Transcript pGLO
Bacterial Transformation AP Biology What does it mean to transform a cell? To insert a foreign piece of DNA into a cell How would a living organism be transformed? Using a cloning vector/ plasmid Don’t Forget… DNA RNA Protein Trait Bacterial Transformation • Step 1 DNA Isolation – Isolation of Your Gene of Interest (GFP) • 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 • Use CaCl2 and heat shock techniques • http://www.dnalc.org/resources/animations/transformation2.html Step 1: DNA Isolation digesting the ”gene of interest” with restriction enzyme •In the lab, this has been done for you! •How did they do it? After Isolating GFP from a jellyfish … Step 2: Make Recombinant DNA Amp Need to use Restriction Enzymes to Make Recombinant DNA • Act as molecular scissors • Naturally found in bacteria – Used to decompose viral & phage DNA • Act as ENDOnucleases (cut WITHIN the DNA strand) • ~3,000 known enzymes Step 3 Transformation RECALL WHAT THE PLASMID (pGLO) LOOKS LIKE… CaCl2 Step 3 Transformation GFP protein Amp resistance Bacteria is ALSO pGLO “Ampicillin Resistance” Transformed Bacteria! When will this happen? What is an operon? -Clusters of genes located together and transcribed from ONE promoter usually found only in bacteria -3 arabinose genes are present in a natural (not recombined) plasmid: araB, araA, araD -All 3 genes dependent on 1 promoter (called pBAD) -Interaction with arabinose (sugar) changes the shape of the promoter & enables RNA polymerase to bind to the DNA coding strand for transcription Visualize the Operon Promoter called Pbad Repressor araB Pbad Polymerase araD = ARABINOSE (a sugar needed to make room for RNA polymerase) Repressor RNA araA araB araA araD Visualize the pGLO recombinant DNA What was changed? Pbad RNA Polymerase araB ara A araD Also on the pGLO plasmid but NOT in place of the ara operon! Repressor Pbad RNA Polymerase GFP gene Pamp amp gene pGLO plasmid contains: 1)ampR gene 2)GFP gene 3)Arabinose operon and promoter (pBAD) operon promoter Materials Lab Procedure-Brief Overview 1. Label micro test tubes (+pGLO and –pGLO) 2. 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 NO chunks! Lab Procedure-Brief Overview LAB PRCEDURE REVISION 5. I will have some pGLO plasmid in a labeled micro test tube… • You (a lab group member) will come to the front of the room and retrieve your 9 μL of pGLO plasmid • I will add plasmid using a micropipette • Cap the +pGLO microtest tube and mix the plasmid into the cell suspension by inverting tube. • Return this test tube to the ice. • DO NOT add plasmid DNA to the –pGLO tube. You are NOT doing this method!!! Lab Procedure-Brief Overview Lab Procedure-Brief Overview -pGLO 10:00 +pGLO 6. Incubate both +pGLO and –pGLO tubes on ice for 10 minutes 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. Lab Procedure-Brief Overview 9. Remove the rack with tubes from ice and place on lab bench. Open a tube and use a new sterile pipet, add 250 µl of LB broth to EACH tube and reclose it. • Use a new sterile pipet for the other tube. Incubate the tubes for 10 minutes at room temperature. 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 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 in Ms. Gaynor’s transfer box. She will put all plates in the 37°C incubator upside down for 24 hours. Reasons for Performing Transformation Step 1. Transformation solution = CaCI2 -Positive charge of Ca++ ions shields negative charge of DNA phosphates & helps neutralize cell membrane so plasmid can get in 2. Incubate on ice -Slows movement of cell membrane so Ca++ can bind & plasmid can slip into bacterial cell 3. Heat-shock -Increases movement of membranes (heat) - Then closes up holes in membranes 4. Nutrient broth incubation -Allows bacteria to be feed Review… What does the following mean? • +pGLO • a cell contains the pGLO plasmid (transformed cell) • pGLO plasmid contains 2 genes: AMP and GTP • -pGLO •a cell without the plasmid (“normal” cell) •LB •Luria Broth (LB or Agar) sugar needed for E.Coli to live (feeds on this sugar solution) •amp • Ampicillin an liquid antibiotic added to the LB •Normally KILLS bacteria by breaking down cell wall peptidoglycan •ara •Arabinose sugar needed to turn on operon containing the GFP gene; needed to make glow protein What do this Hypothesis: Will Hypothesis: Will Petri Dish dish have on it? the bacteria the bacteria Label +pGLO LB/amp +pGLO LB/amp/ara -pGLO LB/amp -pGLO LB grow on the dish? Y or N GLOW green on the dish? Y or N What do this Hypothesis: Will Hypothesis: Will Petri Dish dish have on it? the bacteria the bacteria Label **they all have E. coli +pGLO LB/amp +pGLO LB/amp/ara -pGLO LB/amp -pGLO LB grow on the dish? Y or N GLOW green on the dish? Y or N pGLO & AMPR), Luria Broth (Agar), ampicillin YES- colony growth NO Plasmid, Luria Broth, ampicillin, arabonose YesColony growth Yes No plasmid, Luria Broth, ampicillin No No Plasmid (with No plasmid, Luria broth NEGATIVE CONTROL Yes- Lawn No growth POSITIVE CONTROL Results + control - control