Transcript What is transformation?

Biotechnology
Explorer Program
Serious About Science Education
Aequorea victoria
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Central Framework of
Molecular Biology
DNA
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RNA Protein Trait
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Transformation is a natural
process that Bacterial have
evolved in order to obtain DNA
from their environment.
Use of the procedure enables
scientists to insert genes by
recombinant techniques and
place the plasmid into a bacteria
for expression
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Links to Real-world
 study of biological processes (ex.
biosynthesis of proteins)
 localization of gene expression
 cell movement
 cell fate during development
 formation of different organs
 screenable marker to identify
transgenic organisms
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What is transformation?

Uptake of foreign DNA, often a circular
plasmid
GFP
Amp Resistance
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What is a plasmid?
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A circular piece of autonomously
replicating DNA
ori
bla
Originally evolved by bacteria
 May express antibiotic resistance gene or
be modified to express proteins of interest
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Steps to Make a
Transgenic Bacterium
1.
2.
3.
DNA Extraction- get genome from organism
containing gene of interest
Splice gene of interest from genome using a
restriction enzyme(RE)
- RE, chemical that cuts DNA at a
recognition site(short sequence of bases)
Splice bacterial plasmid using same RE
- plasmid: circular piece of DNA in bacteria
video clip
Plasmid
may not be
exactly
circular
Making recombinant
plasmid(rDNA)
Red= bacteria
DNA
Blue= human
insulin gene
- Details making recombinant
plasmid (rDNA)
a.
Due to sticky ends,
gene can be
spliced in
b. DNA ligase help
nuclotides bond
c.
Combined DNA
called rDNA or
chimera
d.
Put rDNA back into
bacteria and grow
in culture
Insertion of Gene of
Interest into Plasmid
http://www.ncbi.nlm.nih.gov
/books/NBK22390/
Using rDNA plasmid to
create a transgenic
plant
A virus is
another
biological
vector also
used to
deliver genes
Transgenic Organisms
GOAT GOODS. A transgenic goat
named Artemis produces in her milk
a human-breast–milk compound
called lysozyme. Lysozyme destroys
bacteria by drilling through their cell
walls.
E. Scharfen
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pGLO Plasmid
 Ampicillin
resistance
gene
 Green Fluorescent
Protein
 Aequorea
pGLO
victoria jellyfish
gene
 ara
ara
Amp-r
GFP
operon
 Regulates
transcription of
genes in operon and GFP
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Bacterial Transformation
Cell wall
GFP
Bacterial
chromosomal DNA
pGLO plasmids
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Beta lactamase
(ampicillin resistance)
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Regulating Expression of GFP
 Lactose
operon
araC
 Arabinose
operon
pGLO
bla
 pGLO
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GFP
plasmid
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Transcriptional Regulation
lac Operon
Lac
Z
Y
ara Operon
A
ara
B
A
D
Effector
(Lactose)
Lac
Z
Y
A
Effector
(Arabinose)
ara
B
RNA Polymerase
Z
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Y
A
A
D
RNA Polymerase
ara
B
A
D
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Gene Regulation
ara GFP Operon
ara Operon
ara
B
A
D
ara
Effector
(Arabinose)
Effector
(Arabinose)
ara
B
A
D
ara
RNA Polymerase
ara
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B
A
D
GFP Gene
GFP Gene
RNA Polymerase
ara
GFP Gene
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Transformation
Procedures
Day 1
Day 2
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Transformation Procedure
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Suspend bacterial colonies in
Transformation Solution
Add pGLO plasmid DNA
Place tubes on ice
Heat shock at 42oC and place on ice
Incubate with nutrient broth
Streak plates
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Reasons for performing each
transformation step?
Ca++
O
Transformation
solution = CaCl2
Ca++
O P O
Base
O
CH2
O
Sugar
Positive charge of Ca+2
ions shields negative
charge of DNA
phosphates
O
Ca++
O P O
Base
O
CH2
O
Sugar
OH
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Why perform each transformation
step?
Incubation on ice slows
fluid cell membranes
Cell wall
GFP
Heat-shock increases
permeability of cell membrane
Nutrient broth incubation
allows beta lactamase expression
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Beta lactamase
(ampicillin resistance)
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What is nutrient broth?
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Luria-Bertani (LB) broth
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Medium that contains nutrients for bacterial
growth and gene expression
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carbohydrates
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amino acids

nucleotides
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salts

vitamins
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Volume Measurement
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Grow? Glow?
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Follow protocol
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On which plates will
colonies grow?
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Which colonies will
glow?
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