PGLO TRANSFORMATION LAB

(AP LAB 7)

BIO-RAD lab book

pGLO ori

bla

GFP

araC

PURPOSE:

To observe gene expression in real time by performing

a genetic transformation procedure on E. coli bacteria

using a plasmid as a vector.

If successful, the plasmid will provide the E. coli with two new traits:

• expression of a gene that codes for Green Fluorescent Protein

(GFP) bioluminescence under the control of an operon

• resistance to the antibiotic Ampicillin

BACKGROUND

OLD CENTRAL DOGMA

OF MOLECULAR

BIOLOGY

WHAT IS TRANSFORMATION?

Uptake of foreign DNA plasmid – that

produces new traits in the bacteria

Bacterial

chromosomal DNA

pGLO plasmids

WHAT IS A PLASMID?

A plasmid is a small circular piece of DNA (about

2,000 to 10,000 base pairs long) that contains

important genetic information for the growth of

bacteria.

In nature, this information is

often a gene that codes for

a protein that will make the

bacteria resistant to an

antibiotic.

• Bacteria can exchange

plasmids with one another.

WHAT IS THE GFP GENE?

GFP is a green fluorescent protein that naturally occurs in some bioluminescent jellyfish.

GLOWING IN NATURE

Many species have the ability to glow

Most are marine: jellyfish, dinoflagellates

Some live on land: firefly, glow worm

Purposes of glowing:

Spook predators

Lure prey

Attract mates

Communicate

Fireflies

Glow worm and glow worm cave

WHY DO SCIENTISTS USE PLASMIDS?

A plasmid is used as a vector. The gene of interest is inserted into the vector plasmid and this newly constructed plasmid is then put into E. coli or some other target.

Vector - Something that is used to transfer something else (a mosquito is a vector for the organism that causes malaria)

pGLO ori

bla

GFP

araC For example: transformed bacteria can be used to make insulin, human growth hormone, and clotting factor cheaply and in great abundance.

Jellyfish Gene put into Other Critters

http://www.technologyreview.com/files/21291/monkey_x600.jpg

ALBA – BUNNY CREATED FOR “ART”

•http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html

Three kittens. Two have been genetically modified to make

red fluorescent protein. All three look similar under normal

light, but when irradiated with blue light only the two

genetically modified kittens glow red.

(Photo courtesy of Biology of Reproduction)

MOUSE UNDER BLUE LIGHT (LEFT) SAME MOUSE UNDER NORMAL LIGHT (RIGHT)

Mouse blood vessels (green-GFP) in tumor (red-DsRed). Mouse with brain tumor

expressing DsRed.

In Brainbow mice, Harvard

researchers have introduced

genetic machinery that randomly

mixes green, cyan and yellow

fluorescent proteins in individual

neurons thereby creating a palette

of ninety distinctive hues and colors.

This is a photograph of the cerebral

cortex. In non-living preserved

brains the outer layers of this

portion of the brain are gray.

(Confocal image by Tamily Weissman.

Mouse by Jean Livet and Ryan Draft.)

REAL-WORLD APPLICATION:

The World Health Organization estimates that 300-500 million cases of malaria

occur each year and more than 1 million people die of malaria. A possible

breakthrough in curtailing the spread of malaria carrying mosquitoes was

reported in October 2005 - the creation of mosquitoes with green

fluorescent testicles. Without green fluorescent gonads it is impossible to

separate mosquito larvae based on their sex, and it is very difficult to separate

the adults. Now male mosquito larvae can easily be separated from female

mosquito larvae.

Malaria is the

world's most

common and

deadly parasitic

disease.

ARE WE GOING TO MAKE CATS GLOW

GREEN? NO, JUST BACTERIA.

