By Lynn More - Olympian High School along with UCSD

Protein Transformation Lab Intro

UCSD: BioBridge Program

E. coliE. coli

What is protein transformation?

Introducing DNA that expresses preferred gene(s) into a host to:

1. Inhibit or silence the expression of a gene

2. Carry out certain functions

3. Used as markers to track the location and function of the gene

i.e. - allows you to determine its function or importance

i.e. - make insulin, clot blood, resist pests, resist antibiotics, eat oil

i.e. - fluorescent proteins

Fluorescent Proteins-ApplicationsFluorescent Proteins-Applications

Transgenic Zebra Fish Zebra Fish

Neuron

•Transgenic Mice

Aequorea victoriaAequorea victoria and Discovery of GFP-and Discovery of GFP-19601960’’ss

OSAMU SHIMOMURA Co-winner of Nobel Prize

There are three amino acids which are critical for

GFP’s green fluorescent color.

Only a 1 amino acid Only a 1 amino acid difference changes green to difference changes green to

blue, and blue, and blue to cyanblue to cyan.

Roger Tsien and Rainbow ProteinsRoger Tsien and Rainbow Proteins

DsRed.T1

Dimer 2

mRFP1

mgrape 1

mHoneydew

mBanana

mOrange mTangerine

mStrawberry

mCherry

17 Mut

33 Mut

6 Mut

6 Mut

8 Mut

3 Mut7 Mut

4 Mut

3 Mut

The rainbow of mFruit Fluorescent ProteinsThe rainbow of mFruit Fluorescent Proteins

Central Dogma Central Dogma http://www.dnai.org/a/index.html

DNA---> mRNA---> Protein---> Trait

What is a plasmid?What is a plasmid?

• What: A small circular piece of DNA naturally occurring in bacteria

Origin

AmpR

GFP

Stop

promoter

PM1GreenBlue

Grape

PM2CherryTangerineBanana

PM means Plasmid Mix

Gene of Interest:AMPR - Ampicillin ResistanceAntibiotic Resistance

Gene of Interest:Fluorescent Protein

• Why: Can be altered in lab to express protein of interest.

What is Transformation?

Uptake of foreign DNA, often a circular plasmid

Bacteria now express cloned fluorescent protein (transcription of gene and translation of mRNA to protein at ribosomes).

Bacterial chromosome Plasmid

Bacterial chromosome

Allow bacteria to grow for 1-3 days on plate with ampicillin.Plasmid

Plasmid = a vector that carries genetically engineered DNA segment into a host cell.

Recombinant DNA

Bacteria cell Bacterial

chromosome

Bacteria plated on LB agar + antibiotic

Only bacteria containingRecombinant DNA grow

cloning

Insert the DNA (plasmid) Using a Heat Shock Method

Collect cultureDNAPurification

X 106

Why Ampicillin?

• Ampicillin inhibits cell growth. Only cells that can inactivate the ampicillin around them will grow.

• Ampicillin resistance is tied to (expressed with) the fluorescent protein gene

• Ampicillin is a selection mechanism that only allows transformed bacteria to grow on the plate

Make two Plasmids

Pick a Fluorescent Protein gene to insert

mCherry mTangerine mBanana GFP

Green Fluorescent

Protein

BFP

Blue Fluorescent

Protein

mGrape mPlum

Insulin gene

Save a diabetic

Factor VIII gene

Save a hemophiliac

Pest Resistance

gene

Save a plant

Oil Spill gene

Save an environment

Use DNA Ligase (tape or glue) to bond the gene of interest

Pick a gene of interest to add to the second plasmid you make

AmpR gene

Ampicillin

resistance

TetraR gene

Tetracycline resistance

KanR gene

Kanamycin resistance

PenR gene

Penicillin resistance

Add an antibiotic resistance gene to both plasmids you make.

Glue/Tape

Tuck Under & Glue/Tape

Glue/Tape

Tuck Under

& Glue/Tape

Time to make the second

Plasmid Model

Congratulations scientists you

have just made recombinant

DNA: genetically engineered DNA with genes that can save lives!

