Lesson 4 Extension Activity: Calculate Transformation Efficiency

Transcription

1 Lesson 4 Extension Activity: Calculate Transformation Efficiency Your next task in this investigation will be to learn how to detem~ne the extent to which you genetically transformed E. coli cells. This quantitative measurement is referred to as the transformation efficiency. In many experiments, it is important to genetically transform as many cells as possible. For example, m some types of gene therapy, cells are collected from the patient, transformed in the laboratory, and then put back into the patient. The more cells that are transformed to produce the needed protein, the more likely that the therapy will work. The transformation efficiency is calculated to help scientists determine how well the transformation is working. The Task You are about to calculate the transformation efficiency, which gives you an indication of how effective you were in getting DNA molecules into bacterial cells. Transformation efficiency is a number. It represents the total number of bacterial cells that express the green protein, divided by the amount of DNA used in the experiment. (It tells us the total number of bacterial cells transformed by one microgram of DNA.) The transformation efficiency is calculated using the following formula: Transformation efficiency = Total number of cells growing on the agar plate Amount of DNA spread on the agar plate (in ~ g ) Therefole, before you can calculate the efficiency of your transfoimation, you will need two pieces of information, (I) The total number of green fluorescent colonies growing on your LBlamplara plate. (2) The total amount of pglo plasmid DNA in the bacterial cells spread on the LBlamplara plate.

2 1. Determining the Total Number of Green Fluorescent Cells Place your LB/amp/ara plate near a W light. Each colony on the plate can be assumed to be derived 6-om a single cell. As individual cells reproduce, more and more cells are formed and develop into what is termed a colony. The most direct way to determine the total number of green fluorescent cells is to count the colonies on the plate. L Enter that number here =, Totzl number of cells = 2. Determining the Amount of pglo DNA in the Bacterial Cells Spread on the LBIamplara Plate Wy need two pieces of information to find out the amount of pglo DNA in the bacterial ceus spread on the LB/amp/ara plate in this experiment. (a) What was the total amount of DNA we began the experiment with, What fraction of the DNA (in the bacteria) actually got spread onto the LB/amp/ara plates. Once you calculate this data, you will need to multiply the atal amount of pglo DNA used in this experiment by the fraction of DNA you spread on the LB/amp/ara plate. The answer to this multiplication will tell you the amount of pglo DNA in the bacterial cells that were spread on the LB/amp/ara plate. a. Determining the Total Amount of pglo plasmid DNA The total amount of DNA we began with is equal to the product of the concentration and the total volume used, or (DNA in pg) = (concentration of DNA in ~g/pl) x (volume of DNA in pl) In this experiment you used 10 p1 of pglo at concentration of 0.08 pg/pl. This means that each microliter of solution contained 0.08 pg of pglo DNA. Calculate the total amount of DNA used in this experiment. Enter that number here 3 Total amount of pglo DNA (pg) used in this experiment = How will you use this piece of information?

3 I b. Determining the fraction of p a 0 plasmid DNA (in the bacteria) that actually got spread onto the LBlamplara plate: Since not au the DNA you added to the bacterial cells wil be transferred to the agar plate, you need to find out what ktion of the DNA was actually spread onto the LBIamplara plate. To do this, divide the volume ofdna you spad on the LBIarnplara plate by the total volume of hquid in the test tube containing the DNA. A formula for this statanent is Fraction of DNA used = v01uitle spread on LBIanlp plate (pl) Total sample volume in test tube (pl) You spread 100 p1 of cells contatning DNA from a test tube containing a total volime of 5 10 p1 of solution. Do you remember why there is 5 10 p1 total solution? Look in the laboratory procedure and locate all the steps where you added liquid to the reaction tube. Add the volumes Use the above formula to calculate the fraction of pglo plasmid DNA you spread on the LB/amp/ara plate. 1 Eraction of DNA = Enter that number here =. How will you use this piece of information? So, how many micrograms of pglo DNA did you spread on the LBIamplara plates? To answer this question, you will need to multiply the total amount of pglo DNA used in this experiment by the fraction of pglo DNA you spread on the LB/amplara plate. pglo DNA spread in pg = Total amount of DNA used r7j in pg x fiaction of DNA used pg-lo DNA spread (pg) = Enter that number here + What will this number tell you?