In this lab, you will “transform” bacteria by making them take up a commercially prepared plasmid that contains three genes of interest:

amp, araC and GFP

Genetically modified organisms are called “transgenic”

BACTERIAL TRANSFORMATION

Plasmids

Chromosomal

DNA

Bacterial Cell

The uptake of plasmid DNA

The bacterium Escherichia coli or E. coli is an ideal organism for the

molecular geneticist to manipulate and has been used extensively in

recombinant DNA research. It is a common inhabitant of the human

colon and can easily be grown in suspension culture in a nutrient

medium such as Luria broth, or in a petri dish of Luria broth mixed

with agar (LB agar) or nutrient agar.

Aequorea victoria Source of “glowing gene” for this experiment

source of GFP

PGLO PLASMID

bla (β-lactamase)

- “on” all the time

- makes protein that breaks down ampicillin

- provides ampicillin resistance

GFP-Green Fluorescent Protein

- Glows green in fluorescent light

Arabinose Operon (inducible)

Turns on (makes a protein)

when arabinose sugar is present

Allows bacteria to “turn on” the

Fluorescence because it has been

linked into the same operon system

pGLO ori

bla

GFP

araC Ori-

Plasmid

Replication

genes

PBAD arabinose promoter

Plasmids can transfer

genes that occur

naturally within them, or

they can act as carriers

for introducing foreign

DNA from other sources

into recipient bacterial

cells.

Restriction

endonucleases can be

used to cut and insert

pieces of foreign DNA

into the plasmid vectors

GENES OF INTEREST: AMP, ARAC, GFP

amp – this gene will give our transgenic bacteria

resistance to the antibiotic ampicillin

araC – this gene will produce a protein in the

presence of arabinose (a sugar that is added to agar)

that will allow the bacteria to turn on the GFP gene

GFP – in the presence of arabinose, this gene will

“turn on” and cause the transformed (transgenic)

bacteria to glow green

ARABINOSE OPERON REGULATION

araC

RNA Polymerase

Effector

(Arabinose)

B

B

B

A

A

A

D

D

D

araC

araC

ara Operon

INDICIBLE OPERON:

The presence of

arabinsoe turns on

genes that make

enzymes (proteins) to

digest the sugar

arabinose

PGLO REGULATION

araC

RNA Polymerase

Effector

(Arabinose)

B

B

B

A

A

A

D

D

D

araC

araC

ara Operon

GFP Gene

GFP Gene

GFP Gene

GFP GENE HAS

BEEN ADDED TO

ara OPERON

WHEN ARABINOSE

IS PRESENT,

OPERON IS

TURNED ON and

GFP GENE

IS EXPRESSED

TOO!

GETTING STARTED

E. coli starter plate

This plate has the

bacteria we will use in

the lab growing in a

luria broth (LB) agar

plate.

These bacteria are

normal (have NOT

been transformed)

EXPLANATION OF AGAR PLATES

LB/amp/

This plate will have E. coli bacteria on LB agar to which ampicillin has been added.

LB/amp/ara

This plate will have bacteria growing on agar that has both ampicillin and arabinose added to it.

WHAT SHOULD YOU EXPECT?

If your technique is good, you should expect to see green glowing bacteria in some plates and not others.

Read the transformation lab

procedure and answer

questions 1 – 4 for Lesson 1.

protocol

BACTERIAL TRANSFORMATION

MAKE OBSERVATIONS!

Be sure to make observation and answer questions on pg.

30 of your packet – before you start the experiment.

The liquid (broth) and solid (agar) nutrient media are made from an extract

of yeast and an enzymatic digest of meat byproducts, which provide a

mixture of carbohydrates, amino acids, nucleotides, salts, and vitamins, as

nutrients for bacterial growth. The foundation, agar, is derived from

seaweed. It melts when heated, forms a solid gel when cooled and

functions to provide a solid support on which to culture bacteria.

Label Tubes

+ pGLO - pGLO

Gather Supplies - 10 groups

What do these

represent?

Use sterile pipette to

add 250µL transformation

solution to pGLO + and – tubes

Transformation

solution (CaCl2)

WHEN USING THE PIPETTE FOR

MEASUREMENTS TAKE INTO ACCOUNT THE

FOLLOWING GRADUATIONS

GET YOUR RACK ON ICE!