Restriction Enzyme

Restriction Enzyme

Restriction Enzyme Restriction Enzyme

Use DNA Ligase (tape or glue) to bond the gene of interest

Restriction Enzyme

Restriction Enzyme

Make a Plasmid Activity #18

1. Cut the DNA with a _______________ (Scissors)

2. My gene of interest was (FP - ________ & __________)

3. My goal is to (FP) - track ____________; save ________

4. The petri dish would have: ___________ antibiotic; ___________ antibiotic

so…I need to make the transformed bacteria resistant to that antibiotic (_____); (_____)

5. What I have made are 2 small circular pieces of DNAwith two genes of interest each & they called

plasmids.

Escherichia coli

• What?• AKA (also known as) E. coli• Prokaryote

• Single-celled organism• No nucleus• No membrane –bound organelles

• Why?• Small, so only need…

• Food (LB Agar)• Little space• Warm temps (37˚C)• Little humidity

• Reproduces fast• Binary fission• x 106

• Can uptake foreign DNA• Clones itself & its contents

Why calcium chloride?

OCH2

O

P O

O

OBase

CH2

O

P

O

O

O

Base

OH

Sugar

Sugar

O

Ca++

Ca++

Ca++

• Calcium Chloride (CaCl2) Transformation solution

• CaCl2 is necessary because: • The positive charge of Ca++   ions neutralizes DNA’s slightly negative charge• increases the diffusion of its  foreign genetic information   through the cell wall and cell  membrane into the bacteria.

The what’s and why’s of Heat Shock

• Incubate on ice (10 minutes) slows fluid cell membrane (constricts pore size)

• Heat-shock (42˚C for 45 seconds) increases permeability of membranes (dilates/opens pores, allowing the plasmid to get inside the bacteria)

• Incubate on ice (2 minutes) slows fluid cell membrane (reduces permeability again, “locking” the plasmid inside the bacteria)

What’s happening in the petri dish?

Ampicillin acts as a __________________ that only allows ___________ bacteria to _____ on the plate

Represent ___________________________________________Ampicillin - an antibiotic that inhibits bacterial growth

Represent ______________Bacteria growth

Represents _________________________________________LB Agar - a nutrient substrate to encourage growth

Represent _________________________________Genetically transformed bacteria that are: 1. Resistant (or shielded) from the effects of ampicillin 2. Marked with a Fluorescent Protein

selection mechanismtransformed grow

______________________Bacteria killed by ampicillin

More Review??More Review??

Bioluminescent organism produces its own light.

A fluorescent organism absorbs light at one wavelength (UV) and a re-emits the light at a visible wavelength= color

Scorpion- UV LightScorpion- Natural Light

http://fireflyforest.net/firefly/2006/11/13/fluorescent-scorpion-in-uv-light/

Natural Light

In the Dark

Bioluminescence Fluorescence

Bioluminescence vs. FluorescenceBioluminescence vs. Fluorescence

How are plasmids engineered?

DNA Plasmid Vector

Cut plasmids open with restriction enzymes

Cut genomic DNA into fragments +

Ligate (paste) fragments into cut DNA vector

End result: Plasmid containing FP gene

Host DNA fragments (i.e. coral or jellyfish FP

coding DNA)

The plasmids we have…The plasmid serves as a carrier or transporter of a genetically engineered DNA segment into a host cell.

EcoRI (pronounced "eco R one") is a commonly used restriction enzyme isolated from certain strains of E. coli used to cut DNA at specific locations.

Gene for antibioticresistance

Restriction Enzyme

Area of Interest - Fluorescent Protein

Restriction EnzymeCuts the DNA

Foreign DNA

Recombinant DNA

DNA Ligase

Sticky ends help attach to the plasmid

Human cell stained with two different fluorescent proteins to visulalize cytoskeletal components. Transfected with GFP-tubulin / mCherry actin (Ben Giepmans)

Cellular organelles “marked” with FPs

C Elegans transfected with GFP tubulin construct (Susan Kline)

Transcription / Translation

Campbell