, cells, and genetic engineering tools for producing products beneficial to humans. It discusses key concepts and processes in recombinant DNA technology, including the roles of restriction enzymes, cloning vectors, and various techniques for gene transfer and transformation. Additionally, it highlights the differences between traditional and modern biotechnology, focusing on innovations such as artificial recombinant DNA and the use of specific enzymes for genetic manipulation.

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BIOTECHNOLOGY: Principles and Processes CHAPTER - 11
Introduction Biotechnology can be defined as the use of microorganisms, plants or animal cells or their components to produce products and processes useful to humans. According to the EFB (European Federation of Biotechnology), biotechnology is the integration of natural science and organisms, cells, parts there of and molecular analogues for products and services. The term ‘Biotechnology’ was coined by Karl Ereky in 1919.
We will learn about Concept of Genetic engineering TOOLS OF RECOMBINANT DNA TECHNOLOGY Restriction Enzymes Cloning Vectors Competent Host (For Transformation with Recombinant DNA) PROCESSES OF RECOMBINANT DNA TECHNOLOGY Isolation of the Genetic Material (DNA) Cutting of DNA at Specific Locations Amplification of Gene of Interest using PCR Insertion of Recombinant DNA into the Host Cell/Organism Obtaining the Foreign Gene Product Downstream Processing Microinjection Electroporation Biolistic gene gun Sticky end Gene therapy Recombinant protein Transformation Selectable marker Electrophoresis Insertional activation Biotechnology Genetic engineering Transgenic Gene cloning Recombinant DNA Restriction Enzymes- Molecular scissors Palindromes Recognition sites/Restriction sites Plasmid Polymerase Chain Reaction
BIOTECHNOLOGY Traditional Biotechnology: Includes the processes that are based on natural capabilities of microorganisms. Curd, vinegar, ghee, wine and beer, idli, dosa, cheese paneer, etc are made using traditional biotechnology. Modern biotechnology: New and highly useful crop varieties, animal breeds are created. Recombinant proteins are produced using techniques of modern biotechnology.
PRINCIPLESOFBIOTECHNOLOGY 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. 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.
Sterile Conditions Autoclaving- of apparatus, glassware, media by high pressure treatment Sterilization- of chemicals through filters Laminar air flow- working space containing sterile air
Why do we need modern Biotechnology? PRODUCING HYBRIDS TRADITIONAL BREEDING OR RECOMBINANT DNA TECHNOLOGY?
Identification of DNA with desirable genes Introduction of identified DNA into the host Maintenance of introduced DNA in the host and transfer of host DNA to it’s progeny Wet lab/ dry lab (Bioinformatics) Gene transfer to vector and then the r-vector to organism Recombinant DNA must have ORI Three basic steps in genetically modifying an organism
E. coli Plasmid Vector
ORI is important !
Construction of First Artificial Recombinant DNA (i) It was achieved by linking a gene encoding antibiotic resistance with a native plasmid (an autonomously replicating circular extrachromosomal DNA) of Salmonella typhimurium. (ii) Stanley Cohen and Herbert Boyer accomplished this in 1972. (iii) They isolated the antibiotic resistance gene by cutting out a piece of DNA from a plasmid. (iv) The cutting of DNA at specific locations was carried out by molecular scissors, i.e. restriction enzymes. (v) The cut piece of DNA was then linked to the plasmid DNA with the enzyme DNA ligase (molecular glue). (vi) The plasmid acts as vectors to transfer the piece of DNA attached to it. (vii) When this DNA is transferred into E. coli, it could replicate using the new host’s DNA polymerase enzyme and make multiple copies. (viii) This ability to multiply copies of antibiotic resistance gene in E. coli was called cloning of antibiotic resistance gene in E. coli. Stanley Cohen of Stanford and Herbert Boyer of UCSF applied for a patent on recombinant DNA technology in 1974; it was granted in 1980.
First rDNA from “different organisms” In 1972, Paul Berg succeeded in inserting DNA from a bacterium into the SV40 virus' DNA. He thereby created the first DNA molecule made of parts from different organisms. Nobel Prize in Chemistry- 1980 (shared the Nobel Prize in Chemistry with Walter Gilbert and Frederick Sanger) The Berg letter: https://wellcomecollection.org/works/ue8p83nr/items?canvas=4
Worksheet 1 20.1.2022 Name two core techniques that enabled birth of modern biotechnology Name the scientists who accomplished formation of first artificial rDNA? Where do you see formation of a natural rDNA? What is the advantage of using genetic engineering over traditional hybridization methods? In a chromosome there is a specific DNA sequence called_______________, which is responsible for initiating replication. To clone a particular gene, it must be linked to a DNA molecule that has a __________ . Define cloning. Define plasmid Three basic steps in genetically modifying an organism —
TOOLS OF RECOMBINANT DNA TECHNOLOGY Vectors (DNA) Gene of interest (DNA) Restriction endonucleases DNA Ligases DNA Polymerases (Alkaline) Phosphatases Reverse Transcriptase Host (Cell/organism)
Restriction enzymes or ‘molecular scissors’ are used for cutting DNA.
Nucleases and its types Restriction enzymes belong to a class of enzymes called nucleases. Nucleases are of two types: Exonucleases They remove nucleotides from the ends. Endonucleases They cut at specific positions within the DNA.