INNOCULATE TUBES WITH

E. COLI BACTERIA

Pick one colony

Twirl loop in +pGLO tube

USE SPECIAL

GARBAGE BAG FOR

DISPOSAL OF USED LOOPS

Get new loop

Pick one colony

Twirl loop in –pGLO tube

EXAMINE PGLO PLASMID DNA

Use UV light to examine pGLO plasmid vial

UV light can be harmful to your eyes! Wear your goggles. Do not shine in eyes.

GFP = Green Fluorescent Protein

isolated from jellyfish

USED AS A GENETIC TOOL http://www.mshri.on.ca/nagy/GFP%20mice.jpg

PLASMID DNA TRANSFER

THIS STEP IS CRUCIAL!

Look closely to make sure you have a film of

solution across the ring.

(Similar to soapy film when you blow bubbles)

ADD PLASMID TO + TUBE

DO NOT ADD PLASMID

TO - TUBE

GET YOUR RACK ON ICE!

10

minutes!

WHILE YOUR TUBES COOL

LABEL YOUR PLATES

UPSIDE DOWN AND WRITE LABELS ON BOTTOM

… NOT ON TOP!

SHOCKING INCREASES UPTAKE OF FOREIGN DNA

(PLASMID)

OSMOTIC SHOCK =Transforming solution

CaCl2

HEAT SHOCK

RAPID TEMPERATURE CHANGE is the key

50 SECONDS!! 2 MINUTES

•Place foam rack with + and – tubes on desktop

•Use new sterile pipette to add 250 µL LB (broth) to + tube

•Use new sterile pipette to add 250 µL LB (broth) to – tube

• Incubate at ROOM TEMPERATURE 10 min

TAP WITH FINGER TO

MIX!

Use NEW STERILE

pipette for each vial

to add 100 µL

bacterial suspension

to CORRECT DISH

(CHECK LABELS!)

Use a NEW STERILE

LOOP FOR EACH PLATE

to spread suspension

evenly on surface of plate

QUICKLY REPLACE LIDS

FLIP PLATES UPSIDE DOWN

STACK AND TAPE

LABEL WITH YOUR GROUP NAME

PLACE IN INCUBATOR

The transformation solution

CaCl2

It is thought that the Ca2+ cation neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane to allow the DNA to enter the cells.

Ca++

Ca++

O CH

2

O

P O

O

O Base

CH2

O

P

O

O

O

Base

OH

Sugar

Sugar

O Ca++

REASONS FOR EACH TRANSFORMATION STEP

Incubation on ice slows fluid cell

membranes

Heat-shock increases permeability of

cell membrane

Nutrient broth incubation allows

beta lactamase expression

Reasons for Each Transformation Step

SELECTION FOR PLASMID UPTAKE

Antibiotic becomes a selecting agent

only bacteria with the plasmid will grow on

antibiotic (ampicillin) plate

LB/amp plate LB plate

all bacteria grow

only transformed

bacteria grow

a

a

a a a

a

a a

a a

a a

a a

a

cloning

a a

Transformation Results

All cells grow since

there is no antibiotic

on the plate

LB PLATE

Luria Broth

+

- PGLO = NO Plasmid

→

Transformation Results

NO GROWTH

Cells without plasmid don’t have

antibiotic resistance. Can’t grow

on media with antibiotic added.

LB/AMP PLATE

Luria Broth with antibiotic

+

- PGLO = NO plasmid

→

only bacteria that have acquired the plasmid can grow on the plate. Therefore, as long as you grow the bacteria in ampicillin, it will need the plasmid to survive and it will continually replicate it, along with your gene of interest that has been inserted to the plasmid.

Selective Pressure - The same as

in evolution - only the organisms

that have a particular trait

(in this case antibiotic resistance)

will survive.

How does ampicillin in the agar act as a “selective pressure”?

