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Biotechnology Flashc

Biotechnology flashcards

Biotechnology definition Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans
EFB (European Federation of Biotechnology) Definition by EFB "The Integration of Natural Science and organisms, cells, parts thereof, and molecular analogies for products and services.
First recombinant DNA In 1972,By Herbert Boyer and Stanley Cohen. Took Antibiotic resistance gene ➡ Insert in cut plasmid of Selmonella Typhi (Hence rDNA Formed) ➡ Transfer this rDNA into E.coli (Host) This rDNA used E.coli's DNA polymerase to make it's multiple copies.
Tools of Recombinant DNA Technology 1) Enzymes (Restriction endonuclease, DNA polymerase, DNA Ligase) 2) Vectors 3) Host
Restriction Enzymes- Natural Defense System of Bacteria against Viruses called Bacteriophage. Discovered by Arber, Smith and Nathans(in 1963) Methylases(cause methylation) , Restriction endonuclease(cuts the phosphodiester bond at specific points in DNA)
Restriction endonuclease (Molecular Scissors, Chemical knives, Chemical Scalpels) Till now 900 Restriction endonucleases have been isolated from 230 different strains of Bacteria. First restriction endonuclease - Hind II.
Restriction endonucleases EcoR1 EcoR1 - from E.Coli , palindrome sequence GAATTC it cut strand between G and A (Form sticky ends) ⭐ EcoR5 - form Blunt ends
DNA Ligase(Molecular Glue) Create Phosphodiester bond. Most commonly used DNA Ligase is T4 Ligase.
DNA Polymerase Create complementary strand on DNA template. Most commonly used - DNA Polymerase I.
Vectors(gene taxi) Carry gene of Interest. Example- Plasmids, Bacteriophage, Cosmids, YAC, BAC, Transposons, Agrobacterium Tumefaciens, Disarmed Retrovirus, Shuttle Vector.
Plasmid features Circular, Double stranded, Autonomously Replicating, Extra chromosomal, Enter Bacteria cell or yeast cell and give them Antibiotic resistance or Virulance(disease causing ability)
PBR322 B and R ➡ Bolliver and Rodriguez (scientists who reconstructed it) Ampicillin gene( Pst I and Pvu 1) Tetracycline gene (BamH1 and sal1) Ori (origin of replication,it control copy number) ROP (Produce proteins involved in replication of plasmid)
Selectable Markers Ampicillin resistance gene and Tetracycline resistance gene.
Ti Plasmid (Tumour inducing plasmid) Agrobacterium Tumefaciens. Gives Tumour inducing ability to it. It causes crown gall tumour in dicot roots.
pUC8 Reconstructed at University of California. Lac Z gene is present. It leads to production of Beta-galactosidase enzyme which convert colourless substrate X-gal into blue colour.
Bacteriophage 1) Lambda phage. 2) M-13 phage. Genetic material of lambda phage is dsDNA (linear) with 2 overhanging Cos sites(Cohesive end sites). Which are complementary to each other.
Cosmids Constructed by joining Cos sites of lambda phage with plasmid. Cosmid can carry larger gene of interest in comparison to plasmid.
YAC (Yeast Artificial Chromosome) BAC (Bacterial Artificial Chromosome) Used in Human Genome Project
Disarmed Agrobacterium Tumefaciens If Host is plant cell. Has T-DNA(Transfer DNA) That gets incorporated with Dicot cell genome. This bacteria is disarmed in labs and used as vector.
Disarmed Retrovirus If host is Animal cell.
Shuttle Vector can be used as a vector in Prokaryotes as well as Eukaryotes because they have 2 ori. Example- 1) YEP [Yeast Episomal Plasmid] 2) Modified Ti Plasmid
Process of rDNA Tech. Isolation of DNA from source cell➡Cutting of DNA➡Isolation of Desired segment ➡amplification of DNA➡Ligation of DNA with vector ➡Transfer into host➡culturing of recombinant ➡downstream processing.
Isolation Of DNA from source First cell wall break plants(Cellulase), Fungi(chitinase), Bacteria(lysozyme). Then nuclear membrane and cell memb. break with Lipases and certain detergent. RNA and histone removed by RNAase and Protease. Chilled Ethanol is added to precipitate DNA
Spooling After precipitation with chilled ethanol, DNA is removed from test tube by spooling process in which a glass stick is used.
Isolation of desired DNA fragment after cutting - Gel Electrophoresis DNA being negatively charged, move towards anode under electric field through Agarose gel. Smaller the fragment, farther it will go(or closer it go to anode) Then DNA fragment ➡stained with Ethidium Bromide. In UV light, they appear Bright orange.
Elution The desired DNA fragment is cut out from Agarose gel by this process.
Amplification of DNA - Polymerase Chain Reaction By Kary Mullis. Denaturation(94/95°C) ➡ Annealing (50-60°C) ➡ Extension (72°C) Taq Polymerase used (Obtained from Thermus aquaticus). It's thermostable enzyme.
Transformation of Bacterial Cell First treated with CaCl², because Ca²+ ions improve binding of rDNA on surface of bacterial cell. now cell incubated on Ice ➡ Heat Shock (42°C) ➡ Again Ice. This lead to opening of transient pores in cell wall and cell membrane of bacteria.
Transformation of Animal Cell Micro-injections
Transformation of Plant Cells Gene Gun / Biolistic Method. Microparticles of Gold or Tungsten, Coated with DNA are bombarded at high velocity on target cell.
Culturing of Recombinants in Large Vessels called Bioreactor/Fermentors 100-1000 litre stainless steel container. Provide optimum temperature, pH,etc. Most commonly used bioreactor are Stirring type.
Downstream Processing It involves Filtration, chromatography, drying, centrifugation, Distillation, etc . After this, preservatives are added and strict quality testing is done before marketing this product.
Created by: Rahul27
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