Amgen Bruce Amgen Bruce Wallace Wallace
Transformation Transformation Labs (2-7)Labs (2-7)
TimelineTimeline
Thursday—LectureThursday—Lecture Tuesday—Finish Lecture, Quiz, lab 2Tuesday—Finish Lecture, Quiz, lab 2 Thursday--Lab 3, 4, 5 (Duffy does lab Thursday--Lab 3, 4, 5 (Duffy does lab
6)6) Monday—Lab 7 Part 1Monday—Lab 7 Part 1 Tuesday—Lab 7 Part 2Tuesday—Lab 7 Part 2 Finals Friday—Packet DueFinals Friday—Packet Due
AssignmentsAssignments Pre-lab notes worksheets: do these before we Pre-lab notes worksheets: do these before we
do that lab by reading the information and do that lab by reading the information and lab procedureslab procedures
Flowcharts—draw on the side or bottom of the Flowcharts—draw on the side or bottom of the procedures pageprocedures page
Complete conclusion questions: do at the end Complete conclusion questions: do at the end of the lab each dayof the lab each day
Draw your lab 4 gel results at the end of the Draw your lab 4 gel results at the end of the conclusion (use a ruler, make it nice!)conclusion (use a ruler, make it nice!)
Entire packet will be due 1/27/2012 (day of Entire packet will be due 1/27/2012 (day of final)—no late work because this goes in final)—no late work because this goes in semester grade!semester grade!
Prep. for LabsPrep. for LabsWeek BeforeWeek Before Make labelsMake labels 10% bleach solution10% bleach solution Aliquot chart Aliquot chart Day before labDay before lab Lab 2—water bath set-up for 37 CLab 2—water bath set-up for 37 C Lab 3--Water baths set-up 70 C Lab 4--Pour 8 gels (35ml each) for lab 4Lab 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 Pour .8% gels, add ethidium bromide (200ng/mL final or 1uL of 10mg/mL stock in gel prepared from 50mL), 6 well comb, SB buffergel prepared from 50mL), 6 well comb, SB buffer(2.4 grams agarose add up to 300mL TBE buffer)(2.4 grams agarose add up to 300mL TBE buffer)
lab 5--ice lab 5--ice Lab 5—water bath set-up at 42C Lab 5—water bath set-up at 42C Lab 6--Start overnight culture for lab 6 (use update instructions)Lab 6--Start overnight culture for lab 6 (use update instructions) Lab 7--Container of 10% at front for wasteLab 7--Container of 10% at front for waste --Set-up chromatography columns--Set-up chromatography columns
Vocab.Vocab. ““transformed cell” – cell has acquired new characteristicstransformed cell” – cell has acquired new characteristics ““characteristics” – due to the characteristics” – due to the expressionexpression of incorporated foreign of incorporated foreign
genetic materialgenetic 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!
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 LabsOverview of LabsLab 2—Restriction Analysis of pARA and pKAN-RLab 2—Restriction Analysis of pARA and pKAN-R Cut the 2 plasmids using restriction enzymesCut the 2 plasmids using restriction enzymesLab 3—Ligation of pARA/pKAN-R Restriction Fragments Producing a Lab 3—Ligation of pARA/pKAN-R Restriction Fragments Producing a
Recombinant Plasmid, pARA-RRecombinant Plasmid, pARA-R Insert the gene of interest into the pARA plasmid from the pKAN-R Insert the gene of interest into the pARA plasmid from the pKAN-R Lab 4—Confirmation of Restriction and Ligation Using Agarose-Gel Lab 4—Confirmation of Restriction and Ligation Using Agarose-Gel
ElectrophoresisElectrophoresis Run a gel to confirm the ligation in lab 3 worked (we want to make sure the 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 gene of interest was inserted into that plasmid, if not, there is no reason to transform the plasmid into the bacteria)transform the plasmid into the bacteria)
Lab 5—Transforming Lab 5—Transforming Escherichia coliEscherichia coli with a Recombinant Plasmid with a Recombinant Plasmid Insert pARA-R (plasmid with our gene of interest) into bacteria using shock Insert pARA-R (plasmid with our gene of interest) into bacteria using shock
treatment, grow bacteria on plates, plasmid will produce proteins from our treatment, grow bacteria on plates, plasmid will produce proteins from our genegene
Lab 6--Preparing an Overnight Culture of Lab 6--Preparing an Overnight Culture of Escherichia coliEscherichia coli Take a colony that has the gene of interest from the plate, put into broth to 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 genereplicate, now we have tons of bacteria (so tons of our geneprotein!)protein!)Lab 7—Purification of mFP from an Overnight CultureLab 7—Purification of mFP from an Overnight Culture Lyse the bacteria cell, isolate the desired protein using chromatographyLyse the bacteria cell, isolate the desired protein using chromatography
The Big PictureThe Big Picture
2005 Pearson Education, Inc.
2005 Pearson Education, Inc
Background Concepts
1) What are Plasmids?