Transformation Results

LAWN

Cells with plasmid have antibiotic

resistance gene so can grow on

media with antibiotic

LB/AMP PLATE

Luria Broth with antibiotic

+

+ PGLO = Plasmid added

→

Transformation Results

Cells with pGLO plasmid

GROW & GLOW

-can grow on media with

antibiotic

GLOW on media with

arabinose (turns on GFP gene)

LB/AMP/ARA PLATE

Luria Broth

+ antibiotic|

+ arabinose

+

+ PGLO = Plasmid added

→

the operon

GENE EXPRESSION IN PROKARYOTES (DIFFERENT IN EUKARYOTES)

GENE REGULATION REVIEW

Organisms regulate expression of their genes and ultimately the amounts and kinds of proteins present within their cells for a myriad of reasons.

Gene regulation not only allows for adaptation to differing conditions, but also prevents wasteful overproduction of unneeded proteins which would put the organism at a competitive disadvantage.

The genes involved in the transport and breakdown (catabolism) of food are good examples of highly regulated genes. For example, the sugar arabinose is both a source of energy and a source of carbon.

E. coli bacteria produce three enzymes (proteins) needed to digest arabinose as a food source. The genes which code for these enzymes are not expressed when arabinose is absent, but they are expressed when arabinose is present in their environment.

Regulation of the expression of proteins often occurs at the

level of transcription from DNA into RNA.

This regulation takes place at a very specific location on the

DNA template, called a promoter, where RNA polymerase

sits down on the DNA and begins transcription of the gene.

In bacteria, groups of related genes are often clustered

together and transcribed into RNA from one promoter.

These clusters of genes controlled by a single promoter are

called operons.

The regulation of gene expression is a

complicated and varied process. One,

more well understood process is the

operon.

An operon is made up of several structural

genes3 arranged under a common

promoter1 and regulated by a common

operator2.

It is defined as “a set of adjacent structural

genes, plus the adjacent regulatory signals

that affect transcription of the structural genes.”

operon

3 1 2

Genes are transcribed together into a mRNA strand and

either translated together, or undergo trans-splicing to

create several strands of mRNA that each encode a single

gene product translated separately.

The result of this is that the genes contained in the

operon are either expressed together or not at all.

BASICS OF TRANSCRIPTION AND TRANSLATION

Transcription is the synthesis of RNA under the direction of DNA

Transcription produces messenger RNA (mRNA)

Translation is the synthesis of a polypeptide, which occurs under the direction of mRNA

Ribosomes are the sites of translation

THE LAC OPERON

The first operon to

be described was

the lac operon in

Escherichia coli.

Provides a typical example of operon function. It consists of

three adjacent structural genes, a promoter, a terminator,

and an operator.

The Lac operon - showing its genes and its binding sites.

The lac operon is required for the

transport and metabolism of lactose in

Escherichia coli and some other enteric

bacteria.

The lac operon is

regulated by several

factors including

availability of glucose and

lactose. (This is an example of the

negative inducible model.)

In the "repressed" state, the repressor IS bound to the operator.

RNA

polymerase

repressor

promoter operator

The gene is essentially turned off.

There is no lactose to inhibit the

repressor, so the repressor binds to

the operator, which obstructs the

RNA polymerase from binding to the

promoter and making lactase. gene sequence for lactase production

The gene is turned on. Lactose is

inhibiting the repressor, allowing

the RNA polymerase to bind with

the promoter, and express the

genes, which synthesize lactase.

Eventually, the lactase will digest

all of the lactose, until there is

none to bind to the repressor. The

repressor will then bind to the

operator, stopping the

manufacture of lactase

off

on

mRNA

ribosome polypeptide

Control of an operon is a type of gene regulation that

enables organisms to regulate the expression of various

genes depending on environmental conditions.

Operon regulation can be either negative or positive by

induction or repression.

APPLICATIONS IN THE “REAL WORLD”

THE PROMOTER

A nucleotide sequence that enables a gene to be

transcribed.

