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Amgen Bruce Wallace Transformation Labs (2

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Amgen Bruce Wallace
Transformation Labs (27)
Timeline
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Thursday—Lecture
Tuesday—Finish Lecture, Quiz, lab 2
Thursday--Lab 3, 4, 5 (Duffy does lab 6)
Monday—Lab 7 Part 1
Tuesday—Lab 7 Part 2
Finals Friday—Packet Due
Assignments
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Pre-lab notes worksheets: do these before we do that
lab by reading the information and lab procedures
Flowcharts—draw on the side or bottom of the
procedures page
Complete conclusion questions: do at the end of the lab
each day
Draw your lab 4 gel results at the end of the conclusion
(use a ruler, make it nice!)
Entire packet will be due 1/27/2012 (day of final)—no
late work because this goes in semester grade!
Prep. for Labs
Week Before
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Make labels
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10% bleach solution
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Aliquot chart
Day before lab
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Lab 2—water bath set-up for 37 C
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Lab 3--Water baths set-up 70 C Lab 4--Pour 8 gels (35ml each) for lab 4
Pour .8% gels, add ethidium bromide (200ng/mL final or 1uL of 10mg/mL stock in gel prepared from 50mL),
6 well comb, SB buffer
(2.4 grams agarose add up to 300mL TBE buffer)
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lab 5--ice
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Lab 5—water bath set-up at 42C
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Lab 6--Start overnight culture for lab 6 (use update instructions)
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Lab 7--Container of 10% at front for waste
--Set-up chromatography columns
Vocab.
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“transformed cell” – cell has acquired new characteristics
“characteristics” – due to the expression of incorporated foreign
genetic material
Gene expression – process by which the information encoded in a
gene is converted into an observable phenotype
Gene regulation – control mechanisms that turn genes on or off
Inducible proteins – synthesis is regulated depending on the
bacterium’s nutritional status
Thank you Francois Jacob and Jacques Monod!
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Prokaryote operon model of gene control
Repressors and activators are “trans-acting” – affect expression of their
genes no matter on which DNA molecule in the cell these are located.
Overview of Labs
Lab 2—Restriction Analysis of pARA and pKAN-R
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Cut the 2 plasmids using restriction enzymes
Lab 3—Ligation of pARA/pKAN-R Restriction Fragments Producing a Recombinant Plasmid,
pARA-R
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Insert the gene of interest into the pARA plasmid from the pKAN-R
Lab 4—Confirmation of Restriction and Ligation Using Agarose-Gel Electrophoresis
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Run a gel to confirm the ligation in lab 3 worked (we want to make sure the gene of interest was
inserted into that plasmid, if not, there is no reason to transform the plasmid into the bacteria)
Lab 5—Transforming Escherichia coli with a Recombinant Plasmid
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Insert pARA-R (plasmid with our gene of interest) into bacteria using shock treatment, grow
bacteria on plates, plasmid will produce proteins from our gene
Lab 6--Preparing an Overnight Culture of Escherichia coli
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Take a colony that has the gene of interest from the plate, put into broth to replicate, now we
have tons of bacteria (so tons of our geneпѓ protein!)
Lab 7—Purification of mFP from an Overnight Culture
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Lyse the bacteria cell, isolate the desired protein using chromatography
The Big Picture
2005 Pearson Education, Inc.
2005 Pearson Education, Inc
Background Concepts
1) What are Plasmids?
2) How can we modify plasmids?
пѓј Restriction Enzymes
3) Origins of restriction enzymes.
4) A close look at restriction enzymes.
5) Understanding plasmid diagrams.
What are Plasmids?
In this Lecture…
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Circular DNA that is used by
bacteria to store their genetic
information.
Modifying plasmids to
include extra genes allows for
the production of new
proteins.
How Can We Modify Plasmids?
In this Lecture…
1)
Restriction Enzymes
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2)
Restriction Enzyme attached to DNA before cleavage
BamHI, HindIII, etc.
Where do they come
from?
How do they work?
Different restriction
enzymes do different
things.
DNA Ligase
Origins of Restriction Enzymes
1)
Bacteria produce restriction enzymes to
protect against invading viral DNA/RNA.
Origins of Restriction Enzymes
2)
The enzymes cut the invading DNA/RNA,
rendering it harmless.
Restriction Enzyme in Action
Sticky Ends
1) DNA strand with EcoRI restriction site highlighted.
2) EcoRI restriction enzyme added (outline of separation about to occur).
3) Restriction fragments separate, with “sticky ends” at each edge.