2) How can we modify plasmids? Restriction Enzymes
3) Origins of restriction enzymes.
4) A close look at restriction enzymes.
5) Understanding plasmid diagrams.
What are Plasmids?What are Plasmids?
Circular DNA that is Circular DNA that is used by bacteria to used by bacteria to store their genetic store their genetic information.information.
Modifying plasmids to Modifying plasmids to include extra genes include extra genes allows for the allows for the production of new production of new proteins.proteins.
In this Lecture…
How Can We Modify How Can We Modify Plasmids?Plasmids?
1)1) Restriction Restriction EnzymesEnzymes
BamBamHI, HI, HinHindIII, etc.dIII, etc. Where do they come Where do they come
from?from? How do they work?How do they work? Different restriction Different restriction
enzymes do enzymes do different things.different things.
2)2) DNA LigaseDNA Ligase
Restriction Enzyme attached to DNA before cleavage
In this Lecture…
Origins of Restriction Origins of Restriction EnzymesEnzymes
1)1) Bacteria produce restriction Bacteria produce restriction enzymes to protect against enzymes to protect against invading viral DNA/RNA.invading viral DNA/RNA.
Origins of Restriction Origins of Restriction EnzymesEnzymes
2)2) The enzymes cut the invading The enzymes cut the invading DNA/RNA, rendering it harmless.DNA/RNA, rendering it harmless.
Restriction Enzyme in Restriction Enzyme in ActionAction
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.
Sticky Ends
Adding DNA LigaseAdding DNA Ligase
DNA ligase bonds sticky ends cut with the same restriction enzyme.
Sticky ends cut with different restriction enzymes will not bond together.
Why?
Because the base pair sequence of the two sticky ends will be different and not match up.
Sticky Ends
Plasmids Can Be Drawn to Plasmids Can Be Drawn to Show the Genes They Show the Genes They
CarryCarryIn this diagram:In this diagram: BlueBlue and and OrangeOrange
are drawn as genes.are drawn as genes. Triangles are Triangles are
indicating the indicating the known restriction known restriction sites for a sites for a restriction enzyme. restriction enzyme. (shapes can vary)(shapes can vary)
Plasmid Maps are Plasmid Maps are more complex. more complex.
Plasmid Name
Bp size
Plasmid Maps Indicate Plasmid Maps Indicate Restriction Sites and Restriction Sites and
GenesGenes
Make Recombinant DNA Make Recombinant DNA Using Restriction Using Restriction
EnzymesEnzymes
Application Exercise
DNA From Two SourcesDNA From Two Sources(Restriction Sites Labeled)(Restriction Sites Labeled)
Circular DNA Linear DNA
Application of Restriction Application of Restriction EnzymesEnzymes
Adding DNA LigaseAdding DNA Ligase
Recombinant DNA Recombinant DNA PlasmidPlasmid
Many possible Many possible recombinant DNA recombinant DNA plasmids can be plasmids can be produced, but this produced, but this was the desired was the desired plasmid for the plasmid for the experiment.experiment.
Many Other Recombinant Many Other Recombinant PossibilitiesPossibilities
…and many more!
Plasmid DNA InsertionPlasmid DNA Insertion
DNA plasmids can be inserted into bacteria using a variety of laboratory processes.
Transgenic Colony Allowed Transgenic Colony Allowed to Growto Grow
How Do We Get the Desired How Do We Get the Desired Plasmid?Plasmid?
Restriction fragments Restriction fragments will ligate randomly, will ligate randomly, producing many plasmid producing many plasmid forms.forms.
Bacterial insertion would Bacterial insertion would be necessary, then be necessary, then colony growth, and colony growth, and further testing to isolate further testing to isolate bacteria with the desired bacteria with the desired plasmid.plasmid.
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.