4 Look at all your calculations above. Decide which of the numbers you calculated belong in the table below. Fill in the following table. Number of colonies on LBIamplara plate = Micrograms of pglo DNA spread on the plates I Now use the data in the table to calculate the efficiency of the pglo transformation Transformation efficiency = Total number of cells growing on the agar plate Amount of DNA spread on the agar plate Enter that number here Transformation efficiency = - transformants/pg Analysis Transformation efficiency calculations result in very large numbers. Scientists often use a mathematical shorthand referred to as scientific notation. For example, if the calculated transformation efficiency is 1,000 bacterialpg of DNA, they often report this number as: lo3 transformantslpg (1 O3 is another way of saying 10 x 10 x 10 or 1,000) How would scientists report 10,000 transformantslpg in scientific notation? Carrying this idea a little farther, suppose scientists calculated an efficiency of 5,000 bacterialpg of DNA. This would be reported as: 5 x lo3 transformantslpg (5 times 1,000) How would scientists report 40,000 transformantslpg in scientific notation?

5 One final example: If 2,600 transformantslpg were calculated, then the scientific notation for this number would be: 2.6 x lo3 transformants/pg Similarly: (2.6 times 1,000) 5,600 = 5.6 x lo3 271,000 = 2.71 x lo5 2,420,000 = 2.42 x lo6 How would scientists report 960,000 transfomants/pg in scientific notation? Report your calculated transformation efficiency in scientific notation. Use a sentence or two to explain what your calculation of transformation efficiency means. Biotechnologists are in general agreement that the transformation protocol that you have just completed generally has atransfonnation efficiency ofbetween 8.0 x lo2 and 7.0 x lo3 transformants per microgram of DNA. How does your transforn~ation efficiency compare with the above? Team In the table be!ow, report the transformation efficiency of several of the teams in the class. 1 - Efficiency How ddcs your transformation efficiency compare with theirs?

6 Calculate the transformation efficiency of the following experiment using the information and the resul~ listed below. DNA plasmid concentration: 0.08 pglpl 250 pl CaCL, transformation solution 10 pl pglo plasmid solution 250 p1 LB broth 100 pl cells spread on agar 227 coloniesof transformants Fill in the following chart and show your calculations to your teacher: Number of colonies on LBIamplara plate = Micrograms cf DNA spread on the plates =.I- Transformation efficiency = Extra Credit Clallenge: If a particular experiment were known to have a transformation efficiency of 3 x lo3 bacterialpg of DNA, how many transformant colonies would be expected to grow on the LBlamplara plate? You can assume that the concentration of DNA and fraction of cells spread on the LB agas are the same as that of the pglo laboratory.

7 4. For which fragment sizes was your graph most accurate? For which fragment sizes was it least accurate? What does this tell you about the resolving ability of agarose-gel electrophoresis? Analysis 1. Discuss how each of the following factors would affect the results of electrophoresis: a. Voltage used b. Running time c. Amount of DNA used d. Reversal of polarity 2. Two small restriction fragments of nearly the same base pair size appear as a single band, even when the sample is run to the very end of the gel. What could be done to resolve the fragments? Why would it work? Questlons 1. What is a plasmid? How are plasmids used in genetic engineering?

8 2. What are restriction enzymes? How do they work? What are recognition sites? 3. What is the source of restriction enzymes? What is their function in nature? 4. Describe the function of electricity and the agarose gel in electrophoresis. 5. A certain restriction enzyme digest results in DNA fragments of the following sizes: 4,000 base pairs, 2,500 base pairs, 2,000 base pairs, 400 base pairs. Sketch the resulting separation by electrophoresis. Show starting point, positive and negative electrodes, and the resulting bands. 6. What are the functions of the loading dye in electrophoresis? How can DNA be prepared for visualization?

