1 Bacterial Transformation Using Fluorescent Protein Teacher Guide sciencebridge
2 PROTOCOL 2 Bacterial Transformation using Fluorescent Protein Central question How does a change in the genotype of an organism affect its phenotype? Overview of experiment In this lab, you will insert a gene that codes for a fluorescent protein into bacteria, changing the genotype. After the bacteria reproduce, transcribe, and translate the gene, you will observe a change in the phenotype (appearance) of the bacteria. How will the addition of a gene for a fluorescent protein affect the phenotype of the bacteria? Hypothesis 3 Student pre-lab questions 1. What is a bacterial colony? How do the genotypes of individual bacteria in the same colony compare to each other? 2. What is a gene? What processes occur to make a protein from a gene? 3. What is a plasmid? 4. What is transformation? 5. What do the + and - on the microtubes indicate about the contents of each tube in the transformation procedure?
3 4 BACTERIAL TRANSFORMATION USING FLUORESCENT PROTEIN 6. What genes are present on the plasmid in this lab, and what is the function of each protein product? 7. What does the red line on two of the plates indicate? 8. What is the purpose of ampicillin (antibiotic) in the transformation procedure? Based on your answers to the questions above, predict the results of your transformation procedure in the table below. Plate # #1 LB/Amp (+)(red line) #2 LB/Amp(-)(red line) #3 LB/No Amp(-)(no red line) Plasmid present? YES NO YES NO YES NO Ampicillin present? YES NO YES NO YES NO Expected Plate Results (Drawing) Expected Plate Results (Description)
4 Bacterial Transformation Group # Assign group roles: Materials: Gathers and organizes group materials, and assists with disposal. Reader: Reads the protocol aloud so group members can follow the steps. Timer: Keeps track of timing for all timed steps. This is especially important for the heat shock! Technician: Carries out the actual steps of the protocol. PROTOCOL 2 5 Role in Group Materials Reader Timer Technician Student Name Materials checklist (1) ScienceBridge Transformation Protocol (1) Bacterial pre-streak plate with bacterial colonies (1) Agar plate containing LB/No Amp (no red line) (2) Agar plates containing LB/Amp (red line) (1) Sterile inoculating loop (4) Plastic transfer pipettes (2) Clear 2.0 ml microtubes (2) Cotton swabs (1) Sharpie Lab procedure NOTE: When labeling plates, write on the BOTTOM of the plate (not the lid) and keep writing small and close to the edges! 1. Label plates all three plates with the plate number, date, class period and your group number or initials as listed below: Plate # Label 1 LB/Amp (+) (red line) 2 LB/Amp ( ) (red line) 3 LB/No Amp ( ) (no red line) (1) Piece of tape for sealing plates after inoculation (1) Waste container (1) Styrofoam cup with ice ON ICE (1) Blue 1.5 ml tube of CaCl 2 (1) Clear 2.0 ml tube of plasmid DNA labeled either PM1 or PM2 Shared materials for class Hot water bath at 42 º C LB/Amp (+) #1 Name/Period LB/No Amp (-) #3 Name/Period LB/Amp (-) Name/Period #2
5 6 BACTERIAL TRANSFORMATION USING FLUORESCENT PROTEIN 2. Close the caps on two microtubes and label each cap with a sharpie: one tube with a +, other tube with a Using a plastic transfer pipette transfer 0.5 ml of CaCl2 to both tubes, close and place them on ice for at least 2 minutes. Discard the pipette in the waste container. CaCl ml Using a sterile loop, gently collect ONE colony ( dot ) of bacteria from the top of your bacterial pre-streak plate. Transfer the collected colony to one tube of CaCl 2. Swirl and twist the loop to make sure all the bacteria mix with the CaCl 2 solution. Mix the contents by inverting the tube or flicking the bottom of the tube. The solution should look cloudy with no chunks. 