1. Bacterial Transformation with ( pGLO Plasmid) Lab #9: Molecular Biology

2. Purpose of this Lab Learn how to insert a gene into bacteria (Heat Shock) Analyze how a gene can transform an organism and express that gene Provide evidence that bacteria can take in foreign DNA in the form of a plasmid Reinforce the following process: DNA  RNA  Protein  Trait Observe how genes are regulated

3. Applications of Genetic Transformation Used in many areas of Biotechnology –Agriculture (pests, frost, & drought) –Bacteria (oil spills) –Gene therapy (sick cells into healthy cells) –Medicine (produce insulin & hormones)

4. Key Terms to Know DNA: Plasmid Bacteria: E. coli (strain: HB101K-12) Growth media: LB Broth (Luria & Bertani) Ampicillin:Antibiotic kills bacteria “amp” Arabinose:Sugar source for energy & carbon Heat shockProcess that increases permeability of the cell membrane to DNA GFP:Green Fluorescent Protein (w/UV)

5. The Genes of Interest Ampicillin resistance Gene regulation proteins-activate the GFP gene when arabinose is present GFP: Green Fluorescent Protein -originally isolated from the jellyfish: Aequorea victoria

6. Supplies for each Group of 4-5 (1) E. Coli-starter plate: has colonies present “LB” S (4) Agar plates: 1-LB 2-LB/amp1-LB/amp/ara (1)Styrofoam holder tray w/ four vials (5) Pipets (7) Yellow inoculation loops: 1 package (1)Cup of crushed ice & water (2)Sharpie marking pen Masking tape

7. Check your Materials-See List Take out what you’ll need ***(leave packages sealed) Label your capped vials as follows: –Yellow: “ - pGlo” –Orange:“ + pGlo” –Green:“TS”- Transformation Soln. –Pink:“LB broth”

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

9. Basic Process - Begin with “Starter” colonies of E. coli -Place a colony into each of the two tubes provided. labeled: -pGlo and +pGlo (yellow loop) -Add the pGlo plasmid to the +pGlo tube only (yellow loop) -Place tubes on ice (10 min) bottom of tubes should be exposed to the ice -Label the four plates as indicated in your guide -Heat Shock the tubes in water bath (50 sec.) -Return to the ice (2 min) -Add 250  L of LB broth to both tubes (sterile pipette each time) -Add 100  L of tube contents to the appropriate plates -Spread the samples (w/yellow loop) & tape all four plates together. Be sure your period and group name is clearly indicated.

10. Adding Transformation Solution

11. Transferring Colonies, Labeling the Plates, & Using Heat Shock

12. The Process of Heat Shock Helps to increase the bacterial uptake of foreign DNA Membrane becomes more permeable to DNA Time is essential: -ice  water bath (42ºC) for 50 sec.  ice The number of transformants should increase

13. Adding the Bacteria to the labeled Plates

14. Expected Results PLATESOBSERVATIONS +pGlo LB/amp Many colonies with white appearance Transformation observed (resistance to amp) NO fluorescence (No arabinose present) +pGlo LB/amp/ara Many transformed white colonies Fluoresce bright green under UV light -pGlo LB/amp (CONTROL) No Bacterial growth present on the plate No transformation -pGlo LB only (CONTROL Bacteria present with whitish colonies (regeneration of the starter plate)