9 7. Use the graph you prepared from your lab data to predict how far (in cm) a fragment of 8,000 bp would migrate. 8. How can a mutation that alters a recognition site be detected by gel electrophoresis?

10 Lesson 2 Inoculation Growing Cell Cultures GFP Purification-Quick Guide 1. Remove the transformation plates from the incubator and examine using the UV light. Identify several green colonies that are not touching other colonies on the LB/amp/ara plate. Identify several white colonies on the LB/amp plate. 2. Obtain two culture tubes containing the growth media LB/amp/ara. Label one "+" and one "-". Using a sterile loop, lightly touch the loop to a green colony and immerse it in the "+" tube. Using a new sterile loop, repeat for a white colony and immerse it in the "-" tube (it is very important to pick only a single colony). Spin the loop between your index finger and thumb to disperse the entire colony. 3. Cap the tubes and place them in the shaking incubator or on the shaking platform and culture overnight at 32 OC or 2 days at room temperature. + or or Cap the tubes and shake vigorously by hand. Place in the incubator horizontally at 32 OC for hours. Remove and shake by hand periodically when possible. Incubate at 32 "C overnight 2 days at room temperature

11 Lesson 3 Purification Phase 1 Bacterial Concentration 1. Label one microtube "+" with your name and class period. Remove your liquid cultures from the shaker and observe with the UV light. Note any color differences between!he two cultures. Using a new pipette, transfer 2 ml of "+" liquid culture into the "+" rnicrotube. Spin the rnicrotube for 5 minutes in the cenhifuge at maximum speed. The pipette used in this step can be repeatedly rinsed in a beaker of water and used for all f~llowing ry period. steps of this laborato- + + I 2. Pour out the supernatant and observe the pellet under W light. 3. Using a rinsed pipette, add 250 yl of TE solution to the tube. Resuspend the pellet thoroughly by rapidly pipetting up and down several times.. 4. Using a rinsed pipette, add 1 drop of 1 drop lysozyme lysozyme to the resuspended bacterial pellet to initiate enzymatic digestion of the bacterial cell wall. Mix the contents gently by flicking the tube. Observe the tube under the W light. 5. Place the microtube in the freezer until the next laboratory period. The freezing causes the bacteria to rupture completely.

12 Lesson 4 Purification Phase 2 Bacterial Lysis 1. Remove the microtube from the freezer and thaw using hand warmth. Place the tube in the centrifuge and pellet the insoluble bacterial debris by spinning for 10 minutes at maximum speed. Thaw I I I Centrifuge 2. While your tube is spinning, prepare the chromatography column. Remove the cap and snap off the bottom from the prefilled HIC column. Allow all of the liquid buffer to drain from the column (-3-5 minutes) Prepare the column by adding 2 ml of Equilibration buffer (2 ml) Equilibration Buffer to the top of the column. This is done by adding two 1 ml aliquots with a rinsed pipette. Drain the buffer to the 1 ml mark on Lhe column. Cap the top and bottom and store the column at room temperature until the next laboratory period. 4. After the 10 minute spin, immediately remove your tube from the centrifuge. Examine the tube with the UV light. Using a new pipette, transfer 250 ~1 of the "+" supernatant into a new microtube labeled "+". Again, rinse the pipette well for the rest of the steps of this lab period. 5. Using a well rinsed pipette, transfer 250 pl of binding buffer to the "+" supernatant. Place the tube in the refngerator until the next laboratory period.

