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Genetic Engineering

Genetic Engineering. Genetic transformation of E. coli bacteria. What is genetic transformation?. Direct manipulation of genes to change an organism’s characteristics Provides a benefit to humans in some way. Cell wall. GFP. pGLO plasmids. Target organism: E. coli.

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Genetic Engineering

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  1. Genetic Engineering Genetic transformation of E. coli bacteria

  2. What is genetic transformation? • Direct manipulation of genes to change an organism’s characteristics • Provides a benefit to humans in some way

  3. Cell wall GFP pGLO plasmids Target organism: E. coli • Prokaryote with circular loop of DNA • Plasmids are small circles of “bonus” DNA Bacterial chromosomal DNA

  4. Genes from bioluminescent jellyfish

  5. Plasmids can be useful tools • Most contain a gene for antibiotic resistance • Scientists can engineer them to also contain genes that code for a desired protein • E. coli (or other bacteria) can be persuaded to “take up” engineered plasmids • Plasmids become part of the bacteria’s genome, and are passed on to future generations

  6. How do we insert the genes we desire into a plasmid?

  7. Bacteria provide the way! • “DNA scissors” produced by bacteria • Adaptation to protect bacteria against viruses

  8. go hang a salami, I'm a lasagna hog Restriction Enzymes • 3000+ known restriction enzymes, or endonucleases • Each cuts at a specific DNA sequence called a restriction site. • Restriction sites are palindromes too hot to hoot war, sir, is raw poor dan is in a droop tango gnat if i had a hi fi step on no pets a man, a plan, a canal, panama

  9. How do restriction enzymes work?

  10. Restriction enzymes at work

  11. How to engineer an organism • Locate a gene that codes for Your Favorite Protein • Cut out the gene and insert it into a plasmid, using restriction enzymes • Put the plasmid into bacteria • Bacteria reproduce exponentially, passing on the new genes • Trillions of bacteria produce Your Favorite Protein

  12. Plasmid BamH1 contains two antibiotic resistance genes (for ampicillin and tetracycline)

  13. Ca++ O Ca++ O P O Base O O CH2 Sugar O Ca++ O O P Base O O CH2 Sugar OH Bacterial transformation procedure • Select E. coli colony (approximally one million cells) and disperse in transformation solution • Transformation solution: Ca2+ cations may neutralize negative charges in phosphate backbone of plasmid DNA as well as of cell phospholipids, allowing DNA to enter • Add engineered plasmid of choice • pGLO: GFP protein; ampicillin resistance, arabinose promoter

  14. Transformation, continued • Heat shock to induce cells to take up plasmids • Increases permeability of cell membrane; mechanism unknown! • Revive survivors with LB broth • Resting period allows bacteria to begin expressing genes; beta-lactamase protein will provide resistance to antibiotic ampicillin • Plate out cells on petri dishes so engineered bacteria will reproduce and form colonies • Successful colonies produce desired protein

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