Genetic Engineering Genetic transformation of E. coli bacteria.
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Transcript of Genetic Engineering Genetic transformation of E. coli bacteria.
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
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 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
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