By Lynn More - Olympian High School along with UCSD Protein Transformation Lab Intro UCSD: BioBridge Program E. 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-Applications Zebra Fish Transgenic Zebra Fish Neuron Transgenic Mice

Aequorea victoria and Discovery of GFP- 1960’s 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 difference changes green to blue, and blue to cyan blue to cyan.

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

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

What is a plasmid? What : A small circular piece of DNA naturally occurring in bacteria Origin Amp R GFP Stop promoter PM1 Green Blue Grape PM2 Cherry Tangerine Banana PM means Plasmid Mix Gene of Interest: AMP R - Ampicillin Resistance Antibiotic 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 containing Recombinant DNA grow cloning Insert the DNA (plasmid) Using a Heat Shock Method Collect culture DNA Purification X 10 6

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 mGrapemPlum Insulin gene Save a diabetic Factor VIII gene Save a hemophiliac Pest Resistance gene Save a plant Oil Spill gene Save an environmen t 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 Amp R gene Ampicillin resistance Tetra R gene Tetracycline resistance Kan R gene Kanamycin resistance Pen R 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     Use DNA Ligase (tape or glue) to bond the gene of interest 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 DNA with 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 10 6 Can uptake foreign DNA Clones itself & its contents

Why calcium chloride? O CH 2 O PO O O Base CH 2 O P O O O Base OH Sugar O Ca ++ Calcium Chloride (CaCl 2 ) Transformation solution CaCl 2 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 mechanism transformedgrow ______________________Bacteria killed by ampicillin

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 Light Scorpion- Natural Light Natural Light In the Dark BioluminescenceFluorescence Bioluminescence 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 enzymerestriction enzyme isolated from certain strains of E. coli used to cut DNA at specific locations.E. coli Gene for antibiotic resistance Restriction Enzyme Area of Interest - Fluorescent Protein Restriction Enzyme Cuts 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