Recombinant plasmids
Running Digested DNA Running Digested DNA Through Gel Through Gel
ElectrophoresisElectrophoresis
Goals of this Hands-On Goals of this Hands-On LabLab Take plasmid DNA that has Take plasmid DNA that has
been previously cut with been previously cut with restriction enzymes and restriction enzymes and compare that to a plasmid compare that to a plasmid NOT cut with restriction NOT cut with restriction enzymes, by running them enzymes, by running them through a gel.through a gel.
Look for different banding Look for different banding patterns and understand patterns and understand how to read them.how to read them.
Predict what kind of Predict what kind of banding pattern a plasmid banding pattern a plasmid will make based on: will make based on: 1.1. The restriction enzyme used.The restriction enzyme used.
2.2. The plasmid’s structural The plasmid’s structural shape.shape.
Gel Box Loading Gel Box Loading TechniquesTechniques
Look directly down the axis Look directly down the axis of the pipette.of the pipette.
Loading dye makes the Loading dye makes the sample heavy, but it can sample heavy, but it can still easily swish out of the still easily swish out of the well.well.
Squirt down slowly.Squirt down slowly. Take the tip out of the Take the tip out of the
buffer.buffer. Then release the plunger.Then release the plunger. If you don’t do that, you will If you don’t do that, you will
suck the sample back up.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 GelAnalyzing Your Gel
What Makes Up the Banding What Makes Up the Banding Pattern in Pattern in Restricted DNARestricted DNA??
The restriction The restriction enzyme cleaves the enzyme cleaves the DNA into fragments DNA into fragments of various sizes. of various sizes.
Each different size Each different size fragment will fragment will produce a different produce a different band in the gel.band in the gel.
Remember that Remember that fragments separate fragments separate into bands based on into bands based on size.size.
Lancer Plasmid
6700 Bp
3300 Bp
2000 Bp
1400 Bp
What Makes Up the What Makes Up the Banding Pattern Banding Pattern After After Adding DNA LigaseAdding DNA Ligase??
Several Several combinations of combinations of plasmids will plasmids will result from the result from the reaction.reaction.
The many forms The many forms will contribute to will contribute to different bands.different bands.
(See following slides for chemical and structural forms)
Different Recombinant Different Recombinant FormsForms
Adding DNA Adding DNA Ligase does not Ligase does not always make the always make the desired plasmid!desired plasmid!
Few if any could be Few if any could be what you wanted.what you wanted.
Think about the Think about the large number of large number of possible possible combinations.combinations.
Different Structural Different Structural FormsForms
circle
“nicked-circle”
“multimer”
Different structural forms produce different bands.
Nicked Circle
SupercoiledLinear
10 K
b L
add
er
5 Kb
Multimer
NickedSuper Coiled
Linear Fragment
A- A+
Linear Fragment
10 K
b L
add
er
10 K
b L
add
er
What Are Some Applications of What Are Some Applications of Recombinant DNA Recombinant DNA
Technology?Technology? Bacteria, Yeasts, and Plants Bacteria, Yeasts, and Plants
can all be modified to can all be modified to produce important produce important pharmaceuticals, enriched pharmaceuticals, enriched foods, and industrial foods, and industrial products.products.
pKAN-R/pARA Sequence
Biotechnology Lab Program
Laboratory Protocols by:
Marty IkkandaPowerpoint by:
Anthony DauloPierce College, Woodland Hills, CA
V.1.2.4
Bruce Wallace
Restriction analysis of pKAN-R and pARA
pKAN-R
pARA
BamH I
Hind III
BamH IHind III
rfp702 bp
5408 bp
4058 bp
40 bp
Bruce Wallace
Restriction analysis of pKAN-R and pARA
BamH I Hind III
702bp
40 bp
BamH I Hind III
BamH IHind III
BamH IHind III
Restriction fragments after digest with Hind III and BamH I
4018 bp
4706 bp
Bruce Wallace
Restriction analysis of pKAN-R and pARA
Prediction for restriction gel
M K+ K- A+ A-M K+ K- A+ A-
500
1000
1500
20003000400050008000
10000
Bruce Wallace
Ligation of pKAN-R/pARA restriction fragments
sticky endBamH I
sticky endHind III
sticky endBamH I
sticky endHind III
3’
5’
5’
3’
3’
3’
5’
5’5’
5’3’
3’
Bruce Wallace
Ligation of pKAN-R/pARA restriction fragments
Recombinant plasmid of interest
pARA-R
BamH I
Hind III
rfp702bp
4720 bp
