Genetic Genetic EngineeringEngineering

Genetic transformation of Genetic transformation of

E. coliE. coli bacteria bacteria

What is genetic What is genetic transformation?transformation?

Direct manipulation of genes to Direct manipulation of genes to change an organism’s change an organism’s characteristicscharacteristics

Provides a benefit to Provides a benefit to humans in some wayhumans in some way

Target organism: Target organism: E. coliE. coli

Prokaryote with circular loop of DNA Prokaryote with circular loop of DNA Plasmids are small circles of “bonus” DNAPlasmids are small circles of “bonus” DNA

pGLO plasmids

Cell wall

GFP

Bacterial chromosomal DNA

Genes from bioluminescent jellyfishGenes from bioluminescent jellyfish

Plasmids can be Plasmids can be useful toolsuseful tools

Most contain a gene for antibiotic resistanceMost contain a gene for antibiotic resistance Scientists can engineer them to also contain Scientists can engineer them to also contain

genes that code for a desired proteingenes that code for a desired protein E. coliE. coli (or other bacteria) can be persuaded (or other bacteria) can be persuaded

to “take up” engineered plasmidsto “take up” engineered plasmids Plasmids become part of the bacteria’s Plasmids become part of the bacteria’s

genome, and are passed on to future genome, and are passed on to future generationsgenerations

How do we insert How do we insert the genes we desire the genes we desire

into a plasmid?into a plasmid?

Bacteria provide the way!Bacteria provide the way!

““DNA scissors” produced by bacteriaDNA scissors” produced by bacteria Adaptation to protect bacteria against virusesAdaptation to protect bacteria against viruses

Restriction EnzymesRestriction Enzymes 3000+ known restriction enzymes, or 3000+ known restriction enzymes, or

endonucleasesendonucleases Each cuts at a specific DNA sequence called Each cuts at a specific DNA sequence called

a a restriction siterestriction site.. Restriction sites are palindromesRestriction sites are palindromes

How do restriction enzymes How do restriction enzymes work?work?

Restriction enzymes at workRestriction enzymes at work

How to engineer How to engineer an organisman organism

Locate a gene that codes for Locate a gene that codes for Your Favorite Your Favorite ProteinProtein

Cut out the gene and insert it into a plasmid, Cut out the gene and insert it into a plasmid, using restriction enzymesusing restriction enzymes

Put the plasmid into bacteria Put the plasmid into bacteria Bacteria reproduce exponentially, Bacteria reproduce exponentially,

passing on the new genespassing on the new genes Trillions of bacteria produce Trillions of bacteria produce

Your Favorite ProteinYour Favorite Protein

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

Bacterial transformation Bacterial transformation procedureprocedure

Select E. coli colony (approximally Select E. coli colony (approximally one million cells) and disperse one million cells) and disperse in transformation solutionin transformation solution Transformation solution: Ca2+ Transformation solution: Ca2+

cations may neutralize negative cations may neutralize negative charges in phosphate backbone of charges in phosphate backbone of plasmid DNA as well as of cell plasmid DNA as well as of cell phospholipids, allowing DNA to enterphospholipids, allowing DNA to enter

Add engineered plasmid of choiceAdd engineered plasmid of choice pGLO: GFP protein; ampicillin pGLO: GFP protein; ampicillin

resistance, arabinose promoterresistance, arabinose promoter

Ca++

Ca++

OCH2

O

P O

O

OBase

CH2

O

P

O

O

O

Base

OH

Sugar

Sugar

OCa++

Transformation, continuedTransformation, continued Heat shock to induce cells to take up plasmidsHeat shock to induce cells to take up plasmids

Increases permeability of cell membrane; mechanism Increases permeability of cell membrane; mechanism unknown!unknown!

Revive survivors with LB brothRevive survivors with LB broth Resting period allows bacteria to begin expressing Resting period allows bacteria to begin expressing

genes; beta-lactamase protein will provide resistance genes; beta-lactamase protein will provide resistance to antibiotic ampicillinto antibiotic ampicillin

Plate out cells on petri Plate out cells on petri dishes so engineered dishes so engineered bacteria will reproduce bacteria will reproduce and form colonies and form colonies

Successful colonies Successful colonies produce desired proteinproduce desired protein