The promoter is recognized by RNA polymerase,

which then initiates transcription. In RNA

synthesis, promoters indicate which genes should

be used for mRNA creation – and, by extension,

control which proteins the cell manufactures.

A segment of DNA that a regulator binds to. It

is classically defined in the lac operon as a

segment between the promoter and the genes

of the operon.

THE OPERATOR

THE REPRESSOR

In the case of a repressor, the repressor

protein physically obstructs the RNA

polymerase from transcribing the genes.

ARABINOSE OPERON The three genes (araB, araA and araD) that code for three digestive

enzymes involved in the breakdown of arabinose are clustered together in what is known as the arabinose operon.3 These three proteins are dependent on initiation of transcription from a single promoter, PBAD.

Transcription of these three genes requires the simultaneous presence of the DNA template (promoter and operon), RNA polymerase, a DNA binding protein called araC and arabinose. araC binds to the DNA at the binding site for the RNA polymerase (the beginning of the arabinose operon).

When arabinose is present in the environment, bacteria take it up. Once inside, the arabinose interacts directly with araC which is bound to the DNA.

The interaction causes araC to change its shape which in turn promotes (actually helps) the binding of RNA polymerase and the three genes araB, A and D, are transcribed.

Three enzymes are produced, they break down arabinose, and eventually the arabinose runs out.

In the absence of arabinose the araC returns to its original shape and transcription is shut off.

PGLO RECOMBINANT DNA The DNA code of the pGLO plasmid has been engineered to

incorporate aspects of the arabinose operon. Both the promoter (PBAD) and the araC gene are present. However, the genes which code for arabinose catabolism, araB, A and D, have been replaced by the single gene which codes for GFP.

Therefore, in the presence of arabinose, araC protein promotes the binding of RNA polymerase and GFP is produced. Cells fluoresce brilliant green as they produce more and more GFP.

In the absence of arabinose, araC no longer facilitates the binding of RNA polymerase and the GFP gene is not transcribed. When GFP is not made, bacteria colonies will appear to have a wild-type (natural) phenotype—of white colonies with no fluorescence.

This is an excellent example of the central molecular framework of biology in action: DNA➜RNA➜PROTEIN➜TRAIT.

The pGLO plasmid, which contains the GFP gene, also contains the gene for beta-lactamase, which provides resistance to the antibiotic ampicillin, a member of the penicillin family. Beta-lactarmase inactivates the ampicillin present in the LB nutrient agar to allow bacterial growth. Only transformed bacteria that contain the plasmid and express beta-lactamase can grow on plates that contain ampicillin.

TRANSFERRING BACTERIAL COLONIES

FROM AGAR PLATES TO MICROTUBES

The process of scraping a single colony off the starter plate leads to the temptation to get more cells than needed.

A single colony that is approximately 1 mm in diameter contains millions of bacterial cells.

To increase transformation efficiency, students should select 2-4 colonies that are 1-1.5 mm in diameter. Selecting more than 4 colonies may decrease transformation efficiency.

Select individual colonies rather than a swab of bacteria from the dense portion of the plate, the bacteria must be actively growing for transformation to be successful.

PLASMID DNA TRANSFER

The transfer of plasmid DNA from its stock

tube to the transformation suspension is

crucial. When dipping the inoculating loop

into the container, you must look carefully at

the loop to see if there is a film of plasmid

solution across the ring, similar to film on

wand when blowing bubbles.

HEAT SHOCK

Since the heat shock increases the permeability of the cell membrane to DNA, it is very important to follow the directions regarding time in the warm bath and the rapid temperature change.

While the mechanism is not known, the duration of the heat shock is critical and has been optimized for the type of bacteria used and the transformation conditions employed.

For optimal results, the tubes containing the cell suspension must be taken directly from ice, placed into the water bath at 42°C for 50 sec and returned immediately to the ice.

RECOVERY

The 10 minutes incubation period following

the addition of LB nutrient broth allows the

cells to recover and to express the ampicillin

resistance protein beta-lactamase so that

the transformed cells survive on the

ampicillin selection plates.