Mrs. Stewart Medical Interventions Central Magnet School Transformation Lab Mrs. Stewart Medical Interventions Central Magnet School

Purpose We will demonstrate the ability to transform bacterial cells with free-floating DNA using a gene that codes for Green Fluorescent Protein (GFP).

Transformation The genetic modification of a bacterium by the incorporation of free-floating DNA from the surrounding environment

GFP – Green Fluorescent Protein Gene found in bioluminescent jellyfish Aequorea victoria Gene causes jellyfish to fluoresce and glow in the dark If transformation is successful, bacteria will produce this protein and fluoresce under UV light

pGlo Plasmid DNA Ori = origin of replication araC = arabinose detection gene that regulates the production of GFP Bla = beta – lactamase enzyme gene (antibiotic resistance to beta-lactam abx) GFP = green fluorescent protein gene inserted into plasmid using restriction enzymes

Introduction to pGlo Gene Regulation Gene expression in all organisms is carefully regulated to allow for adaptation to differing conditions and to prevent wasteful overproduction of unneeded proteins. The genes involved in the breakdown of different food sources are good examples of highly regulated genes. For example, the simple sugar arabinose is both a source of energy and a source of carbon for bacteria. The bacterial genes that make digestive enzymes to break down arabinose for food are not expressed when arabinose is not in the environment. But when arabinose is present, these genes are turned on. When the arabinose runs out, the genes are turned off again. Arabinose initiates transcription of these genes by promoting the binding of RNA polymerase. In the genetically engineered pGLO plasmid DNA, some of the genes involved in the breakdown of arabinose have been replaced by the jellyfish gene that codes for GFP. When bacteria that have been transformed with pGLO plasmid DNA are grown in the presence of arabinose, the GFP gene is turned on and the bacteria glow brilliant green when exposed to UV light.

Day One – Materials Student Stations Common Materials LB agar plate Alcohol burner Permanent marker Inoculation loop Common Materials E. coli Transformation solution

Day One – Streak a Starter Plate The purpose of this starter plate is to have SINGLE COLONIES Streak for colonies

Day Two – Materials Student Stations Group E. coli starter plate 2 – 1.5 ml micro test tubes + tube rack 4 - agar plates – LB, 2 LB/amp, LB/amp/ara Transformation solution LB broth Inoculation loops Micropipet (.5-10 µl) + tips 3 - Sterile disposable pipets Permanent marker

Step 1: Labeling Label the 2 micro test tubes “+ pGlo” on one tube Add group name to both Place tubes in tube rack

Step 2 Transfer 250 µl of transformation solution (CaCl2)into each tube* Place tubes on ice to maintain sterility *Purpose: Both DNA and a bacterial cell wall have a negative charge. CaCl2 will help neutralize those opposing charges to increase change of DNA uptake

Step 3 Use a sterile loop to pick up 3 or 4 LARGE colonies with a uniformly circular shape and smooth edges. Do not use a swab of bacteria from the dense growth – we need actively growing bacteria for optimum transformation Transfer loop w/ bacteria into +pGlo tube and spin until colony is dispersed. Repeat for –pGlo tube

Step 4 Examine the pGlo DNA solution with the UV light Record observations Transfer 10 µl pGlo DNA solution into +pGlo tube DO NOT add pGlo plasmid DNA to –pGlo tube Incubate on ice for 10 minutes 10 minutes

Step 5 Label the 4 agar plates as follows: Add group name to all 4 of them Label 2 plates +pGlo - LB/amp and LB/amp/ara Label 2 plates –pGlo – LB/amp and LB

Step 6 - HEAT SHOCK Transfer both tubes from ice to heat block (42oC) for exactly 50 seconds Immediately transfer back to ice after the 50 seconds has ended Incubate on ice for 2 minutes

Step 7 Remove tubes from ice Add 250 µl of LB nutrient broth to each tube using a sterile pipet Incubate at room temp for 10 minutes

Step 8 Gently flick/tap tubes with your finger to mix and resuspend bacteria Pipet 100 µl of transformation of each tube onto the corresponding plates

Step 9 Use a new sterile loop for each plate Spread the suspensions evenly around the plate surface DO NOT puncture the agar by pressing too deep

Step 10 Stack plates and tape together Incubate 3rd period – countertop 2nd period – incubator Store them upside down!