Pre-Lab: pGLO Bacterial Transformation AP Biology Ms. Day

Bacterial Transformation Step 1 DNA Isolation Isolation of the “Gene of Interest” (foreign DNA) Step 2 Recombinant DNA Insertion of foreign DNA into bacterial plasmid using restriction enzymes and DNA ligase http://www.dnalc.org/resources/animations/transformation1.html Step 3 Transformation Insertion of recombinant DNA into bacteria by making bacteria competent (weaken) Use CaCl2 and heat shock techniques

How do you make Bacteria competent? Step 1: Add Calcium Chloride (CaCl2) CaCl2 is in a solution (creates Ca+2 and Cl- ions) DNA in plasmid is negatively charged due to phosphate groups in the backbone Cell membrane of E. coli also is negatively charged because phospholipids are made of same phosphate groups (PO4-3) Ca+2 ions neutralize charges so plasmid can get near (and inside) bacterial cell.

How do you make Bacteria competent? Step 1: Use Heat Shock Heat Shock is a process that uses warm water (bath) and ice to help get plasmid inside cell Add recombinant plasmid + host cell + CaCl2 solution to ice then heat then back on ice Heat = increases kinetic energy of matter Molecules/atoms move faster Ice = decreases kinetic energy of matter Molecules/atoms move slower http://www.dnalc.org/resources/animations/transformation2.html

2 types of Bacterial Growth in this lab Colony growth Lawn growth

GFP

Step 1: DNA Isolation  digesting the ”gene of interest” with restriction enzyme In the lab, this has been done for you! Gene of Interest = GFP

After Isolating GTP from a jellyfish … Step 2: Make Recombinant DNA Amp

RECALL WHAT THE PLASMID (pGLO) LOOKS LIKE… Step 3 Transformation RECALL WHAT THE PLASMID (pGLO) LOOKS LIKE… CaCl2

Step 3 Transformation Bacteria is ALSO “Ampicillin Resistance” pGLO GFP protein Bacteria is ALSO “Ampicillin Resistance” Amp resistance pGLO

Transformed Bacteria! When will this happen?

What is the ARA operon? -Clusters of genes located together and transcribed from ONE promoter. -Ara operon = Inducible operon -3 arabinose structural genes present natural (not recombined) plasmid: araB, araA, araD -All 3 genes dependent on promoter (pBAD) -Arabinose (sugar) changes the shape of the promoter (INACTIVATES REPRESSOR) INDUCES transcription by allowing RNA polymerase to bind to the DNA

= ARABINOSE (needed to make room for RNA polymerase) Visualize the Operon Promoter called Pbad araB araD araA Repressor = ARABINOSE (needed to make room for RNA polymerase) Repressor Pbad araA araB RNA Polymerase araD

Visualize the pGLO recombinant DNA What was changed? Pbad araB araD araA RNA Polymerase Repressor Pbad GFP gene RNA Polymerase

Some of your Materials

Lab Procedure-Brief Overview Label micro test tubes (+pGLO and –pGLO) Transfer 250 μL (0.25 mL) of transformation solution (CaCl2) to each tube  place on ice Transformation Solution (CaCl2)

Lab Procedure-Brief Overview 3. Use sterile inoculating loop to transfer ~2 “fat” colonies of bacteria to +pGLO tube  spin loop to remove bacteria from loop to CaCl2 solution 4. Use a DIFFERENT sterile inoculating loop to transfer ~2 “fat” colonies of bacteria to -pGLO tube DO NOT GET TOO MUCH BACTERIA NO chunks!

Lab Procedure-Brief Overview 5. You will need pGLO plasmid… Your TEACHER will micropipette 10 μL of plasmid into your +pGLO tube Mix plasmid into the cell suspension of the by tapping the CLOSED microtube on your desk! Return the tube it to on ice. DO NOT add plasmid DNA to the –pGLO tube.

Lab Procedure-Brief Overview

Lab Procedure-Brief Overview 6. Incubate both +pGLO and –pGLO tubes on ice for 10 minutes -pGLO +pGLO 10:00

Lab Procedure-Brief Overview While the tubes are sitting on ice… 7. Label your 4 LB Nutrient agar plates on the bottom (not the lid) as follows:        Label one LB/amp plate: + pGLO       Label the LB/amp/ara plate: + pGLO        Label the other LB/amp plate: - pGLO      Label the LB plate: -pGLO

Lab Procedure-Brief Overview TIME TO HEAT SHOCK… 8. Use foam rack as a holder, transfer both the +pGLO and -pGLO tubes into the water bath, set at 42°C, for exactly 50 seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water. When the 50 seconds are done, RAPIDLY place both tubes back on ice. Incubate tubes on ice for 2 minutes

Lab Procedure-Brief Overview 9. Remove the rack with tubes from ice and place on lab bench. Open a tube and, using a new sterile pipet, add 250 µl of LB nutrient broth to EACH tube and reclose it. Use a new sterile pipet for the other tube. Incubate tubes for 10 minutes at room temperature. LB = FOOD

Lab Procedure-Brief Overview 10. After 10 min have passed, tap the closed tubes with your finger to mix. Using a new sterile pipet for each tube, pipet 100 µl  of liquid onto the appropriate LB agar plates

Lab Procedure-Brief Overview 11. Use a new sterile loop for each plate. Spread liquid evenly around surface of LB agar using streaking method. DO NOT PRESS TOO DEEP INTO THE AGAR.

Streaking Plates with bacteria

Put all plates in the 37°C incubator upside down for 24 hours. Lab Procedure 12. Stack up your plates and tape them together. Put your group name and class period on the tape and place the stack of plates upside down Put all plates in the 37°C incubator upside down for 24 hours.

Petri Dish Label LB/amp LB/amp/ara LB What do this dish have on it? Hypothesis: Will the bacteria grow on the dish? Y or N Hypothesis: Will the bacteria GLOW green on the dish? Y or N +pGLO LB/amp LB/amp/ara -pGLO LB

Petri Dish Label LB/amp LB/amp/ara LB What do this dish have on it? Hypothesis: Will the bacteria grow on the dish? Y or N Hypothesis: Will the bacteria GLOW green on the dish? Y or N +pGLO LB/amp Plasmid (with pGLO & AMPR), Luria Broth (Agar), ampicillin YES-colony growth NO LB/amp/ara Plasmid, Luria Broth, ampicillin, arabonose Yes- Colony growth Yes -pGLO No plasmid, Luria Broth, ampicillin No LB No plasmid, Luria broth Yes- Lawn growth NEGATIVE CONTROL POSITIVE CONTROL

Results + control - control