Restriction endonuclease- History (i) Two enzymes from E. coli that were responsible for restricting the growth of bacteriophage were isolated in 1963, one of them added methyl group to DNA and the other cut DNA into segments. The later was called restriction endonuclease. (ii) The first isolated was Hind II in 1968. They found that it always cut DNA molecules at a particular point by recognising a specific sequence of six base pairs known as recognition sequence. (iii) Discovery of Restriction Endonuclease: Arber, Nathans and Smith (Nobel Prize 1978). (iv) Besides Hind II, more than 900 restriction enzymes have been isolated now, from over 230 strains of bacteria, each of which recognises different recognition sequences. 1972- first rDNA
Restriction Endonuclease: Nomenclature (a) The first letter is derived from the genus name and the next two letters from the species name of the prokaryotic cell from which enzymes are extracted. (b) The Roman numbers after name show the order in which the enzymes were isolated from the bacterial strain. For example, Eco RI comes from Escherichia coli RY13 and Eco RII comes from E. coli R 245, etc. For example HindIII comes from Haemophilus influenzae.
Source book: Principles of Gene Manipulation and Genomics, Primrose and Twyman
Restriction Enzymes: How do they act? Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the DNA. This site is called the Recognition or restriction site. Palindrome in DNA is a. sequence of base pairs that reads same on the two strands when orientation of reading is kept same. For example, the following sequences reads the same on the two strands in 5′ -> 3′ direction as well as 3′ -> 5′ direction. 5′ — GAATTC — 3′ 3′ — CTTAAG — 5′ Eco RI
RE which produce sticky ends are useful in RDT
Mechanism of Action of Restriction Enzymes (a) Restriction enzymes recognize restriction site, bind and cut each of the two strands of DNA at specefic points. (b) This leaves single stranded portions at the ends. (c) There are overhanging stretches called sticky ends on each strands. These are named so, because they form hydrogen bonds with their complementary cut counterparts. (d) The stickiness of the ends facilitates the action of the enzyme DNA ligase. (e) These sticky ends are complementary to each other when cut by same restriction enzyme, therefore can be joined together (end-to-end) using DNA ligases. Restriction endonucleases and DNA Ligases are used in genetic engineering to form recombinant molecules of DNA, which are composed of DNA from different sources/genomes.
DNA Ligase: Molecular Glue
RECOMBINANT DNA MOLECULE Recombinant DNA Molecule Gene of interest
How do we confirm that the DNA has been cut? Or joined.. GEL ELECTROPHORESIS
Separation and Isolation of DNA Fragments (i) The cutting of DNA by restriction endonucleases results in the fragments of DNA. (ii) The technique, which separates DNA fragments based on their size is called gel electrophoresis. (iii) 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. (iv) The most common medium used is agarose, a natural polymer extracted from sea weeds. Hence is also referred to agarose gel electrophoresis. (v) The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel. (vi) The smaller the fragment size, the farther it moves.
How to visualize the DNA on gel? (vi) 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. (vii) The DNA fragments can be seen as bright orange coloured bands. Isolation/ Purification of DNA fragments from the gel: These separated bands are cut out from the agarose gel and extracted from the gel piece. This is called elution. The purified DNA fragments can be used in constructing recombinant DNA by joining them with cloning vectors.
Ladder Plasmid
Worksheet 2 22.1.2022 Mention 5 tools of rDNA technology Name the enzyme: Hemophilus influenzae, strain d, 3rd enzyme Source of EcoRI - Differentiate between endonuclease and exonuclease - What is a palindromic nucleotide sequences - What is ‘recombinant’ molecule of DNA - Gel image: Which band represents smaller DNA fragment. Give basis for your inference. Why were ‘restriction’ enzymes named so ? How many cuts do you need in a circular plasmid to make it linear?
There are two sites of EcoRI on a plasmid. After digesting it with the enzyme, only one band is seen on the gel? Why?
Plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA. Cloning vectors PLASMIDS AND BACTERIOPHAGES HAVE THE ABILITY TO REPLICATE WITHIN BACTERIAL CELLS INDEPENDENT OF THE CONTROL OF CHROMOSOMAL DNA.
Vectors used in RDT (a) Plasmids Autonomously replicating circular extra-chromosomal DNA. (b) Bacteriophages Viruses infecting bacteria. (c) Cosmids Hybrid vectors derived from plasmids which contain cos site of X phage. (d) BAC: Bacterial Artificial Chromosome (e) YAC: Yeast Artificial Chromosome (f) MAC: Mammalian Artificial Chromosome Cloning vectors are the DNA molecules that can carry a foreign DNA segment into the host cell. The vectors used in recombinant DNA technology can be:
CLASS XI NCERT
PLASMIDS 1. Autonomously replicating ds circular extra-chromosomal DNA molecules 2. They are found naturally in some bacteria and yeast. They can be made artificially in lab too. 3. Copy number per cell: They can be high or low copy number. Some plasmids may have only one or two copies per cell whereas others may have 15-100 copies per cell. Their numbers can go even higher. 4. The plasmids are by far the most widely used, versatile, easily manipulated vectors. 5. E.g. pBR322, pUC 19 (artificially created)
Features that are required to facilitate cloning into a Vector/Plasmid. Characteristics of a Vector/ Plasmid (a) Origin of replication (Ori) (b) Selectable marker (c) Cloning sites Vectors for cloning genes in plants and animals.
Origin of replication (ori) 1. 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. 2. This sequence is also responsible for controlling the copy number of the linked DNA. 3. If a genetic engineer wants to recover many copies of the target DNA(insert) it should be cloned in a vector whose origin support high copy number.