Bruce Wallace
Restriction analysis of pKAN-R and pARA
M K+ K- A+ A-
500
1000
1500
20003000400050008000
10000
Confirmation of restriction and ligation
L M K+ K- A+ A- L
Bruce Wallace
Preparing competent cells for transformation
Lipid bilayer(inner)
Lipid bilayer(outer)
Peptidoglycanlayer
Adhesion zone
Calcium ions
Bruce Wallace
Transforming Escherichia coli with pARA-R
Recombinant Plasmids
Competent Cells
pARA-R
Bruce Wallace
Calcium ions
pARA-R
Transforming Escherichia coli with pARA-R
Adhesion zone
Lipid bilayer(inner)
Lipid bilayer(outer)
Peptidoglycanlayer
Bruce Wallace
Growth of transformed bacteria on various plates
P+ plates
P- plates
LB LB/amp LB/amp/ara
LB LB/amp
No growth
Bruce Wallace
Why don’t we see the red protein in any LB growth media?Cells conserve energy and resources
The rfp gene requires a specific substrate (arabinose) to be turned on (expressed))
Colony isolation and culture
Preparing an overnight culture of E. Coli for RFP expression
LB/amp/arabroth
Bruce Wallace
Many of the red colonies picked Many of the red colonies picked from a Lab 5 plate appear to from a Lab 5 plate appear to contain cells that are interfering contain cells that are interfering with with rfp rfp expression.expression.
When there is a mixed culture of When there is a mixed culture of red and white (nonexpressing) red and white (nonexpressing) cells, the white cells will grow cells, the white cells will grow faster than those that are using faster than those that are using their resources producing mFP.their resources producing mFP.
= less mFP produced for purification.= less mFP produced for purification.
RFP expression
araC gene rfp genePBAD
Transcription
mRNA
Translation
araC protein
Bruce Wallace
RFP expression
rfp genePBAD
araC protein
araC gene
araC protein prevents RFP transcription by causinga loop to form in the region of the fp gener
Bruce Wallace
RFP expression
araC protein
arabinose
araC gene rfp genePBAD
arabinose – araC proteincomplex
RNA polymerase
Arabinose – araC protein complex prevents DNA loopingand helps to align RNA polymerase
on the promoter site (PBAD).
mRNA
Transcription
Translation
RFP(red fluorescent
protein)
Bruce Wallace
RFP
Bruce Wallace
Purification of RFP from an overnight culture
Overnightculture
Cell pelletwith RFP
Lysedcells
Pelletcell debris
RFP withbinding buffer
Bruce Wallace
Lab Tips/RemindersLab Tips/Reminders
Add initials or group # to tubesAdd initials or group # to tubes Anything that touches bacteria must go in Anything that touches bacteria must go in
sterilizer sterilizer Sterile technique Sterile technique
Using bacteriaUsing bacteria Contamination may affect resultsContamination may affect results
Carefully READ and FOLLOW the lab Carefully READ and FOLLOW the lab protocol. protocol. Be sure lab partners Be sure lab partners communicatecommunicate
No Food or DrinksNo Food or Drinks
Agar Plate tips (Lab 5)Agar Plate tips (Lab 5) Label the bottom of the plates at the Label the bottom of the plates at the
edgesedges Note the plate markings: I=LB, Note the plate markings: I=LB,
II=LB/amp, III=LB/amp/araII=LB/amp, III=LB/amp/ara Samples go on the agar, not the lidSamples go on the agar, not the lid Open like clam shellsOpen like clam shells Agar is like finger jello, firm but not Agar is like finger jello, firm but not
invincible, be gentleinvincible, be gentle Turn the plates upside down Turn the plates upside down
(lids down) for incubation(lids down) for incubation
Sterile technique tipsSterile technique tips Always Always follow the protocol follow the protocol carefully carefully
– know what you’re doing– know what you’re doing Work quickly – less time = less Work quickly – less time = less
opportunities for contaminationopportunities for contamination Do not leave any container (tube, Do not leave any container (tube,
plate) open any longer than neededplate) open any longer than needed Watch what your equipment touches – Watch what your equipment touches –
there is no “5 second rule” here.there is no “5 second rule” here. All tips, tubes and spreaders in the All tips, tubes and spreaders in the
“contaminated waste” container at the “contaminated waste” container at the end of the lab.end of the lab.
Look at labelsLook at labels
Clam shell techniqueClam shell technique
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) the cells.
Science for LIFEScience for LIFE
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