Adding DNA Ligase
Sticky Ends
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DNA ligase bonds sticky ends cut with the same restriction enzyme.
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Sticky ends cut with different restriction enzymes will not bond together.
пѓ� Why?
пѓ� Because the base pair sequence of the two sticky ends will be
different and not match up.
Plasmids Can Be Drawn to Show
the Genes They Carry
Plasmid Name
Bp size
In this diagram:
пЃ® Blue and Orange are
drawn as genes.
пЃ® Triangles are indicating
the known restriction sites
for a restriction enzyme.
(shapes can vary)
пЃ® Plasmid Maps are more
complex.
Plasmid Maps Indicate
Restriction Sites and Genes
Application Exercise
Make Recombinant DNA Using
Restriction Enzymes
DNA From Two Sources
(Restriction Sites Labeled)
Circular DNA
Linear DNA
Application of Restriction Enzymes
Adding DNA Ligase
Recombinant DNA Plasmid
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Many possible
recombinant DNA
plasmids can be
produced, but this was
the desired plasmid for
the experiment.
Many Other Recombinant
Possibilities
…and many more!
Plasmid DNA Insertion
DNA plasmids can be inserted into bacteria
using a variety of laboratory processes.
Transgenic Colony Allowed to Grow
How Do We Get the Desired Plasmid?
Recombinant
plasmids
Transformation of
bacterial cells through
electroporation.
Bacteria are then moved to a growth
plate, and grown on selective media
to “weed out” cells that have not
picked up the desired plasmid.
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Restriction fragments will
ligate randomly, producing
many plasmid forms.
Bacterial insertion would
be necessary, then colony
growth, and further testing
to isolate bacteria with the
desired plasmid.
Running Digested DNA
Through Gel Electrophoresis
Goals of this Hands-On Lab
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Take plasmid DNA that has been
previously cut with restriction
enzymes and compare that to a
plasmid NOT cut with restriction
enzymes, by running them
through a gel.
Look for different banding
patterns and understand how to
read them.
Predict what kind of banding
pattern a plasmid will make based
on:
1.
2.
The restriction enzyme used.
The plasmid’s structural shape.
Gel Box Loading Techniques
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Look directly down the axis of
the pipette.
Loading dye makes the sample
heavy, but it can still easily swish
out of the well.
Squirt down slowly.
Take the tip out of the buffer.
Then release the plunger.
If you don’t do that, you will
suck the sample back up.
Add DNA samples and ladder to the wells and “run to red!”
10 kb
8 kb
6 kb
5 kb
4 kb
3 kb
2 kb
1 kb
.5 kb
Sample fragments move toward positive end.
Analyzing Your Gel
What Makes Up the Banding Pattern in
Restricted DNA?
1400 Bp
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2000 Bp
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Lancer Plasmid
6700 Bp
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3300 Bp
The restriction enzyme
cleaves the DNA into
fragments of various
sizes.
Each different size
fragment will produce a
different band in the gel.
Remember that
fragments separate into
bands based on size.
What Makes Up the Banding
Pattern After Adding DNA Ligase?
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Several combinations of
plasmids will result from
the reaction.
The many forms will
contribute to different
bands.
(See following slides for chemical and structural forms)
Different Recombinant Forms
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Adding DNA Ligase
does not always make the
desired plasmid!
Few if any could be what
you wanted.
Think about the large
number of possible
combinations.
Different Structural Forms
circle
“multimer”
Nicked Circle
Linear
“nicked-circle”
Supercoiled
Different structural forms
produce different bands.
A+
Multimer
Nicked
Super Coiled
5 Kb
Linear Fragment
Linear Fragment
10 Kb Ladder
10 Kb Ladder
10 Kb Ladder
A-
What Are Some Applications of
Recombinant DNA Technology?
Bacteria, Yeasts, and Plants can all be
modified to produce important
pharmaceuticals, enriched foods, and
industrial products.