(ii) Selectable marker The vector requires a selectable marker, which helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants. (Transformation is a procedure through which a piece of DNA is introduced in a host bacterium) 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. (Only transformed cells do)
(iii) Multiple cloning site (MCS) DNA region within a Plasmid that contains multiple unique recognition sites for the commonly used restriction enzymes. For each enzyme, the vector needs to have very few, preferably single, recognition site. The vector can be cut at these sites so as to enable ligation of the foreign gene (gene of interest). The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes
Are these sites randomly present on the vector? IF not, where are these specifically present? How do we cut the DNA at these sites? How do we insert the DNA? How do we check the DNA is inserted or not ?
Inserting gene within a AbR Gene! How does it work? Ligate a foreign DNA at the BamH I site of tetracycline resistance gene in the vector pBR322. The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA Recombinants will still have the ampicillin resistance. The transformants growing on ampicillin containing medium are then transferred on a medium containing tetracycline. The recombinants will grow in ampicillin containing medium but not on that containing tetracycline. Non-recombinants will grow on the medium containing both the antibiotics. Tet X Amp ✓ Tet ✓ Amp ✓ What about non transformants? Tet- No Amp- No
But the process of selecting recombinants by simultaneous analysis of two plates… It’s a cumbersome process! Let us look for an alternative…. (one step process) β-galactosidase IPTG (Inducer) Blue coloured product Colonies appear blue LacZ gene X-Gal LacZ gene X-Gal is a Substrate (like Lactose)
Using LacZ gene – (instead of selectable marker gene) DNA is inserted within the coding sequence of β-galactosidase. This results into inactivation of the enzyme Principle: Differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. INSERTIONAL INACTIVATION Blue colonies White colonies Non recombinant Recombinant ✓ X Blue White Screening -Insertional Inactivation DNA was not inserted and plasmid self ligated
https://www.sigmaaldrich.com/
Vectors for cloning genes in plants and animals
Ti (Tumor inducing) plasmid in Plants In plants, the Tumour inducing (Ti) plasmid of Agrobacterium tumefaciens is used as a cloning vector. Agrobacterium tumefaciens is a pathogen of several dicot plants. It delivers a piece of DNA known as T-DNA in the Ti plasmid which transforms normal plant cells into tumour cells to produce chemicals required by pathogens. How we use it as vector: ◦ Remove T-DNA region- This makes the vector disarmed ◦ Insert the gene of interest in disarmed Ti plasmid ◦ Unaware Agrobacterium, transfers our gene to the plant genome instead.
Example of a plasmid map
Vectors for animals Retrovirus : PATHOGEN Retroviruses can transform normal cells into cancerous cells Useful vector for delivering gene of interest to humans Retroviruses have been disarmed and are used to deliver desirable genes into animal cells
addgene.org
Competent Host (For Transformation with Recombinant DNA)
Why do we need a ‘competent’ host? Competent host organism (for transformation with recombinant DNA) is required because DNA being a hydrophilic molecule, cannot pass through cell membranes, Hence, the bacteria should be made competent to accept the DNA molecules. (i) Competency is the ability of a cell to take up foreign DNA. (ii) Methods to make a cell competent are as follows- ◦Chemical method- Heat shock method ◦Physical methods- Gene gun, microinjection, electroporation ◦Disarmed pathogen vectors: when allowed to infect the cell, transfer the recombinant DNA(desired gene) into the host.
Chemical method of TRANSFORMATION: Heat shock method Chemical method In this method, the cell is treated with a specific concentration of | a divalent cation such as calcium to increase pore size in cell wall. The cells are then incubated with recombinant DNA on ice, followed by placing them briefly at 42°C and then putting it back on ice. This is called heat shock treatment. This enables the bacteria to take up the plasmid (recombinant) DNA.
Physical method of gene transfer In this method, a recombinant DNA is directly injected into the nucleus of an animal cell by microinjection method. In plants, cells are bombarded with high velocity microparticles of gold or tungsten coated with DNA called as biolistics or gene gun method. Gene gun Microinjecton
PROCESSES OF RECOMBINANT DNA TECHNOLOGY Isolation of Genetic Material Cutting of DNA at specefic location Amplification of Gene of Interest using cloning in vector/PCR Insertion of Recombinant DNA into host cell/organism Obtaining foreign gene product Important
ISOLATIONOF GENETIC MATERIAL Break the cell open- Lysis of cell by treatment of- 1.bacterial cells with lysozyme 2. plant tissue with cellulase 3. Fungal cells with chitinase. Release of DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids. RNA can be removed by treatment with ribonuclease Proteins can be removed by treatment with protease. Addition of chilled ethanol Purified DNA ultimately precipitates out Seen as collection of fine threads in the suspension
http://nobel.scas.bcit.ca/ https://www.sciencelearn.org.nz/
CUTTINGOF DNA AT SPECEFICLOCATION Restriction enzyme digestions Under optimal conditions, purified DNA is cut by restriction enzyme. This step is done to vector as well Agarose gel electrophoresis is used to check the digested DNA. Cut out gene of interest is then incubated with the cut out vector DNA along with DNA Ligase rDNA is prepared
NPT II RB LB NOS Pro SlMBD2 RIP1 Promoter NOS ter pCAMBIA2301 Rip-1-amiRNA SlMBD-Nos XbaI SacI NOS ter 21bp S 21bp AS mir164 Loop Example of a prepared rDNA
Confirmation of insert (SlMBDs) by digestion of back transformed constructs isolated from Agrobacterium. pCambia 2301 digested with XbaI and SacI 1.9 kb 750 bp pCambia 2301 © Bhoomika, 2017
