PGLO Transformation LAB AP LAB 7

PGLO TRANSFORMATION LAB

(AP LAB 7)

BIO-RAD lab book

pGLOori

blaGFP

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

MECHANISMS OF GENE REGULATION

a wide range of mechanisms are used by cells to

increase or decrease the production of specific gene

products

Prokaryotes regulate gene expression by controlling

the amount of transcription.

Eukaryotic gene expression is controlled at the levels of epigenetics,

transcription, post-transcription, translation, and post-translation.

GENE REGULATION IN EUKARYOTES

The latest estimates are that a human cell, a eukaryotic cell,

contains some 21,000 genes.

• Some of these are expressed in all cells all the time. These

“housekeeping genes” are responsible for the routine

metabolic functions (e.g. respiration) common to all cells.

• Some are expressed as a cell enters a particular pathway of

differentiation.

• Some are expressed all the time in only those cells that have

differentiated in a particular way. For example, a plasma

cell expresses continuously the genes for the antibody it

synthesizes.

• Some are expressed only as conditions around and in the cell

change. For example, the arrival of a hormone may turn on (or

off) certain genes in that cell.

Altering the rate of transcription of the gene is the

most important and widely-used strategy

There are several methods used by eukaryotes to regulate gene expression.

Promotors – sequence of DNA before the gene of interest where

transcription factors can bind and begin transcription

Enhancers – increase the rate of transcription of the gene

Silencers – can repress the transcription of the gene they control

GENE REGULATION IN PROKARYOTES

Bacteria adapt to changes in their surroundings

by using regulatory proteins to turn groups of

genes on and off in response to various

environmental signals.

Prokaryotes are sensitive to their environment, and their genetic

activity is controlled by specific proteins that interact directly with their

DNA to quickly adjust to environmental changes.

When the genes in an operon are transcribed, a single mRNA is produced for all

the genes in that operon.

An operon is a self-regulating series of genes that work in concert. An

operon includes a special segment of genes that are regulators of the

protein synthesis, but do not code for protein, called the promoter and

operator. These segments overlap, and their interaction determines

whether the process will start and when it will stop.

The DNA of Escherichia coli can encode about 4000

proteins, but only a fraction of these are made at any one

time.

E. coli regulates the expression of many of its genes

according to the food sources that are available to it.

The operator is a short region of DNA that lies partially

within the promoter and that interacts with a regulatory

protein that controls the transcription of the operon.

The regulatory gene lacI produces a mRNA that

produces a Lac repressor protein, which can bind to the

operator of the lac operon.

The Lac regulatory protein is called a repressor because it

keeps RNA polymerase from transcribing the structural genes.

Thus the Lac repressor inhibits transcription of the lac operon.

In the absence of lactose, the Lac repressor binds to

the operator and keeps RNA polymerase from

transcribing the lac genes.

When lactose is present, the lac genes are expressed

because allolactose binds to the Lac repressor protein

and keeps it from binding to the lac operator.

RNA polymerase can then bind to the promoter and

transcribe the lac genes.

GENE REGULATION IN BACTERIA

pGlo lab

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.

WHAT IS TRANSFORMATION?

Uptake of foreign DNA plasmid – that

produces new traits in the bacteria

Bacterial

chromosomal DNA

pGLO plasmids

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)

pGLOori

blaGFP

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

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

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.

APPLICATIONS IN THE “REAL WORLD”

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

Jellyfish Gene put into Other Critters

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

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.

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)

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:

Fluorescence can be used to

“tag” specific cells – in this case

tumor cells. Scientists hope this

could be a noninvasive way to

identify the location of certain

tumors and cancers in humans.

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

(transmitted by the female Anopheles mosquito’s bite) 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.

BACK TO OUR LAB…

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(Crystal Jelly) Source of “glowing gene” for this experiment

source of GFP

The Crystal jelly (Aequorea victoria) is a jellyfish that is found off the west coast of North America. The species is best known as the source of two proteins involved in bioluminescence; aequorin and green fluorescent protein which led to their discoverers winning the Nobel Prize in Chemistry.

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 systempGLOori

blaGFP

araCOri-

Plasmid

Replication

genes

PBAD arabinose promoter

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 wild

type (“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.

protocol

BACTERIAL TRANSFORMATION

MAKE OBSERVATIONS!

Be sure to make observation and fill in the observations

you can make today – 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

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

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

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++

OCH2

O

P O

O

OBase

CH2

O

P

O

O

O

Base

OH

Sugar

Sugar

OCa++

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 plateLB plate

all bacteria grow

only transformed

bacteria grow

a

a

a aa

a

aa

aa

aa

aa

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

→