making cells glow: bacterial transformation with pglo plasmid dna

Making Cells Glow:Bacterial Transformation with pGLO Plasmid DNA

BACTERIAL TRANSFORMATION,GENETIC ENGINEERING,AND RECOMBINANT PROTEINS

Essential Components of Genetic Engineering

bacterial

animal

HOST CELLSwith their own genomic DNA

A VECTORa plasmid or virus DNA used to • assemble the recombinant

construct• maintain it in its temporary and

permanent host cells• introduce the transgene into

cells• ensure expression of the

transgene in the new host cell

A TRANSGENEintended to give the host cell or organism new or altered traits.

Plasmids are frequently used as vectors.

Extrachromosomal bacterial DNAs

Small 4000 bp; compared to bacterial chromosome of ~4 million base pairs

Easy to work withCan be removed, altered, then returned to cells.

Replicate independently of bacterial chromosomeSingle or multiple copies per cell

Discovered in nature as a source of antibiotic resistance

http://commons.wikimedia.org/wiki/File:Plasmid_episome.png

plasmidshost chromosome

Some plasmids integrate into the host genome

Origin of Replicationensures replication of DNA in a host cell

Selectable Markerallows for selection of transformants; usually confers antibiotic resistance

Reporter Genegene whose phenotype changes depending on whether a foreign transgene has been inserted into the plasmid

Promoter(s)promotes transcription of selectable marker and transgene/reporter gene

Polylinker or Multiple Cloning Site (MCS)series of closely spaced, unique restriction sites at which the plasmid can be cut (linearized) to allow insertion (ligation) of the transgene into the plasmid

polylinker

origin of replicationselectable

marker

reporter gene

promoters

Features of a TypicalCloning Vector

Ampicillin Beta-Lactamase

bla: a common selectable marker

An antibioticPrevents the growth of bacteria by

inhibiting an enzyme that is needed for building new cell wall peptidoglycan

Chemical structure based on a Beta-lactam ringBeta-lactam antibiotics include

penicillins (amoxycillin) and cephalosporins

An enzymeChemically breaks the beta-lactam

ring, inactivating the enzyme

The bla geneEncodes the beta-lactamase

enzymeMakes bacteria resistant to

ampicillin (ampr)

Prokaryotes Eukaryotes

Issues in Moving Transgenes Between Hosts

BacteriaSimple cell structures

No nucleus; DNA spread throughout the cell

Simple gene structuresProtein-coding DNA sequence (open

reading frame, or ORF) is contiguousNo machinery for RNA splicing

Simple promotersFewer transcription factor proteins

Fungi, Protists, Plants, AnimalsComplex cell structures

Internal membrane-enclosed organelles, including a nucleus

Complex gene structuresProtein-coding DNA sequence (exons)

is interrupted by non-coding sequences introns

Requires RNA splicing to convert pre-mRNA (primary transcript; exons + introns) into mRNA (exons)

Complex promotersMany transcription factor proteins

Eukaryotic Gene Structure

http://commons.wikimedia.org/wiki/File:DNA_exons_introns.gif

cDNA Cloning of Eukaryotic Genes into Prokaryotic HostsSince eukaryotic genes have introns that prokaryotic cells can’t remove, a cDNA transgene is created from a DNA copy of the mRNA with introns removed.

cDNA: complementary DNA

Transgene must be attached to a prokaryotic promoter to ensure transcription in new bacterial host.

Regulation of Transcription in Prokaryotes at the Para and Plac PromotersIn the absence of inducer (lactose or arabinose), transcription is turned OFF

Repressor protein binds to the operator, blocking the RNA polymerase from the promoter.

In the presence of inducer (lactose or arabinose), transcription is turned ON

Inducer binds to the repressor protein, causing a change in its shape. The repressor falls off the operator, allowing RNA polymerase to bind to the promoter and transcribe the gene.

Recombinant Proteins

Proteins that are produced through genetic engineering.

Encoded by the introduced transgene.

Produced upon the transgene’s transcription and translation.

Can be purified from the transgenic cells or organisms.

Can be produced in much higher quantities that protein available from natural sources.

Medicine Industry & Consumer Products

Examples of Recombinant Proteins

Human insulin (1982): Used to treat diabetes.

rHGH or human growth hormone (1985): Stimulates growth (height) and development of muscle.Cadaver-derived natural HGH transferred Creutzfeldt-Jacob

disease (1985)

rBST or bovine somatotropin (1994): Stimulates milk production in cowsRequired or permissible labeling of rBST milk or non-rBST milk is

debated

EPO or erythropoeitin: Stimulates creation of red blood cells. Used to treat anemia in cancer chemotherapy patients.

Common blood doping agent for athletes.

tPA or tissue plasminogen activator: Enzyme given to heart attack patients to dissolve blood clots blocking arteries.