Amplificationof Geneof Interest usingPCR PCR stands for Polymerase Chain Reaction. It was developed by Kary Mulis for which he was warded Nobel Prize in 1993 With this technique, we can get multiple copies of the DNA or gene of interest in vitro by using set of primers and enzyme DNA polymerase. PCR components: 1. A DNA template: which contains the region to be amplified. 2. A set of Primers (Forward and Reverse) :small chemically synthesised oligonucleotides that are complementary to the regions of DNA 3. dNTPs : deoxyribonucleotide TriPhosphates 4. A heat stable DNA Polymerase- Taq Polymerase : isolated from a bacterium, Thermus aquaticus, which remains active during the high temperature induced denaturation of double stranded DNA
PCR : Each cycle has three steps (A) Denaturation (B) Primer annealing (C) Extension of primers ~30 cycles
Steps of PCR Cycle
Applications of PCR 1. Diagnosis of Pathogens 2. Diagnosis of specefic mutations 3. DNA Fingerprinting 4. Detection of specefic microorganism 5. In prenatal diagnosis
PCR result of 15 samples- out of which 4 are positive
Insertionof RecombinantDNA intohost cell/organism – andscreening Recipient cells is made ‘competent’ to receive, take up DNA present in its surrounding The recombinant DNA bearing gene for resistance to an antibiotic is transferred into E.coli cells Host cell become transformed into ampicillin-resistance cells Only transformants will grow on Ampicillin containing medium Screening of transformants with selectable marker gene- Antibiotic resistance genes Insertion screening
This is - AMPLIFICATON OF GENE INVIVO AMPLIFICATON OF GENE INVITRO PCR
Gene of interest Recombinant DNA Host Cell containing recombinant Vector PCR/Genome Vector Transfer to plant Transgenic plant Expression of foreign gene in host cells Optimized condition for Induction RE RE Stored in E. coli for further outsourcing
Obtainingforeigngene product After having cloned the gene of interest and having optimised the conditions to induce the expression of the target protein, the protein can be produced on a large scale. 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. But, small volume cultures cannot yield appreciable quantities of products. To produce in large quantities of recombinant protein, bioreactors are required, where large volumes (100- 1000 litres) of culture can be processed. BIOREACTORS Bioreactors re large 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). Small scale Large scale: Industrial Biotechnology
(a) Simple stirred-tank bioreactor (b) Sparged stirred-tank bioreactor through which sterile air bubbles are sparged Bioreactor: Stirred type, the most common 1. A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. 2. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. 3. Alternatively air can be bubbled through the reactor. 4. 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.
DownstreamProcessing After completion of the biosynthetic stage, downstream processing is required. Downstream Processing involves processes that make the product obtained ready for marketing. This process includes separation and purification. Suitable preservatives are added to it and send for clinical trial in case of drugs before releasing to market for public use Strict quality control testing for each product is also required. Steps of downstream processing (for understanding purpose)
Image Sources: http://theupturnedmicroscope.com/
THANKYOU © Bhoomika
1. What is a recombinant protein? 2. Name the components a bioreactor must possess to achieve the desired product? 3. What is a continuous culture system? What are its advantages? 4. Explain the role of enzymes in the extraction of DNA from Rhizopus in its purest form. 5. Since DNA is a hydrophillic moelcule, it cannot pass through cell membranes. Name and explain the technique with which the DNA is forced into (ii) a bacterial cell (ii) a plant cell (iii) an animal cell. 6. Describe two methods of selection of recombinants (bacteria). 7. A selectable marker is used in the section of recombinants on the basis of their ability to produce colou*r in presence of chromogenic substrate. (a) Mention the name of mechanism involved. (b) Which enzyme is involved in production of colour? (c) How is it advantageous over using antibiotic resistant gene as a selectable marker?
1. How is copy number of the plasmid vector related to yield of recombinant protein? 2. Restriction enzymes should not have more than one site of action in the cloning site of a vector. Comment. 3. A plasmid without a selectable marker was chosen as vector for cloning a gene. How does this affect the experiment? 4. A mixture of fragmented DNA was electrophoresed in an agarose gel. After staining the gel with ethidium bromide, no DNA bands were observed. What could be the reason? 5. Describe the role of Agrobacterium tumefaciens in transforming a plant cell. 6. What would happen when one grows a recombinant bacterium in a bioreactor but forget to add antibiotic to the medium in which the recombinant is growing? 7. Make a chart (with diagrammatic representation) showing a restriction enzyme, the substrate DNA on which it acts, the site at which it cuts DNA and the product it produces. 8. A plasmid DNA and a linear DNA (both are of the same size) have one site for a restriction endonuclease. When cut and separated on agarose gel electrophoresis, plasmid shows one DNA band while linear DNA shows two fragments. Explain.
Describe the various steps involved in Recombinant DNA technology with the help of a well labeled. Diagram? 5
BLOTTING
PCR- AND DETECTION
PROBES AND HYBRIDIZATION
What after infection with Agrobacterium mediated transfer of gene? Tissue culture ----> Transgenic

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Biotechnology: Principles and Processes Class XII Chapter 11.pptx

  • 1.
  • 2.
    Introduction Biotechnology can bedefined as the use of microorganisms, plants or animal cells or their components to produce products and processes useful to humans. According to the EFB (European Federation of Biotechnology), biotechnology is the integration of natural science and organisms, cells, parts there of and molecular analogues for products and services. The term ‘Biotechnology’ was coined by Karl Ereky in 1919.
