pGLO Transformation LAB AP LAB 6 BIO-RAD lab book pGLO ori bla GFP araC

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

Jellyfish Gene put into Other Critters

PLASMID Extrachromosomal DNA Often carry genes for antibiotic resistance Can be passed from one bacterium to another

Bacterial Transformation Plasmids Chromosomal DNA Bacterial Cell The uptake of DNA

pGLO plasmid bla (beta-lactamase) - On all time - Makes protein that breaks down ampicillin - Provides ampicillin resistance GFP-Green Fluorescent Protein - Glows green in fluorescent light ARABINOSE OPERON (INDUCIBLE) Turns on when arabinose sugar is present Allows bacteria to digest this sugar pGLO ori bla GFP araC Ori- Plasmid Replication genes

Gene Regulation RNA Polymerase araC ara GFP Operon GFP Gene araC GFP Gene araC GFP Gene Effector (Arabinose) BAD araC BAD RNA Polymerase Effector (Arabinose) araC BAD ara Operon -GFP gene has been added to operon -When arabinose is present, operon is turned and GFP gene is expressed -Cells “glow” on media with arabinose

Bacterial Transformation Beta lactamase (ampicillin resistance) pGLO plasmids Bacterial chromosomal DNA Cell wall GFP

What is Nutrient Broth? Luria-Bertani (LB) broth (helps for E. coli specificity) Medium that contains nutrients for bacterial growth and gene expression –Carbohydrates –Amino acids –Nucleotides –Salts –Vitamins

Types of agar = LB nutrient agar grows the E. coli with or without the plasmid = LB nutrient agar + ampicillin grows E. coli with the plasmid only = LB nutrient agar + ampicillin + arabinose grows E. coli with the plasmid and turns on the GFP gene Amp = ampicillin = antibiotic Ara = arabinose = sugar

Using the Pipet Properly Find the markings for each graduated volume on the pipet

Adding Transformation Solution

Reasons for Performing Each Transformation Step? 1.Transformation solution = CaCI 2 Positive charge of Ca ++ ions shields negative charge of DNA phosphates Ca ++ O CH 2 O PO O O Base CH 2 O P O O O Base OH Sugar O Ca ++

Transferring Colonies, Labeling the Plates, & Using Heat Shock

Why Perform Each Transformation Step? 2. Incubate on ice slows fluid cell membrane 3. Heat-shock Increases permeability of membranes 4. Nutrient broth incubation Allows beta-lactamase expression Beta-lactamase (ampicillin resistance) Cell wall GFP

Adding the Bacteria to the labeled Plates

LABEL TUBES Purple = + pGLO pink = - pGLO

Use sterile pipette to add 250µL transformation solution to pGLO + and – tubes Transformation solution (CaCl 2 )

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 USE SPECIAL GARBAGE BAG FOR DISPOSAL OF USED LOOPS

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 USED AS A GENETIC TOOL

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!

WHILE YOUR TUBES COOL LABEL YOUR PLATES FLIP UPSIDE DOWN AND WRITE LABELS ON BOTTOM … NOT ON TOP!

LB (Luria and Bertani) – broth & agar provides nutrients for bacterial growth LB/amp Luria agar + ampicillin (antibiotic) LB/amp/ara Luria agar + ampicillin + arabinose sugar

SHOCKING INCREASES UPTAKE OF FOREIGN DNA (PLASMID) OSMOTIC SHOCK =Transforming solution –CaCl 2 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 Luria broth to + tube Use new sterile pipette to add 250 µL Luria broth to – tube Incubate a ROOM TEMPERATURE 10 min

TAP 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 DO NOT DIG INTO AGAR! QUICKLY REPLACE LIDS

FLIP PLATES UPSIDE DOWN STACK AND TAPE LABEL WITH YOUR GROUP NAME PLACE IN INCUBATOR

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 →

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 →