Factor VIII: Blood clotting factor missing in hemophiliacs

In Laundry DetergentsProtease for proteins, lipases for greases, and amylases

for carbohydrates

Amylases and MaltasesFor production of high fructose corn syrup from corn

starch

Cellulases and LigninasesEnzymes that digest cellulose into sugars to be fermented

in ethanol for biofuels

PectinasesClarify fruit juices

RenninUsed in cheese production

SESSION 1:TRANSFORMING THE BACTERIA

Mixing bacterial cells and DNA under transformation conditions.

Introduces DNA into cells.

The pGLO Plasmid

bla gene: a selectable

marker; encodes beta-lactamase enzyme; confers ampr phenotype

gfp gene: a transgene;

encodes Green Fluorescent

Protein (GFP); confers glowing

phenotype

Para promoter: allows

transcription of the gfp gene

when cells are treated with

arabinose

Origin of replication

araC gene: encodes the

repressor protein that blocks

transcription at Para promoter in

absence of arabinose

Label Your Transformation Culture Tubes

Use a lab marker to label two 15ml round-bottom culture tubes:

-DNA & your initials+DNA & your initials

Place these tubes in your ice cup to chill. It is very important that the transformation reactions be keep cold. Don’t handle these tubes or have them out of the ice for more than a few seconds at a time.

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Making the Transformation Mixtures

To each tube, add 100µl of competent E. coli cells. Provided in a microtube in your ice cupPipet slowly (the cells are fragile)Carefully deposit the drop of cells to the very bottom of the tube.Keep tubes on ice.Promptly replace the snap-on caps to avoid contamination by bacteria and fungi in the air.Don’t forget: always use a fresh pipet tip each time!

To the –DNA tube, add 10µl of TE Buffer directly to the drop of cells

To the +DNA tube, add 10µl of 5ng/µl pGLO plasmid DNA directly to the drop of cells.

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100µl cells

10µl TE10µl DNA

Cold-Incubating the Transformation Mixtures

Gently tap the bottom of each tube to gently mix the cells and solutions.

Incubate on ice for 15 minutes.During this time, DNA becomes attached to the outer surface of the cells.

Heat-Shocking the Transformation MixturesBring your ice cup with the two culture tubes to the 42°C heat block.

Quickly place your pair of tubes into the heat block. Note the time.

After exactly 45 seconds, quickly remove your pair of tubes and immediately place them back in your ice cup for at least one minute.

This “heat-shock” step opens pores in the cell’s membranes, allowing the DNA to enter some cells. The heat shock requires instantaneous transitions between cold to hot to cold.

Initial Cell Culture:Recovery and bla Gene ExpressionAdd 800µl of LB broth to each culture tube.

Don’t forget: always use a fresh pipet tip each time!Replace the caps promptly to avoid contamination.

Tap the bottom of the tube to mix.

Place the tubes into the foam adapter mounted on a vortex mixer.

Your samples will be agitated at room temperature for about 45 minutes. This gives the new genes (on the plasmid DNA) introduced into the cells time to be transcribed and translated into proteins.

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ls800µl LB broth

800µl LB broth

Teacher Note

After about a class period of incubation (40-50 minutes), transfer the tubes to a lab refrigerator (without food!) for overnight storage.

SESSION 2:SPREAD-PLATING THE TRANSFORMATION CULTURES

Growing the transformation cultures on non-selective, selective, and indicator plates.

Culture Media

LBLuria-Bertani medium: a rich medium that provides a complete mixture of nutrients (sugars, amino acids) and vitamins in which bacteria can grow.

agara substance added to media that makes it semi-solid

Culture Media Additivesamp: ampicillin

an antibiotic that kills bacteria, except those cells that contain genes that provide resistance (such as the beta-lactamase or bla gene, sometimes called an ampicillin-resistance or ampr gene)

selective mediuma growth medium that causes the

death, or prevent the growth, of some cells but not others

ara: arabinosea sugar that induces transcription

of a gene by removing the repressor protein from the gene’s specific “ara” promoter

indicator mediuma growth medium that causes

some cells to appear differently than other cells, indicating the presence or absence of certain traits

Why Grow Transformation Cultures On Selective Media?The transformation process is very inefficient.

Only a tiny fraction of the cells actually take up the DNA.

We face a “finding a needle in a haystack” problem.How do we detect, and obtain, only the cells that have been successfully transformed (the “transformants”)?

“Burn down the haystack!” Kill off all the non-transformants on selective media.Cells lacking the pGLO plasmid will lack its bla gene, and thus will be sensitive to ampicillin.

To possess a trait, you need to both possess the gene and express it. The gfp gene encodes the green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria.