  • 3.
    We will learnabout Concept of Genetic engineering TOOLS OF RECOMBINANT DNA TECHNOLOGY Restriction Enzymes Cloning Vectors Competent Host (For Transformation with Recombinant DNA) PROCESSES OF RECOMBINANT DNA TECHNOLOGY Isolation of the Genetic Material (DNA) Cutting of DNA at Specific Locations Amplification of Gene of Interest using PCR Insertion of Recombinant DNA into the Host Cell/Organism Obtaining the Foreign Gene Product Downstream Processing Microinjection Electroporation Biolistic gene gun Sticky end Gene therapy Recombinant protein Transformation Selectable marker Electrophoresis Insertional activation Biotechnology Genetic engineering Transgenic Gene cloning Recombinant DNA Restriction Enzymes- Molecular scissors Palindromes Recognition sites/Restriction sites Plasmid Polymerase Chain Reaction
  • 4.
    BIOTECHNOLOGY Traditional Biotechnology: Includes theprocesses that are based on natural capabilities of microorganisms. Curd, vinegar, ghee, wine and beer, idli, dosa, cheese paneer, etc are made using traditional biotechnology. Modern biotechnology: New and highly useful crop varieties, animal breeds are created. Recombinant proteins are produced using techniques of modern biotechnology.
  • 5.
    PRINCIPLESOFBIOTECHNOLOGY 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. 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.
  • 6.
    Sterile Conditions Autoclaving- ofapparatus, glassware, media by high pressure treatment Sterilization- of chemicals through filters Laminar air flow- working space containing sterile air
  • 7.
    Why do weneed modern Biotechnology? PRODUCING HYBRIDS TRADITIONAL BREEDING OR RECOMBINANT DNA TECHNOLOGY?
  • 8.
    Identification of DNAwith desirable genes Introduction of identified DNA into the host Maintenance of introduced DNA in the host and transfer of host DNA to it’s progeny Wet lab/ dry lab (Bioinformatics) Gene transfer to vector and then the r-vector to organism Recombinant DNA must have ORI Three basic steps in genetically modifying an organism
  • 9.
  • 10.
  • 11.
    Construction of FirstArtificial Recombinant DNA (i) It was achieved by linking a gene encoding antibiotic resistance with a native plasmid (an autonomously replicating circular extrachromosomal DNA) of Salmonella typhimurium. (ii) Stanley Cohen and Herbert Boyer accomplished this in 1972. (iii) They isolated the antibiotic resistance gene by cutting out a piece of DNA from a plasmid. (iv) The cutting of DNA at specific locations was carried out by molecular scissors, i.e. restriction enzymes. (v) The cut piece of DNA was then linked to the plasmid DNA with the enzyme DNA ligase (molecular glue). (vi) The plasmid acts as vectors to transfer the piece of DNA attached to it. (vii) When this DNA is transferred into E. coli, it could replicate using the new host’s DNA polymerase enzyme and make multiple copies. (viii) This ability to multiply copies of antibiotic resistance gene in E. coli was called cloning of antibiotic resistance gene in E. coli. Stanley Cohen of Stanford and Herbert Boyer of UCSF applied for a patent on recombinant DNA technology in 1974; it was granted in 1980.
  • 12.
    First rDNA from“different organisms” In 1972, Paul Berg succeeded in inserting DNA from a bacterium into the SV40 virus' DNA. He thereby created the first DNA molecule made of parts from different organisms. Nobel Prize in Chemistry- 1980 (shared the Nobel Prize in Chemistry with Walter Gilbert and Frederick Sanger) The Berg letter: https://wellcomecollection.org/works/ue8p83nr/items?canvas=4
  • 13.
    Worksheet 1 20.1.2022 Nametwo core techniques that enabled birth of modern biotechnology Name the scientists who accomplished formation of first artificial rDNA? Where do you see formation of a natural rDNA? What is the advantage of using genetic engineering over traditional hybridization methods? In a chromosome there is a specific DNA sequence called_______________, which is responsible for initiating replication. To clone a particular gene, it must be linked to a DNA molecule that has a __________ . Define cloning. Define plasmid Three basic steps in genetically modifying an organism —
  • 15.
    TOOLS OF RECOMBINANTDNA TECHNOLOGY Vectors (DNA) Gene of interest (DNA) Restriction endonucleases DNA Ligases DNA Polymerases (Alkaline) Phosphatases Reverse Transcriptase Host (Cell/organism)
  • 16.
  • 17.
    Nucleases and itstypes Restriction enzymes belong to a class of enzymes called nucleases. Nucleases are of two types: Exonucleases They remove nucleotides from the ends. Endonucleases They cut at specific positions within the DNA.
  • 18.
    Restriction endonuclease- History (i)Two enzymes from E. coli that were responsible for restricting the growth of bacteriophage were isolated in 1963, one of them added methyl group to DNA and the other cut DNA into segments. The later was called restriction endonuclease. (ii) The first isolated was Hind II in 1968. They found that it always cut DNA molecules at a particular point by recognising a specific sequence of six base pairs known as recognition sequence. (iii) Discovery of Restriction Endonuclease: Arber, Nathans and Smith (Nobel Prize 1978). (iv) Besides Hind II, more than 900 restriction enzymes have been isolated now, from over 230 strains of bacteria, each of which recognises different recognition sequences. 1972- first rDNA
  • 19.
