Download 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
Target organism: E. coli
 Prokaryote with circular loop of DNA
 Plasmids are small circles of “bonus” DNA
Cell wall
GFP
Bacterial
chromosomal
DNA
pGLO plasmids
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
 3000+ known restriction enzymes, or
endonucleases
 Each cuts at a specific DNA sequence called
a restriction site.
 Restriction sites are palindromes
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++
 Select E. coli colony (approximally
Ca++
 Add engineered plasmid of choice
 pGLO: GFP protein; ampicillin
resistance, arabinose promoter
O P O
O
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
O
CH2
Base
O
Sugar
O
Ca++
O P O
Base
O
CH2
O
Sugar
OH
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