For a cell to have GFP, it must transcribe and translate the gfp gene.

Arabinose induces the transcription of the gfp gene.The gfp gene is expressed when transformed cells are treated with arabinose.

http://en.wikipedia.org/wiki/File:Aequorea_victoria.jpg

http://en.wikipedia.org/wiki/File:GFP_structure.png

Labeling the Plates

Period __Team ___or initials

Non-selective plate

Selective plate

Selective & Indicator plate

Keep plates

agar-side UP!

Spread-Plating the Transformation Cultures

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200µl

The other partner

does these three.One partner does these three.

Spread-PlatingYour foil packet contains two sterile yellow spreaders. Feel the foil packet and find the end shaped like a triangle. Carefully open the foil at the stick (not the triangle) end, keeping the triangle ends covered with foil. Keep the spreader in the opened pack for now.

Turn your three –DNA plates over (agar side on bottom) and apply 200µl of your –DNA culture to each of the three –DNA plates: LB, LB amp, and LB amp ara. Use a fresh pipet tip each time.

Remove one spreader from the pack (keep the other spreader covered) and use it to gently spread the liquid across the entire surface of each plate, turning the plate as you spread. Don’t press too hard, or the agar will tear. Place the used spreader in the collection bin.

Repeat using the other spreader to apply the +DNA culture to each of the three +DNA plates.

Incubating Your Cultures

Allow your plates to sit, agar side down, for a few minutes to allow the liquid to absorb into the agar.

Tape your set of six plates together using colored lab tape.

Label the tape with your class period.

Place you set of six plates into the 37°C incubator for an overnight incubation.

Predict Whether Cells Will Grow On Each Plate, and What They Will Look Like

- DNA +DNA

Grow? Lawn or Colonies?

Glow under UV

light?Grow? Lawn or

Colonies?Glow

under UV light?

LBamp ara

Teacher Note

After overnight incubation, if the students will not be observing their results the following day, wrap the plates in parafilm and store them in a refrigerator (with no food!).

SESSION 3:INTERPRETING RESULTS

Examining for evidence of transformation and recombinant gene expression.

Imagining Mind Thinking Mind

Use Your “Two Minds”

Imagines what’s possible

Finds all alternatives

Decides what’s realtrue or false

Eliminates alternatives

Scientific Thinking is Critical Thinking

E A C+ =Evidence

“Facts we SEE”Assumptions“Things we THINK”

Conclusions“Claims we MAKE”

• Observations• Data• Results

• Materials• Procedures• Experimental

Design• CONTROLS 34

Words of Wisdom from Sherlock Holmes

“It is an old maxim of mine that when you have excluded the impossible, whatever remains, however improbable, must be the truth.”

DESIGNING AN EXPERIMENT:EXPERIMENTAL VARIABLES

•Manipulated Variables

•Controlled Variables

•Responding Variables

•Uncontrolled Variables

Manipulated Variables

Also called the Independent Variable.

The condition or treatment that is changed or manipulated during the experiment.

Each sample is subjected to different conditions for the manipulated variable: treatment, amount, time, duration, etc.

The manipulated variable is the "cause" for which we wish to identify an "effect".

Controlled Variables

Conditions and treatments that are identical for all samples within the experiment

Conditions that are to be ruled out as affecting the outcome.

Responding Variables

Also called the Dependent Variable.

The properties to be observed or measured.

The "effect(s)" associated with changes in the manipulated variable.

Uncontrolled Variables

Factors which may impact experimental samples or subjects differently, resulting in effects that are not due to the manipulated variable.

Experimenter errorBiasEnvironmental conditionsNon-random sampling

ANALYSIS OF RESULTS

Arrange your plates like this.

What do you conclude from THIS plate ALONE?

What ELSE might you conclude?

Critical Thinking involves identifying and considering ALL alternatives!

Negative results have no meaning

EXCEPT in comparison

to a POSITIVE CONTROL.

What do you conclude from THIS plate ALONE?

What is the Manipulated Variable? Controlled Variables? Responding Variable? Conclusion?

Examining for Production of Green Fluorescent Protein

Turn out the room lights.

Hold the UV lamp over your plates.Do not look directly into the UV lamp.

Record which of your plates have colonies that glow green.

GOOD EXPERIMENTAL DESIGN

•Seeks to control variables.

•Confirms all of our assumptions about materials and procedures.

•Allows us to conclude a clear CAUSE-and-EFFECT relationship between the MANIPULATED VARIABLE and the RESPONDING VARIABLE.

Natural Light UV Light

Sample Results

What Questions Do YOU Have?

making cells glow: bacterial transformation with pglo plasmid dna

new host cell

host cell selectable

pglo plasmid dna slide

host genome slide

replication of dna

bla gene

nucleus dna

permanent host cells

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