pGLO Transformation LAB AP BIO LAB 6 ori bla GFP araC BIO-RAD lab book http://www.mshri.on.ca/nagy/GFP%20mice.jpg

PLASMID Extrachromosomal DNA Often carry genes for antibiotic resistance Can be passed from one bacterium to another http://www.agen.ufl.edu/~owens/age2062/OnLineBiology/OLBB/www.emc.maricopa.edu/faculty/farabee/BIOBK/14_1.jpg

pGLO plasmid Contains genes for: Image from: BIO-RAD lab book pGLO plasmid Contains genes for: - GFP = Green fluorescent protein taken from jellyfish (Aequorea victoria) glows green in UV- light - ori = Origin of replication allows plasmid to replicate itself - araC = codes for arabinase = enzyme to break down arabinose sugar Turns on when arabinose sugar is present (INDUCIBLE) - bla On all time makes enzyme beta-lactamase that breaks down ampicillin provides antibiotic resistance (=ampR)

pGLO plasmid genetically engineered plasmid Image from: BIO-RAD lab book pGLO plasmid genetically engineered plasmid used in biotechnology as a vector creating genetically modified organisms. contains several “reporter“ genes green fluorescent protein (GFP) – glows under UV light ampicillin resistance gene (ampR) – allows growth on media containing ampicillin produces an observable phenotype to help identify cells that contain the plasmid (AND ANY OTHER GENES THAT SCIENTISTS ATTACH TO THE PLASMID!)

Source of “glowing gene” for this experiment Aequorea victoria: Source of “glowing gene” for this experiment

put into Other Critters Jellyfish Gene put into Other Critters http://www.lafuga.de/GFP_pig.jpg http://www.technologyreview.com/files/21291/monkey_x600.jpg

Bacterial Transformation Uptake of DNA from environment Changes phenotype Image modified from BIORAD pGLO lab manual

pGLO LAB SUPPLIES FOAM tube holder/float 4 - flip top microtubes Blue- Transforming solution (CaCl2) Yellow- LB nutrient broth Pink- label + Purple- label - 1- colored eraser (to ID your tubes in water bath) 1-pkg yellow innoculating loops 2- Sterile pipettes 4 poured agar plates 1 - LB 2 - LB/amp 1- LB/amp/ara PERMANENT MARKER Cup with crushed ice

PLATE ABBREVIAIONS LB (LURIA & BERTANI) BROTH contains nutrients for bacterial growth AMPICILLIN (amp) Antibiotic that kills bacteria ARABINOSE (ara) = sugar used for food if glucose is unavailable

LABEL TUBES purple = + pGLO pink = - pGLO Images from BIORAD pGLO lab manual

solution to pGLO + and – tubes Transformation solution (CaCl2) Use sterile pipette to add 250µL transformation solution to pGLO + and – tubes Images from BIORAD pGLO lab manual

Get your TUBES on ICE! Images from BIORAD pGLO lab manual

INNOCULATE TUBES WITH E. coli BACTERIA Images from BIORAD pGLO lab manual 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 USE SPECIAL GARBAGE BAG FOR DISPOSAL OF USED SUPPLIES FOR THIS LAB

EXAMINE pGLO plasmid DNA Use UV light to examine pGLO plasmid vial DOES IT GLOW? UV light can be harmful to your 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 Images from BIORAD pGLO lab manual

Put rack on ICE for 10 MIN! Images from BIORAD pGLO lab manual

WHILE TUBES COOL CHECK LABELS ON PLATES LB (Luria and Bertani) – broth & agar provides nutrients for bacterial growth LB/amp Nutrients + ampicillin (antibiotic) LB/amp/ara Nutrients + ampicillin + arabinose sugar + plasmid will be added - NO plasmid added Images from BIORAD pGLO lab manual

MAKING CELLS COMPETENT “COMPETENT” cells have the ability to pick up plasmids TRANSFORMATION SOLUTION (CaCl2) Positive charge of Ca++ ions neutralizes: ~ negative charge of DNA phosphates ~ negative charge of membrane phospholipids HEAT SHOCK Increases permeability of cell membranes so plasmid can enter Image modified from BIORAD pGLO lab manual

