Fluorescent Protein BACTERIAL TRANSFORMATION

What is genetic transformation…and why do it? 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

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 Light Scorpion- Natural Light Natural Light In the Dark BioluminescenceFluorescence Bioluminescence vs. Fluorescence

Many organisms have the ability to fluoresce Many organisms have the ability to fluoresce Jellyfish Amphipod Spider’s palps Mushroom Sea Anemone Coral

Jellyfish- Bioluminescence and Fluorescene

Aequorea victoria and Discovery of GFP- 1960’s OSAMU SHIMOMURA Co-winner of Nobel Prize

Fluorescent Proteins-Applications Fish Transgenic Fish Neuron Transgenic Mice

xxx GFP-chromatin met ana pro telo GFP-membrane cyto met ana telo GFP-tubulin met ana pro telo Visualizing FPs in Live Worms ……analyzing mitosis from 3 perspectives:

xxx MITOSIS a similar process in diverse species ……...using various organisms to understand humans: Frog Egg Extract + sperm DNA A. Desai Frog Cell C.E. Walczak Marsupial Cell S.L. Kline Fly Embryo T. Megraw Frog Egg Extract + DNA-coated beads R. Heald Human Cell J. Waters Worm Embryo I.M. Cheeseman

Green Fluorescent Protein (GFP) is produced by a number of organisms, such as the jellyfish. There are three amino acids which are critical for GFP’s green fluorescent color. Only a 1 amino acid difference changes green to blue, and blue to cyan Only a 1 amino acid difference changes green to blue, and blue to cyan. Aequoria victoria

Roger Tsien and Rainbow Proteins DsRed.T 1 Dimer 2 mRFP1 mgrape 1 mHoneydew mBanana mOrange mTangerine mStrawberry mCherry 17 Mut 33 Mut 6 Mut 8 Mut 3 Mut 7 Mut 4 Mut 3 Mut

The rainbow of mFruit Fluorescent Proteins

E. coli

Central Dogma Central Dogma DNA--->mRNA--->Protein--->Trait

What is a plasmid? A small circular piece of DNA Naturally occurring in bacteria & yeast Can be altered in lab to express protein of interest Origin Amp R GFP Stop promoter PM1PM2 GreenCherry BlueTangerine GrapeBanana

Vector - any agent that acts as a carrier or transporter (ie. a virus or plasmid) that conveys 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 antibiotic resistance produces ß-lactamase EcoRI Area of Interest - Fluorescent Protein EcoRI Foreign DNA Recombinant DNA DNA Ligase Sticky ends help attach to the plasmid End result = a plasmid containing the FP gene From GFP: From RFP: PM1PM2 GreenCherry BlueTangerine GrapeBanana How are plasmids engineered? (aka: genes transformed)

What is Transformation? Plasmid Uptake of foreign DNA, often a circular plasmid Bacterial chromosome

What is Transformation? Bacterial chromosome Allow bacteria to grow for 1-3 days on plate with ampicillin. Plasmid Uptake of foreign DNA, often a circular plasmid Bacterial chromosome

What is Transformation? Bacteria now express cloned fluorescent protein… Bacterial chromosome Allow bacteria to grow for 1-3 days on plate with ampicillin. Plasmid Uptake of foreign DNA, often a circular plasmid Bacterial chromosome

How does the plasmid get in? O CH 2 O PO O O Base CH 2 O P O O O Base OH Sugar O Ca ++ CaCl 2 - Calcium Chloride (Transforming Sol’n) Positive charge of Ca ++ ions shields negative charge of DNA phosphates

How does the plasmid get in? Incubate on ice = slows fluid cell membrane Heat-shock = increases permeability of membranes, allowing plasmids to get inside bacteria (42˚C for 45 Seconds) Leave in heat 45 seconds! Too short, and bacteria won't let in plasmid. Too long, and the bacteria will die. Incubate immediately on ice = slows fluid cell membrane again, “locking the plasmids inside.”

Antibiotic Resistance Ampicillin - is an antibiotic. Kills any normal bacteria. The regular bacteria would compete for food and space. We want the antibiotic to eliminate them. Origin Amp R GFP Stop promoter Amp R - the gene for Ampicillin Resistance This gene makes a protein (enzyme) that breaks down the ampicillin and makes it edible for the transformed bacteria

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

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 mechanism transformedgrow ______________________Bacteria killed by ampicillin

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 containing Recombinant DNA grow cloning DNA (plasmid) insertion Using a Heat Shock Method Collect culture DNA Purification Why is that? Why use bacteria? Do the two genes do?

Go to: Protein Transformation Lab Preview.ppt UCSD: BioBridge Program

Students make bags in groups of four:  2 empty microcentrifuge tubes  4 disposable transfer pipette  Inoculating loops OR sterile tips  2 cotton swabs Teacher will have at lab tables for each group of four:  1 waterproof pen - for labeling  1 LB plate  2 LB/AMP plates  1 microcentrifuge tube of CaCl2 on ice  Ice bucket (cup with ice and water)  1 tube of plasmid labeled either PM1 or PM2 on ice.  2 Lab station waste containers - one with 10% bleach, other empty  Tape for sealing plates after inoculation FP Transformation materials checklist

FP transformation procedure + LB/Amp + - LB/Amp - Control - Colonies of bacteria With plasmids Testing effectiveness Of Ampicillin “Lawn” testing viability of the bacteria CaCl 2 + Bacteria + Plasmid (PM1 or PM2) CaCl 2 + Bacteria + Control (TE or dH 2 O) Purpose of Each plate

FP transformation procedure

DNA Transformation by BioBridge Online resources: