1. Genetic transformation of E. coli bacteria
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

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

5. Genes from bioluminescent jellyfish

6. 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

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

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

9. Restriction Enzymes go hang a salami, I'm a lasagna hog
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

10. How do restriction enzymes work?

11. Restriction enzymes at work

12. 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

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

17. Bacterial transformation procedure
Ca++ O
CH2 P
Base OH
Sugar
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

18. 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