Genetic transformation of E. coli bacteria 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

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

Genes from bioluminescent jellyfish

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

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

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

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

How do restriction enzymes work?

Restriction enzymes at work

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

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

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

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