13 Lesson 5 Purification Phase 3 Protein Chromatography 1. Label 3 collection tubes 1-3 and place the tubes in the foam rack or in a rack supplied in your laboratory. Remove the caps from the top and bottom of the column and place the column in collection tube 1. When the last of the buffer has reached the surface of the HIC matrix proceed to the next step below. 2. Using a new pipette, carefully and gently load 250 ~1 of the "+" supernatant onto the top of the column. Hold the pipette tip against the side of the column wall, just above the upper surface of the matrix and let the supernatant dnp down the side of the column wall. Examine the column using a TJV light. Note your observations. After it stops dripping transfer the column to collection tube pl) + supernatant 3. Using the rinsed pipette, add 250 pl of wash buffer and let the entire volume flow into the column. Examine the column using the UV light. Note you- observations. After the column stops dripping, transfer it to tube 3. ' n Wash buffer (250 pl), TE buffer (750 pl) 4. Using the rinsed pipette, add of TE Buffer and let the entire volume flow into the column. Examine the column using the UV light. Note your observations. 5. Examine all three collection tubes and note any differences in color between the tubes. Parafilm or Saran Wrap the tubes and place in the refrigerator until the next laboratory period.

14 Lesson 1 Finding the Green Fluorescent Molecule Genetic Transformation Review In Bio-Rad Kit 1, you performed a genetic transformation of E. coli bacterial cells. The results of this procedure were colonies of cells that fluoresced when exposed to ultraviolet light. This is not a normal phenotype (characteristic) for E.coli. You were then asked to figure out a way to determine which molecule was becoming fluorescent under UV light. After determining that the pglo plasmid DNA was not responsible for the fluorescence under the W light, you concluded that it was not the plasmid DNA that was fluorescing in response to the ultraviolet light within the cells. This then led to the next hypothesis that if it is not the DNA fluorescing when exposed to the UV light, then it must be a protein that the new DNA produces with the cells. 1. Proteins. a. What is a protein? b. List three examples of proteins found in your body. c. Explain the relationshp between genes and proteins. 2. Using your own words, describe cloning. 3. Describe how the bacterial cloned cells on your LB/amp plate differ from the cells on your LB/amp/ara plate. Can you design an experiment to show that both plates of cloned cells behave similarly and do contain the same DNA? 4. Describe how you might recover the cancer-curing protein from the bacterial cells.

15 Lesson 2 Review Questions 1. What is a bacterial colony? Name 2. Why did you pick one green colony and one white colony from your agar plate@)? Why do you think you picked one of each color? What could this prove? 3. How are these items helpful in this cloning experiment? a. ultraviolet (UV) light - b. incubator - c. shaking incubator - 4. Explain how placing cloned cells in nutrient broth to multiply rslates to your overall goal of purifying the fluorescent protein.

16 Lesson 3 Review Questions Name 1. You have used a bacterium to propagate a gene that produces a green fluorescent protein. Identify the function of these items you need in Lesson 3. a. Centrifuge - b. Lysozyme - c. Freezer - 2. Can you explain why both liquid cultures fluoresce green? 3. Why did you discard the supernatant in this part of the protein purification procedure? 4. Can you explain why the bacterial cells' outer membrane ruptures when the cells are frozen. What happens to an unopened soft drink when it freezes? 5. What was the purpose of rupturing or lysing the bacteria?

17 Lesson 4 Review Questions Name 1. What color was the pellet in this step of the experiment? What color was the supernatant? What does ;this tell you? 2. Why did you discard the pellet in this part of the protein purification procedure? 3. Briefly describe hydrophobic interaction chromatography and identify its purpose in this lab.

18 Lesson 5 Review Questions Name 1. List your predictions and observations for the sample and what happens to the sample when the following buffers are added to rhe HIC column. Observations Under UV Light Collection Tube Number Prediction (column and collection tube) Tube 1 Sample in Binding Buffer Tube 2 Sample with Wash Buffer Tube 3 Sample with Elution Buffer 2. Using the data table above, compare how your predictions matched up with your observations for each buffer. a. Binding Buffer- b. Wash Buffer- c. Elution Buffer- 3. Based on your results, explain the roles or functions of these buffers. Hint: how does the name of the buffer relate to its function. a. Equilibration Buffer- b. Binding Buffer- c. Wash Buffer- d. TE (Elution) Buffer- 4. Whlch buffers have the highest salt content and which have the least? How can you tell? 5. Were you successful in isolating and purifying GFP from the cloned bacterial cells? Identify the evidence you have to support your answer.

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