GFP Transformation Genetic Manipulations

Transcription

1 MODULE 2 Objective 2.1 Lesson E GFP Transformation Genetic Manipulations Course Advanced Biotechnology Unit DNA Technology Essential Question How is foreign DNA genes taken up by organisms and expressed? TEKS A-K, 2E-J, 7A, 7B, 7F, 7G, 9A, 9C, 9F, 10A, 10B,11A, 11B Rationale Is it possible to manipulate bacteria to become protein production factories? Can diabetics control blood glucose with insulin produced by bacteria? How cool would it be to take advantage of this microorganism s sophisticated makeup, short doubling times and cheap growth media to mass-produce medically and commercially useful proteins? All of these are possible with a few simple genetic manipulations. Objectives Students will: Practice formulating hypotheses, predictions, and experimental design. Describe the principles of bacterial transformation. Explain the procedure for gene transfer using plasmid vectors. Induce the transfer of the GFP gene (in a plasmid) into E. coli. Describe the traits carried by the GFP gene. Describe how to activate ( turn on ) the GFP gene. Describe how to recognize the transformed cells (from this lab). Perform a bacterial transformation. Determine the degree of success in your efforts to genetically alter an organism. TAKS Science 1A, 2A, 2B, 2C, 2D, 6A Prior Student Learning Recombinant DNA, bacterial structure, protein expression Estimated Time 4 days

2 Key Points I. Dolan Center Information and Expected Results: II. Videos on Lab Procedure and Expected Results: o Part 1: o Part 2: Xe9rZh8&feature=related Engage 1. Assign each lab group one of the recombinant product below: Human Insulin Recombinant Growth Hormone Spider Silk Golden Rice Edible Vaccine Bt Crops 2. Give each lab group 20 minutes to use the Internet to research the science behind each product, its uses and common characteristics. Example: Golden Rice Science behind it Genes for making beta carotene are taken from daffodils and inserted into the genome of a strain of rice. Characteristics Contains beta-carotene which forms Vitamin A Uses To protect undernourished people caused by a lack of Vitamin A 3. Allow groups to present to the class.

3 Activity There are several green fluorescent protein transformation kits available through your district approved science vendor. 1. Pre-Lab Set Up: Have students set up the lab stations, materials and equipment the day before for a lab grade. An Instructor s Advance Preparation will come with any GFP Bacterial Transformation Kit. Below are sample lab group assignments to perform: a. Set up Student Workstation b.set up Instructor s Workstation c. Prepare Agar Plates d. Label Agar Plates e. Aliquot Transformation solution f. Prepare Starter Plates 2. Students complete Virtual Bacteria Transformation worksheet. This activity will give student s background information about how scientist create and identify recombinant plasmids. The lab procedure is similar to the wet lab they will perform in the classroom. Answer key provided. 3. Student will need some specific background information and the green fluorescent protein and how it is selected for in the lab before beginning this lab. Kits should come with student background reading, teacher instructions. Some even provide power points for direct teach. Dolan Science also has a background reading section at There are two approaches that can be used for this lab: I. Experimental Design Approach resulting in a Formal Lab Report. a. Have students use Lab Write to guide them through the writing process. This software has many different features to help students write effective lab reports. There is also a teacher section which provides materials that can be used to introduce students to Lab Write and how to use it. b. Direct students to DNALC Lab Center: Bacterial Transformation. At this site students have access to background readings on GFP, student protocols, pre-lab worksheets, post lab worksheets, applications and further exploration exercises. Allow students to explore this site on their own to find the information and materials needed to complete a lab report using Lab Write.

4 c. Steps to implement: Assign student the pre-lab section in Lab Write. Students complete the lab protocol provided by the transformation kit. Assign students the lab section in Lab Write. Students record results using post-lab section in Lab Write. Students peer grade using Lab Write grading rubric. II. Descriptive Approach resulting in completion of a handout. Students complete all parts of the Bacterial Transformation Handout. This handout pre-lab, lab, analyzing the results, applications and an extension section. Materials Handout: Virtual Bacterial Transformation Handout: Virtual Bacterial Transformation Answer Key Handout: LAB: bacterial Transformation Handout: Formal Lab Report Guidelines Science Kit: GFP Transformation from district approved science supply vendor. Accommodations for Learning Difference Visit the Special Populations section of the CTE Career and Technical Education Website: Texas College and Career Readiness Standards I. Nature of Science: Scientific Ways of Learning and Thinking A4, B1, C1, C2, C3, D3, E1, E2 II. Foundation Skills: Scientific Applications of Mathematics A1, A2, A5, A6, A7, B1, B2, F1, F2, F3 III. Foundation Skills: Scientific Applications of Communication B1, B2, B3, C1 VI. Biology A1, D3

5 Virtual Bacterial Transformation Name: Go to site below to help you complete this document. ia/chapter9virtuallab02/lessonp.html Overview 1. Define biotechnology- 2. List the four steps involved with molecular cloning. 3. What are the three techniques used commonly in biotech labs described by the video? 4. Restriction digestion is a laboratory technique in which proteins called are used to cut DNA at specific sequences of nucleotides. 5. is a laboratory technique in which macromolecules or fragments of macromolecules are separated according to their migration in an electric field. 6. What sized molecules move faster in the electric field? 7. Define bacterial transformation-

6 8. Create a recombinant plasmid by completing the following steps: A. Draw a plasmid. B. Draw a general diagram of a plasmid after a small DNA fragment has been cut from the plasmid by a restriction enzyme. In the diagram, be sure to include the DNA fragment that will eventually get replaced by foreign DNA. Label the plasmid, the DNA fragment, and all sticky ends.

