Genetic Engineering - DVBiology.org

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Dec 14, 2012 (4 years and 10 months ago)

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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


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


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


Ca
++

Ca
++

O

CH
2

O

P

O

O

O

Base

CH
2

O

P

O

O

O

Base

OH

Sugar

Sugar

O

Ca
++

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