SHOCKING INCREASES UPTAKE OF FOREIGN DNA (PLASMID) OSMOTIC SHOCK = Transformation solution CaCl2 HEAT SHOCK increases the permeability of the cell membrane to DNA RAPID TEMPERATURE CHANGE is the key 2 MINUTES 50 SECONDS Images from BIORAD pGLO lab manual

Place foam rack with + and – tubes on desktop Images from BIORAD pGLO lab manual 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 for 10 min

TAP TUBE WITH FINGER TO MIX! Use NEW STERILE pipette for each vial to add 100 uL bacterial suspension to CORRECT DISH (CHECK LABELS!) Use a NEW STERILE LOOP FOR EACH PLATE to spread suspension evenly on surface of plate DON’T DIG INTO AGAR! QUICKLY REPLACE LIDS Images from BIORAD pGLO lab manual

HOW TO INNOCULATE A PLATE Images from BIORAD pGLO lab manual http://www.cdn.sciencebuddies.org/Files/620/7/MicroBio_img_006.jpg

FLIP PLATES UPSIDE DOWN STACK AND TAPE LABEL WITH YOUR GROUP NAME PLACE IN INCUBATOR Images from BIORAD pGLO lab manual

MAKE A PREDICTION WHAT WILL HAPPEN?

Selection for plasmid uptake Slide from Kim Foglia http://explorebiology.com Selection for plasmid uptake Antibiotic becomes a selecting agent only bacteria with the plasmid will grow on antibiotic (ampicillin) plate only transformed bacteria grow all bacteria grow a a a a a a a a a a a a a a a a a LB plate LB/amp plate cloning

+pGLO LB/amp +pGLO LB/amp/ara -pGLO LB/amp -pGLO LB http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/Bio%20101/Bio%20101%20Laboratory/Bacterial%20Transformation/results.htm

Transformation Results LB PLATE Luria Broth + - PGLO = NO Plasmid → All cells grow since there is no antibiotic on the plate

Transformation Results LB/AMP PLATE Luria Broth with antibiotic + - PGLO = NO plasmid → NO GROWTH Cells without plasmid don’t have antibiotic resistance. Can’t grow on media with antibiotic added.

Transformation Results LB/AMP PLATE Luria Broth with antibiotic + + PGLO = Plasmid added → LAWN Cells with plasmid have antibiotic resistance gene so can grow on media with antibiotic

Transformation Results Cells with pGLO plasmid GROW & GLOW -can grow on media with antibiotic ONLY GLOW on media with arabinose (turns on GFP gene) LB/AMP/ARA PLATE Luria Broth + antibiotic| + arabinose + + PGLO = Plasmid added →

Inducible operon: lactose Slide from Kim Foglia http://explorebiology.com Inducible operon: lactose Digestive pathway model GLUCOSE is food of choice Don’t need lactose digesting enzymes Gene is turned off RNA polymerase repressor TATA gene1 gene2 gene3 gene4 DNA promoter operator repressor ACTIVE repressor protein Slide from Kim Foglia http://explorebiology.com

Inducible operon: lactose Slide from Kim Foglia http://explorebiology.com Inducible operon: lactose lac Digestive pathway model When lactose is present, binds to lac repressor protein & triggers repressor to release DNA induces transcription RNA polymerase repressor TATA lac gene1 gene2 gene3 gene4 DNA promoter operator 1 2 3 4 mRNA enzyme1 enzyme2 enzyme3 enzyme4 repressor repressor protein lactose lac conformational change in repressor protein makes it INACTIVE! repressor lactose – repressor protein complex lac

Lactose operon What happens when lactose is present? Slide from Kim Foglia http://explorebiology.com Lactose operon What happens when lactose is present? Need to make lactose-digesting enzymes Lactose is allosteric regulator of repressor protein Slide from Kim Foglia

promoter operator Slide from Kim Foglia http://explorebiology.com RNA polymerase TATA gene1 gene2 gene3 gene4 DNA promoter operator

ARABINOSE OPERON REGULATION Slide from Kim Foglia http://explorebiology.com ARABINOSE OPERON REGULATION = INDUCIBLE OPERON PRESENCE OF ARABINOSE TURNS ON GENES WHICH MAKE ENZYMESTO DIGEST ARABINOSE (along with pGLO gene) Adding ARABINOSE to media makes bacteria GLOW