    Restriction Endonuclease: Nomenclature (a)The first letter is derived from the genus name and the next two letters from the species name of the prokaryotic cell from which enzymes are extracted. (b) The Roman numbers after name show the order in which the enzymes were isolated from the bacterial strain. For example, Eco RI comes from Escherichia coli RY13 and Eco RII comes from E. coli R 245, etc. For example HindIII comes from Haemophilus influenzae.
  • 20.
    Source book: Principlesof Gene Manipulation and Genomics, Primrose and Twyman
  • 21.
    Restriction Enzymes: Howdo they act? Each restriction endonuclease recognises a specific palindromic nucleotide sequences in the DNA. This site is called the Recognition or restriction site. Palindrome in DNA is a. sequence of base pairs that reads same on the two strands when orientation of reading is kept same. For example, the following sequences reads the same on the two strands in 5′ -> 3′ direction as well as 3′ -> 5′ direction. 5′ — GAATTC — 3′ 3′ — CTTAAG — 5′ Eco RI
  • 22.
    RE which producesticky ends are useful in RDT
  • 24.
    Mechanism of Actionof Restriction Enzymes (a) Restriction enzymes recognize restriction site, bind and cut each of the two strands of DNA at specefic points. (b) This leaves single stranded portions at the ends. (c) There are overhanging stretches called sticky ends on each strands. These are named so, because they form hydrogen bonds with their complementary cut counterparts. (d) The stickiness of the ends facilitates the action of the enzyme DNA ligase. (e) These sticky ends are complementary to each other when cut by same restriction enzyme, therefore can be joined together (end-to-end) using DNA ligases. Restriction endonucleases and DNA Ligases are used in genetic engineering to form recombinant molecules of DNA, which are composed of DNA from different sources/genomes.
  • 25.
  • 27.
    RECOMBINANT DNA MOLECULE RecombinantDNA Molecule Gene of interest
  • 28.
    How do we confirmthat the DNA has been cut? Or joined.. GEL ELECTROPHORESIS
  • 29.
    Separation and Isolationof DNA Fragments (i) The cutting of DNA by restriction endonucleases results in the fragments of DNA. (ii) The technique, which separates DNA fragments based on their size is called gel electrophoresis. (iii) 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. (iv) The most common medium used is agarose, a natural polymer extracted from sea weeds. Hence is also referred to agarose gel electrophoresis. (v) The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel. (vi) The smaller the fragment size, the farther it moves.
  • 31.
    How to visualizethe DNA on gel? (vi) 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. (vii) The DNA fragments can be seen as bright orange coloured bands. Isolation/ Purification of DNA fragments from the gel: These separated bands are cut out from the agarose gel and extracted from the gel piece. This is called elution. The purified DNA fragments can be used in constructing recombinant DNA by joining them with cloning vectors.
  • 32.
  • 34.
    Worksheet 2 22.1.2022 Mention5 tools of rDNA technology Name the enzyme: Hemophilus influenzae, strain d, 3rd enzyme Source of EcoRI - Differentiate between endonuclease and exonuclease - What is a palindromic nucleotide sequences - What is ‘recombinant’ molecule of DNA - Gel image: Which band represents smaller DNA fragment. Give basis for your inference. Why were ‘restriction’ enzymes named so ? How many cuts do you need in a circular plasmid to make it linear?
  • 35.
    There are twosites of EcoRI on a plasmid. After digesting it with the enzyme, only one band is seen on the gel? Why?
  • 36.
    Plasmids and bacteriophageshave the ability to replicate within bacterial cells independent of the control of chromosomal DNA. Cloning vectors PLASMIDS AND BACTERIOPHAGES HAVE THE ABILITY TO REPLICATE WITHIN BACTERIAL CELLS INDEPENDENT OF THE CONTROL OF CHROMOSOMAL DNA.
  • 37.
    Vectors used inRDT (a) Plasmids Autonomously replicating circular extra-chromosomal DNA. (b) Bacteriophages Viruses infecting bacteria. (c) Cosmids Hybrid vectors derived from plasmids which contain cos site of X phage. (d) BAC: Bacterial Artificial Chromosome (e) YAC: Yeast Artificial Chromosome (f) MAC: Mammalian Artificial Chromosome Cloning vectors are the DNA molecules that can carry a foreign DNA segment into the host cell. The vectors used in recombinant DNA technology can be:
  • 38.
  • 39.
    PLASMIDS 1. Autonomously replicatingds circular extra-chromosomal DNA molecules 2. They are found naturally in some bacteria and yeast. They can be made artificially in lab too. 3. Copy number per cell: They can be high or low copy number. Some plasmids may have only one or two copies per cell whereas others may have 15-100 copies per cell. Their numbers can go even higher. 4. The plasmids are by far the most widely used, versatile, easily manipulated vectors. 5. E.g. pBR322, pUC 19 (artificially created)
  • 41.
    Features that arerequired to facilitate cloning into a Vector/Plasmid. Characteristics of a Vector/ Plasmid (a) Origin of replication (Ori) (b) Selectable marker (c) Cloning sites Vectors for cloning genes in plants and animals.
  • 42.
    Origin of replication(ori) 1. 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. 2. This sequence is also responsible for controlling the copy number of the linked DNA. 3. If a genetic engineer wants to recover many copies of the target DNA(insert) it should be cloned in a vector whose origin support high copy number.
  • 43.
