Plasmid showing the operon for ampicilin resistance (ori) and the gene for ampicillin resistance (amp R )

Transcription

1 AP Biology Name AP Lab 8: Biotechnology (Bacterial Transformation) The bacterium Escherichia coli (E. coli) is a common inhabitant of the human colon and can be easily grown in inexpensive suspension culture. E. coli contains only around 5 million DNA base pairs (1/600 th the size of the human genome) located within a plasmid and can be easily manipulated in a lab. For these reasons, and others, E. coli is often used for molecular biology labs and labs testing the translation of enzymes from sequences of DNA. Bacteria transfer genes amongst themselves in three different ways. Conjugation, which is when genetic material is transferred from one bacterium to another of a different mating type; Transduction, which is when a virus acts as a vector (carrier) to transfer small pieces of DNA from one bacterium to another; and transformation, which involves a cell directly uptaking DNA from the environment into its own genome. Although effective, transformation only occurs at the end of logarithmic growth of a single parental strain. In a lab setting, restriction enzymes are used to insert and remove specific sections of DNA from plasmids. In this lab, you will use the concept of transformation to insert genes for antibiotic resistance and UV glowing into E. coli cells and observe whether translation occurs for these genes. Plasmid showing the operon for ampicilin resistance (ori) and the gene for ampicillin resistance (amp R )

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3 Procedure In this lab, you will transform a gene for resistance to an antibiotic called ampicillin and another gene called pglo, which creates a protein that glows under UV light. Ampicillin is lethal to most strains of E. coli, so the gene should allow E. Coli to grow even in the presence of ampicillin. You will then observe the growth of E. coli in the presence of ampicillin and UV lights to see if the transformation was successful. DAY 1 1. Obtain one micro test tube labeled + for pglo and another - for no pglo. Place them in the foam tube rack. 2. Open the tubes and using a sterile transfer pipet, transfer 250 µl of ice cold 0.05 M transformation solution (CaCl 2 ). The CaCl 2 opens the plasma membrane of the E. coli. 3. Place the tubes on ice. 4. Use a sterile loop to pick up a single colony of bacteria from your starter plate. Pick up the +pglo tube and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Place the tube back in the tube rack in the ice. 5. Using a new sterile loop, repeat step 4 for the pglo tube. Try to get a colony of approximate similar size to each tube. 6. Examine the pglo plasmid DNA solution with the UV lamp. Note your observations. Immerse a new sterile loop into the plasmid DNA stock tube. Withdraw a loopful. There should be a film of plasmid solution across the ring. This is similar to seeing a soapy film across a ring for blowing soap bubbles. Mix the loopful into the cell suspension of the +pglo tube. 7. Close the tube and return it to the rack on ice. Also close the pglo tube. Do not add plasmid DNA to the pglo tube. 8. Incubate the tubes on ice for 10 minutes. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the ice. 9. While the tubes are on ice, label your four agar plates on the bottom (not the lid) as follows a. LB/amp + pglo b. LB/amp/ara + pglo c. LB/amp pglo d. LB pglo i. *amp stands for Ampicillin. These plates have agar that has been laced with ampicillin. ii. **ara stands for Arabinose. Arabinose is a sugar that serves as a promoter for the pglo operon. 10. The heat shock step. Using the 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 bottom of the tubes stick out and make contact with the warm water. When the 50 seconds are done, place both tubes back on ice. For the best transformation results the change form the ice to 42 C and then back to the ice must be rapid.

4 a. Heat-shocking is essential because it will increase the permeability of the cell membrane, allowing the plasmid to enter the E. coli cell by creating a vacuum. 11. Incubate tubes on ice for 2 minutes. This will give the cells enough time to read the new ampicillin-resistance and pglo genes (if they have it) and translate it into a protein to hydrolyze ampicillin and build GFP, the gene from the pglo. 12. Remove the rack from the ice and place on the table. Open a tube and, using a new sterile pipet, add 250 µl of LB nutrient broth to the +pglo tube and close it. Mix by tapping with your finger. Repeat with a new sterile pipet for the pglo tube. Incubate at room temperature for 10 minutes. a. These 10 minutes are essential because it gives the E. coli cells time to transcribe and translate the ampicillin-hydrolyzing proteins and the pglo operon proteins. 13. Using a new sterile pipet for each tube, pipet 100 µl of the suspensions into their appropriate plates according to your labels. 14. Immediately spread the cells evenly by using a new sterile spreading rod for each plate. 15. Allow plates to set for several minutes. Then, stack your plates bottom-side up and tape them together. Put your group name and class period on the side facing up and place the stack in the 37 incubator. 16. Fill in the first column in Table 8.1. DAY When time to observe the cells, pull out of the incubator but DO NOT OPEN THE PLATES. Count the number of individual colonies and record in Table 8.1. Use a marker to mark each colony as it is counted. If the cell growth is too dense to count individual colonies, record lawn. 18. Complete the transformation efficiency analysis in Table 8.2. Post-Lab Questions 1. Indicate how each plate actually appeared compared to your prediction. 2. Which combination of plates best shows the transformation efficiency? Explain your reasoning. 3. Name two factors that influenced transformation efficiency? Explain the effect of each. 4. What are restriction enzymes, and how do they work? 5. What is the function of restriction enzymes in nature? Lab Book Requirements 1. Date of Lab 2. Title of Lab 3. Objective of Lab 4. Manipulated and Responding variables 5. Three Control Variables 6. Hypothesis 7. Table Table Conclusion 10. What is the next experiment you could perform? 11. Answers to Post-Lab Questions

5 Table 8.1: Colony Count. Plate Prediction: # of Colonies and UV Glowing? Number of Colonies Glowing under UV Light? LB/amp +pglo LB/amp/ara +pglo LB/amp pglo LB pglo Table 8.2: Transformation Efficiency. Transformation efficiency is expressed as the number of antibiotic-resistant colonies per microgram of pamp. Because transformation is limited to only those cells that are competent, increasing the amount of plasmid used does not necessarily increase the probability that a cell will be transformed. A sample of competent cells is usually saturated with small amounts of plasmid and excess DNA may actually interfere with the transformation process. a. Determine the total mass of pglo used by multiplying 10 µl of pglo at a concentration of µg/µl. Total mass = volume x concentration b. Review your procedure and calculate the total volume of cell suspension prepared c. Calculate the fraction of the total cell suspension that was spread on the plate by dividing the volume pipetted onto the plate by total volume (B). d. Determine the mass of pglo in cell suspension by multiplying the total mass of pglo (A) and the fraction of suspension spread (C). e. Determine the number of colonies per mg of plasmid by dividing the number of colonies observed by the mass of the pglo spread (D). This is your transformation efficiency.