Chapter 9 - Biotechnology Principles and Processes
Biology - Class 12
1. Principles - Recombinant DNA and Boyer-Cohen
Recombinant DNA (rDNA) means DNA made by joining DNAs from two different sources. Like cut and paste.
Story: Boyer and Cohen 1972 - Cohen had plasmid, Boyer had EcoRI scissors. Both cut plasmid and frog DNA with same scissors, joined with ligase (fevicol), made first rDNA. This started biotechnology!
Principle = scissors + fevicol + vehicle. Scissors = Restriction Enzyme, Fevicol = DNA Ligase, Vehicle = Vector plasmid that carries gene into host bacteria.
Host is E.coli where rDNA makes many copies.
rDNA formed by joining genetic material from different origins. Boyer (EcoRI) + Cohen (pSC101) created first rDNA 1972. Requires RE, ligase, vector, competent host. Vector = cloning vehicle, Host = factory for amplification.
2. Molecular Scissors - Restriction Enzymes
Restriction Enzymes are molecular scissors that cut DNA at specific sequence. Bacteria make them to cut virus DNA and protect themselves.
EcoRI cuts at GAATTC. Cuts between G and A. Makes sticky ends AATT hanging like tape, easy to join. This is fevicol tape.
EcoRV cuts GATATC in middle makes blunt ends - straight cut, no overhang.
Type II enzymes used in lab, need Mg2+ cofactor, work at 37°C.
Nomenclature: EcoRI = E = genus Escherichia, co = species coli, R = strain RY13, I = first enzyme. Simple!
Palindrome: MALAYALAM reads same forward backward. DNA palindrome GAATTC reads same 5' to 3' on both strands opposite direction. Symmetry helps enzyme bind.
EcoRI 5' GAATTC 3' -> G^AATTC sticky 5' overhang AATT. EcoRV GAT^ATC blunt flush. Type II palindromic recognition, cut within site, Mg2+ dependent, 37C. Naming: Genus first letter + species two letters + strain + order I II. Palindrome DNA reads same 5'->3' both strands.
3. Cloning Vectors - pBR322 and Other Vectors
Vector = vehicle auto that carries our gene into host. Without auto, gene cannot enter bacteria.
Most used vector pBR322 - 4361 base pairs circular. Parts: ori origin where replication starts, ampR ampicillin resistance, tetR tetracycline resistance for selection, rop helps replication.
Many cutting sites: HindIII, EcoRI, BamHI, PstI. If we insert gene inside tetR, tet gene breaks - bacteria becomes tet sensitive but amp resistant. So recombinant = amp resistant only. This is insertional inactivation.
Better selection = blue-white: lacZ gene, if insert inside white colonies = recombinant, blue = non-recombinant.
Other vectors: Ti plasmid of Agrobacterium natural genetic engineer for dicots, disarmed Ti used. Lambda phage carries big DNA. Retrovirus for animal cells. Ri plasmid makes root.
pBR322 4361bp ori ampR tetR rop unique RE sites. Insertional inactivation inactivates marker, recombinant loses one resistance. Blue-white lacZ alpha complementation white recombinant. Ti plasmid Agrobacterium tumefaciens T-DNA disarmed for dicots, lambda >10kb, retrovirus mammals, Ri root.
4. Competent Host and Transformation
Bacteria normally do not take outside DNA. We make them competent - door open.
CaCl2 method: Treat with ice-cold CaCl2 makes wall porous, then heat shock 42°C for 90 seconds then ice - plasmid enters!
Microinjection: For animal cells - directly inject DNA with fine needle under microscope.
Biolistics / Gene Gun: Gold or tungsten bullets coated with DNA, shoot into plant cells. Works for monocot wheat where Agrobacterium fails because Agrobacterium only works for dicots.
Disarmed pathogen: Make pathogen weak and use it to deliver gene.
Selection: grow on ampicillin - only transformed grow. Blue-white white = recombinant.
Competent by CaCl2 divalent cation + heat shock 42C 90s creates transient pores. Microinjection direct for animal cells. Biolistics high-velocity gold/tungsten microparticles for monocots. Disarmed Agrobacterium Ti lacking tumor genes delivers T-DNA. Selection markers + reporter.
5. Processes - Isolation, Gel, PCR, Bioreactor
Step 1 Isolation: Break cell to get DNA. Enzymes: lysozyme bacteria, cellulase plants, chitinase fungus to break wall. Then RNase removes RNA, protease removes protein. Then ethanol makes DNA precipitate as white fibers spooling on spool.
Step 2 Cutting: Cut vector and gene with same RE to make same sticky ends.
Step 3 Ligation: Join with ligase - fevicol, makes rDNA.
Step 4 Gel Electrophoresis: Check DNA. Agarose gel, electric field, small pieces move farther. EtBr stain + UV shows orange bands. Separates by size.
Step 5 PCR: Photocopy machine! Makes billions copies. 3 steps: Denaturation 94°C double to single, Annealing 50-65°C primers 18-22 bases bind, Extension 72°C Taq polymerase from Thermus aquaticus heat stable adds dNTPs. Formula 2^n exponential, 25-35 cycles = billion copies. Machine = thermocycler.
Step 6 Transform into competent host, select.
Step 7 Bioreactor: Big factory tank to grow bacteria large scale. Stirred tank sparged - impeller mixes, sparger gives air bubbles, sensors pH, dissolved O2, temperature, sampling port, jacket for cooling. Types: batch one time, continuous fresh media added.
Step 8 Downstream: Purify product by filtration, chromatography.
Isolation wall enzymes + RNase + protease + ethanol spool. Cutting same RE sticky. Ligation ligase rDNA. Gel agarose size separation EtBr UV. PCR denaturation 94C single, annealing 50-65C primers 18-22mer, extension 72C Taq Thermus aquaticus thermostable dNTPs 2^n 25-35 cycles billion copies thermocycler. Transformation + selection. Bioreactor stirred-tank sparged agitation impeller aeration sparger pH DO temp sampling foam jacket batch vs continuous. Downstream purification.