    (ii) Selectable marker Thevector requires a selectable marker, which helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants. (Transformation is a procedure through which a piece of DNA is introduced in a host bacterium) 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. (Only transformed cells do)
  • 44.
    (iii) Multiple cloningsite (MCS) DNA region within a Plasmid that contains multiple unique recognition sites for the commonly used restriction enzymes. For each enzyme, the vector needs to have very few, preferably single, recognition site. The vector can be cut at these sites so as to enable ligation of the foreign gene (gene of interest). The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes
  • 45.
    Are these sitesrandomly present on the vector? IF not, where are these specifically present? How do we cut the DNA at these sites? How do we insert the DNA? How do we check the DNA is inserted or not ?
  • 46.
    Inserting gene withina AbR Gene! How does it work? Ligate a foreign DNA at the BamH I site of tetracycline resistance gene in the vector pBR322. The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA Recombinants will still have the ampicillin resistance. The transformants growing on ampicillin containing medium are then transferred on a medium containing tetracycline. The recombinants will grow in ampicillin containing medium but not on that containing tetracycline. Non-recombinants will grow on the medium containing both the antibiotics. Tet X Amp ✓ Tet ✓ Amp ✓ What about non transformants? Tet- No Amp- No
  • 48.
    But the processof selecting recombinants by simultaneous analysis of two plates… It’s a cumbersome process! Let us look for an alternative…. (one step process) β-galactosidase IPTG (Inducer) Blue coloured product Colonies appear blue LacZ gene X-Gal LacZ gene X-Gal is a Substrate (like Lactose)
  • 49.
    Using LacZ gene– (instead of selectable marker gene) DNA is inserted within the coding sequence of β-galactosidase. This results into inactivation of the enzyme Principle: Differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. INSERTIONAL INACTIVATION Blue colonies White colonies Non recombinant Recombinant ✓ X Blue White Screening -Insertional Inactivation DNA was not inserted and plasmid self ligated
  • 50.
  • 51.
    Vectors for cloning genes inplants and animals
  • 52.
    Ti (Tumor inducing)plasmid in Plants In plants, the Tumour inducing (Ti) plasmid of Agrobacterium tumefaciens is used as a cloning vector. Agrobacterium tumefaciens is a pathogen of several dicot plants. It delivers a piece of DNA known as T-DNA in the Ti plasmid which transforms normal plant cells into tumour cells to produce chemicals required by pathogens. How we use it as vector: ◦ Remove T-DNA region- This makes the vector disarmed ◦ Insert the gene of interest in disarmed Ti plasmid ◦ Unaware Agrobacterium, transfers our gene to the plant genome instead.
  • 53.
    Example of aplasmid map
  • 55.
    Vectors for animals Retrovirus: PATHOGEN Retroviruses can transform normal cells into cancerous cells Useful vector for delivering gene of interest to humans Retroviruses have been disarmed and are used to deliver desirable genes into animal cells
  • 56.
  • 57.
  • 58.
    Why do weneed a ‘competent’ host? Competent host organism (for transformation with recombinant DNA) is required because DNA being a hydrophilic molecule, cannot pass through cell membranes, Hence, the bacteria should be made competent to accept the DNA molecules. (i) Competency is the ability of a cell to take up foreign DNA. (ii) Methods to make a cell competent are as follows- ◦Chemical method- Heat shock method ◦Physical methods- Gene gun, microinjection, electroporation ◦Disarmed pathogen vectors: when allowed to infect the cell, transfer the recombinant DNA(desired gene) into the host.
  • 59.
    Chemical method ofTRANSFORMATION: Heat shock method Chemical method In this method, the cell is treated with a specific concentration of | a divalent cation such as calcium to increase pore size in cell wall. The cells are then incubated with recombinant DNA on ice, followed by placing them briefly at 42°C and then putting it back on ice. This is called heat shock treatment. This enables the bacteria to take up the plasmid (recombinant) DNA.
  • 60.
    Physical method ofgene transfer In this method, a recombinant DNA is directly injected into the nucleus of an animal cell by microinjection method. In plants, cells are bombarded with high velocity microparticles of gold or tungsten coated with DNA called as biolistics or gene gun method. Gene gun Microinjecton
  • 62.
    PROCESSES OF RECOMBINANTDNA TECHNOLOGY Isolation of Genetic Material Cutting of DNA at specefic location Amplification of Gene of Interest using cloning in vector/PCR Insertion of Recombinant DNA into host cell/organism Obtaining foreign gene product Important
  • 63.
    ISOLATIONOF GENETIC MATERIAL Breakthe cell open- Lysis of cell by treatment of- 1.bacterial cells with lysozyme 2. plant tissue with cellulase 3. Fungal cells with chitinase. Release of DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids. RNA can be removed by treatment with ribonuclease Proteins can be removed by treatment with protease. Addition of chilled ethanol Purified DNA ultimately precipitates out Seen as collection of fine threads in the suspension
  • 64.
  • 65.
    CUTTINGOF DNA ATSPECEFICLOCATION Restriction enzyme digestions Under optimal conditions, purified DNA is cut by restriction enzyme. This step is done to vector as well Agarose gel electrophoresis is used to check the digested DNA. Cut out gene of interest is then incubated with the cut out vector DNA along with DNA Ligase rDNA is prepared
  • 66.
    NPT II RB LB NOSPro SlMBD2 RIP1 Promoter NOS ter pCAMBIA2301 Rip-1-amiRNA SlMBD-Nos XbaI SacI NOS ter 21bp S 21bp AS mir164 Loop Example of a prepared rDNA
  • 67.