Biotechnology Lab Program
Bruce Wallace
pKAN-R/pARA Sequence
Laboratory Protocols by:
Marty Ikkanda
Powerpoint by:
Anthony Daulo
Pierce College, Woodland Hills, CA
V.1.2.4
Restriction analysis of pKAN-R and pARA
Bruce Wallace
pKAN-R
5408 bp
rfp
702 bp
pARA
4058 bp
40 bp
Restriction analysis of pKAN-R and pARA
Bruce Wallace
Restriction fragments after digest with Hind III and BamH I
BamH I
Hind III
4018 bp
BamH I
Hind III
4706 bp
BamH I
Hind III
702bp
Hind III
BamH I
40 bp
Restriction analysis of pKAN-R and pARA
Bruce Wallace
Prediction for restriction gel
M K+ K- A+ A-
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1000
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M K+ K- A+ A-
Ligation of pKAN-R/pARA restriction fragments
Bruce Wallace
BamH I
sticky end
3’
5’
3’
5’
sticky end
5’
5’
3’
3’
Hind III
BamH I
sticky end
Hind III
sticky end
3’
5’
3’
5’
Ligation of pKAN-R/pARA restriction fragments
Bruce Wallace
Recombinant plasmid of interest
pARA-R
4720 bp
rfp
702bp
Restriction analysis of pKAN-R and pARA
Bruce Wallace
Confirmation of restriction and ligation
M K+ K- A+ A- L
10000
8000
5000
4000
3000
2000
1500
1000
500
M K+ K- A+ A- L
Preparing competent cells for transformation
Bruce Wallace
Lipid bilayer
(inner)
Adhesion zone
Peptidoglycan
layer
Lipid bilayer
(outer)
Calcium ions
Transforming Escherichia coli with pARA-R
Bruce Wallace
Competent Cells
pARA-R
Recombinant Plasmids
Transforming Escherichia coli with pARA-R
Bruce Wallace
Lipid bilayer
(inner)
Peptidoglycan
layer
Adhesion zone
Lipid bilayer
(outer)
Calcium ions
pARA-R
Growth of transformed bacteria on various plates
Bruce Wallace
P+ plates
LB
LB/amp
P- plates
No growth
LB
LB/amp
LB/amp/ara
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Why don’t we see the red protein in any
LB growth media?
пЃ® Cells conserve energy and resources
пЃ® The rfp gene requires a specific substrate
(arabinose) to be turned on (expressed)
Preparing an overnight culture of E. Coli for RFP expression
Bruce Wallace
Colony isolation and culture
LB/amp/ara
broth
Many of the red colonies picked from a
Lab 5 plate appear to contain cells that are
interfering with rfp expression.
пЃ® When there is a mixed culture of red and
white (nonexpressing) cells, the white cells
will grow faster than those that are using
their resources producing mFP.
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= less mFP produced for purification.
RFP expression
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araC gene
PBAD
Transcription
mRNA
Translation
araC protein
rfp gene
RFP expression
Bruce Wallace
araC protein prevents RFP transcription by causing
a loop to form in the region of the r fp gene
araC gene
araC protein
PBAD
rfp gene
RFP expression
Bruce Wallace
arabinose
Arabinose – araC protein complex prevents DNA looping
and helps to align RNA polymerase
on the promoter site (PBAD).
RFP
(red fluorescent
protein)
Translation
RNA polymerase
arabinose
araC protein
araC–protein
complex
mRNA
Transcription
araC gene
PBAD
rfp gene
Bruce Wallace
RFP
Purification of RFP from an overnight culture
Bruce Wallace
Overnight
culture
Cell pellet
with RFP
Lysed
cells
Pellet
RFP with
cell debris binding buffer
Lab Tips/Reminders
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Add initials or group # to tubes
Anything that touches bacteria must go in sterilizer
Sterile technique
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Carefully READ and FOLLOW the lab protocol.
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Using bacteria
Contamination may affect results
Be sure lab partners communicate
No Food or Drinks
Agar Plate tips (Lab 5)
Label the bottom of the plates at the edges
пЃ® Note the plate markings: I=LB, II=LB/amp,
III=LB/amp/ara
пЃ® Samples go on the agar, not the lid
пЃ® Open like clam shells
пЃ® Agar is like finger jello, firm but not invincible,
be gentle
пЃ® Turn the plates upside down
(lids down) for incubation
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Sterile technique tips
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Always follow the protocol carefully – know
what you’re doing
Work quickly – less time = less opportunities for
contamination
Do not leave any container (tube, plate) open
any longer than needed
Watch what your equipment touches – there is
no “5 second rule” here.
All tips, tubes and spreaders in the
“contaminated waste” container at the end of
the lab.
Look at labels
Clam shell technique
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You need to aliquot and centrifuge twice to get a
sufficient number of cells = product.
Be sure the centrifuge has a balanced number of
tubes.
Be careful not to disturb the resultant pellets. When
“wicking” don’t let the towel touch the pellet
Supernatant and wicking towel go in disinfectant
(10% bleach solution) containing beaker
Incubate in 37 C water bath (60 min.) instead of
overnight at room temperature.
Freeze – ice crystals also help to lyse (break open)
Science for LIFE
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