5. Transfer ONE additional colony to the second tube and repeat instruction #4. Place the tubes back on ice. 6. Using a clean plastic pipette, add all of the plasmid mix solution (labeled either PM1 or PM2 ) into the positive (+) tube with CaCl 2 BE SURE THAT THE PLASMID IS ONLY TRANSFERRED TO THE + TUBE. Mix. Discard the used pipette. DNA (plasmid) + 7. Incubate both tubes on ice for 10 minutes. Make sure the tubes are immersed in the ice. 10 minutes
6 PROTOCOL 2 8. The timer and one other group member will 9. Check the water bath temperature to ensure it is at 42ºC. 42o Hold the tubes in the hot water for exactly 45 seconds. Make sure that the tubes are in contact with the hot water. Immediately return the tubes to the ice for 2 minutes. 2 minutes 45 seconds Plate #3 Invert your tubes gently to mix. Using a new pipette, transfer 0.25 ml of the cell mixture from the (-) negative tube to the LB/No Amp (-) agar plate. Spread the mixture around the plate gently with a clean cotton swab. LB/No Amp (-) 0.25 ml - Plate #2 10. With the same pipette, transfer 0.25 ml of the cell mixture from the (-) negative tube to the LB/Amp (-) agar plate. Spread the mixture around the plate gently with the same cotton swab. Make sure to completely finish the two - (negative) plates before spreading the cells on the + (positive) plate. LB/Amp (-) 0.25 ml - Plate #1 LB/Amp (+) 11. With a new clean pipette, transfer 0.25 ml of the cell mixture from the (+) positive tube to the LB/Amp (+) plate. Spread the mixture around the plate gently with a clean cotton swab ml 37o 12. Stack your plates, tape them together, put plates UPSIDE DOWN to grow overnight in 37ºC incubator. Amp (-) LB/No #3 Name/Perio d 7
7 8 BACTERIAL TRANSFORMATION USING FLUORESCENT PROTEIN Post-Lab Questions Now that you have performed a transformation, fill out the table below to describe your results. Plate # #1 LB/Amp (+)(red line) #2 LB/Amp(-)(red line) #3 LB/No Amp(-)(no red line) Plasmid present? YES NO YES NO YES NO Ampicillin present? YES NO YES NO YES NO Actual Plate Results (Drawing) Actual Plate Results (Description) 1. Estimate the number of fluorescent colonies that grew on your experimental plates. 2. Which plasmid mix did you have (PM1 or PM2) and which three colors of fluorescent colors of bacteria did you observe? (Yes, three colors are present! Look again if you did not see them all!) 3. What made it possible for the colonies to be different colors?
8 PROTOCOL Describe how the plasmid was able to enter the cell. (Hint: There are two processes in the protocol that were designed to help the plasmid get into the cell. What are they?) 5. There are two controls in this experiment: the LB/Amp (-) plate and the LB/No Amp (-) plate. What are they testing? Assuming the lab procedure was performed correctly, what would it mean if bacteria grew on the LB/Amp (-) plate? What would it mean if no bacteria grew on the LB/No Amp (-) plate? 6. How can transformation be used in the medical industry or in research? Conclusion / summary (revisit hypothesis)
9 10 BACTERIAL TRANSFORMATION USING FLUORESCENT PROTEIN Transformation Protocol Flow Chart Positive tube (+) Negative Tube (-) 500 μl CaCl 2 ON ICE! 500 μl CaCl 2 ON ICE! + - One (1) Bacteria Colony Vortex or Shake to Mix One (1) Bacteria Colony Vortex or Shake to Mix FP gene 100 μl Plasmid (PM1 or PM2) No Plasmid Tap Tube Gently Amp R Ampicillin resistance gene ICE HEAT ICE ICE HEAT ICE 45 Second Heat Shock 45 Second Heat Shock 100 μl on LB/Amp (+) Plate 100 μl on LB/Amp (-) and LB Only (-) Plates LB/Amp (+) LB/Amp (-) LB Only (-)