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