    Confirmation of insert(SlMBDs) by digestion of back transformed constructs isolated from Agrobacterium. pCambia 2301 digested with XbaI and SacI 1.9 kb 750 bp pCambia 2301 © Bhoomika, 2017
  • 68.
    Amplificationof Geneof InterestusingPCR PCR stands for Polymerase Chain Reaction. It was developed by Kary Mulis for which he was warded Nobel Prize in 1993 With this technique, we can get multiple copies of the DNA or gene of interest in vitro by using set of primers and enzyme DNA polymerase. PCR components: 1. A DNA template: which contains the region to be amplified. 2. A set of Primers (Forward and Reverse) :small chemically synthesised oligonucleotides that are complementary to the regions of DNA 3. dNTPs : deoxyribonucleotide TriPhosphates 4. A heat stable DNA Polymerase- Taq Polymerase : isolated from a bacterium, Thermus aquaticus, which remains active during the high temperature induced denaturation of double stranded DNA
  • 69.
    PCR : Eachcycle has three steps (A) Denaturation (B) Primer annealing (C) Extension of primers ~30 cycles
  • 72.
  • 73.
    Applications of PCR 1.Diagnosis of Pathogens 2. Diagnosis of specefic mutations 3. DNA Fingerprinting 4. Detection of specefic microorganism 5. In prenatal diagnosis
  • 74.
    PCR result of15 samples- out of which 4 are positive
  • 75.
    Insertionof RecombinantDNA intohostcell/organism – andscreening Recipient cells is made ‘competent’ to receive, take up DNA present in its surrounding The recombinant DNA bearing gene for resistance to an antibiotic is transferred into E.coli cells Host cell become transformed into ampicillin-resistance cells Only transformants will grow on Ampicillin containing medium Screening of transformants with selectable marker gene- Antibiotic resistance genes Insertion screening
  • 76.
    This is - AMPLIFICATONOF GENE INVIVO AMPLIFICATON OF GENE INVITRO PCR
  • 77.
    Gene of interest RecombinantDNA Host Cell containing recombinant Vector PCR/Genome Vector Transfer to plant Transgenic plant Expression of foreign gene in host cells Optimized condition for Induction RE RE Stored in E. coli for further outsourcing
  • 78.
    Obtainingforeigngene product After havingcloned the gene of interest and having optimised the conditions to induce the expression of the target protein, the protein can be produced on a large scale. 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. But, small volume cultures cannot yield appreciable quantities of products. To produce in large quantities of recombinant protein, bioreactors are required, where large volumes (100- 1000 litres) of culture can be processed. BIOREACTORS Bioreactors re large 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). Small scale Large scale: Industrial Biotechnology
  • 79.
    (a) Simple stirred-tankbioreactor (b) Sparged stirred-tank bioreactor through which sterile air bubbles are sparged Bioreactor: Stirred type, the most common 1. A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents. 2. The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. 3. Alternatively air can be bubbled through the reactor. 4. 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.
  • 80.
    DownstreamProcessing After completion ofthe biosynthetic stage, downstream processing is required. Downstream Processing involves processes that make the product obtained ready for marketing. This process includes separation and purification. Suitable preservatives are added to it and send for clinical trial in case of drugs before releasing to market for public use Strict quality control testing for each product is also required. Steps of downstream processing (for understanding purpose)
  • 82.
  • 83.
  • 84.
    1. What isa recombinant protein? 2. Name the components a bioreactor must possess to achieve the desired product? 3. What is a continuous culture system? What are its advantages? 4. Explain the role of enzymes in the extraction of DNA from Rhizopus in its purest form. 5. Since DNA is a hydrophillic moelcule, it cannot pass through cell membranes. Name and explain the technique with which the DNA is forced into (ii) a bacterial cell (ii) a plant cell (iii) an animal cell. 6. Describe two methods of selection of recombinants (bacteria). 7. A selectable marker is used in the section of recombinants on the basis of their ability to produce colou*r in presence of chromogenic substrate. (a) Mention the name of mechanism involved. (b) Which enzyme is involved in production of colour? (c) How is it advantageous over using antibiotic resistant gene as a selectable marker?
  • 85.
    1. How iscopy number of the plasmid vector related to yield of recombinant protein? 2. Restriction enzymes should not have more than one site of action in the cloning site of a vector. Comment. 3. A plasmid without a selectable marker was chosen as vector for cloning a gene. How does this affect the experiment? 4. A mixture of fragmented DNA was electrophoresed in an agarose gel. After staining the gel with ethidium bromide, no DNA bands were observed. What could be the reason? 5. Describe the role of Agrobacterium tumefaciens in transforming a plant cell. 6. What would happen when one grows a recombinant bacterium in a bioreactor but forget to add antibiotic to the medium in which the recombinant is growing? 7. Make a chart (with diagrammatic representation) showing a restriction enzyme, the substrate DNA on which it acts, the site at which it cuts DNA and the product it produces. 8. A plasmid DNA and a linear DNA (both are of the same size) have one site for a restriction endonuclease. When cut and separated on agarose gel electrophoresis, plasmid shows one DNA band while linear DNA shows two fragments. Explain.
  • 86.
    Describe the varioussteps involved in Recombinant DNA technology with the help of a well labeled. Diagram? 5
  • 87.
  • 88.
  • 89.
  • 90.
    What after infectionwith Agrobacterium mediated transfer of gene? Tissue culture ----> Transgenic