7 C. In the left section below, draw a snapshot figure just before DNA ligase joins the foreign DNA segment to the plasmid. Label DNA ligase, foreign DNA, plasmid, and ALL sticky ends. In the right section below, draw the recombinant (completed) plasmid after DNA ligase has finished its job. For each section, highlight the foreign DNA and the plasmid with different colors. I. II.

8 Plasmid Analysis Fill in the data table with the correct values from the Gel Electrophoresis step. Gel Electrophoresis Data Lane Sample Fragments (kb) 1 Ladder Using the table below and the gel electrophoresis data, identify which plasmids are present in each sample. Sample 1= Sample 2 = Sample 3 =

9 Bacterial Transformation 10. Calcium ions are used to make cells. This treatment enhances the uptake of DNA for these cells. 11. What additional procedure is necessary to allow bacteria to take up plasmid DNA? 12. Only some competent bacterial cells have successful uptake of the plasmid DNA. How is ampicillin used to identify these desired bacterial cells? 13. An indicator called X-GAL is used to identify which bacterial cells do and do not contain the insulin gene within their plasmid. Specify which color identifies the bacterial colony containing the insulin gene and the color for the colony without the insulin gene? 14. Define transformation efficiency-

10 Virtual Bacterial Transformation Answer KEY Overview Define biotechnology- The use of living things or parts of living things to create products useful to society. List the four steps involved with molecular cloning. 1) Isolate the insulin gene. 2) Insert insulin gene into another piece of DNA. 3) Insert recombinant DNA into bacterial cell. 4) Grow bacteria and allow them to produce insulin. What are the three techniques used commonly in biotech labs described by the video? Restriction digestion, gel electrophoresis, and bacterial transformation Restriction digestion is a laboratory technique in which proteins called restriction enzymes are used to cut DNA at specific sequences of nucleotides. Gel electrophoresis is a laboratory technique in which macromolecules or fragments of macromolecules are separated according to their migration in an electric field. What sized molecules move faster in the electric field? Smaller DNA fragments and molecules Define bacterial transformation-the process by which bacteria are changed by the uptake of foreign DNA. Create a recombinant plasmid by completing the following steps:

11 1) Draw a plasmid. OR 2) Draw a general diagram of a plasmid after a small DNA fragment has been cut from the plasmid by a restriction enzyme. In the diagram, be sure to include the DNA fragment that will eventually get replaced by foreign DNA. Label the plasmid, the DNA fragment, and all sticky ends. Sticky ends DNA fragment Sticky ends Plasmid OR Same but using simple inner/outer circle plasmid

12 3) In the left section below, draw a snapshot figure just before DNA ligase joins the foreign DNA segment to the plasmid. Label DNA ligase, foreign DNA, plasmid, and ALL sticky ends. In the right section below, draw the recombinant (completed) plasmid after DNA ligase has finished its job. For each section, highlight the foreign DNA and the plasmid with different colors. I. Foreign DNA Sticky II. Sticky ends ends DNA Ligase DNA Ligase Plasmid Plasmid Analysis Fill in the data table with the correct values from the Gel Electrophoresis step. Gel Electrophoresis Data Lane Sample Fragments (kb) 1 Ladder Using the table below and the gel electrophoresis data, identify which plasmids are present in each sample.

13 Sample 1= Sample 2 = Sample 3 = B C A Bacterial Transformation Calcium ions are used to make competent cells. This treatment enhances the uptake of DNA for these cells. What additional procedure is necessary to allow bacteria to take up plasmid DNA? Heat shock Only some competent bacterial cells have successful uptake of the plasmid DNA. How is ampicillin used to identify these desired bacterial cells? Only the plasmid DNA contains the ampicillin resistance gene amp. The bacteria that do not contain the plasmids will die from ampicillin because they do not have the ampicillin resistance gene. When ampicillin is applied, only the bacterial cells that have successfully accepted the plasmid DNA will survive. An indicator called X-gal is used to identify which bacterial cells do and do not contain the insulin gene within their plasmid. Specify which color identifies the bacterial colony containing the insulin gene and the color for the colony without the insulin gene? White-Colony with insulin gene Blue-Colony without insulin gene Define transformation efficiency-the ratio of the number of colonies formed to the amount of plasmid DNA used

14 LAB: Bacteria Transformation I. Pre-Lab Name : Answer the questions and fill in the table in the space provided below. Complete the Hypothesis, Predictions, and Experimental Design sections during the first lab period. The Results section will be completed after we analyze the data next week. Use the DNALC Lab Center: Bacterial Transformation Website to help you answer these questions. Hypothesis Formulate a hypothesis on which this investigation is based, of how E. coli cells can be transformed by the pglo plasmid. Predictions Prepare and complete the table below to indicate what you predict will happen on each of the four agar plates. (Will E. coli grow on these plates? Will the E. coli have any special properties compared to wild type?) Plate Plasmid? Growth (G) No Growth (NG) Other properties? LB/amp LB/amp/ara LB/amp LB +DNA +DNA -DNA -DNA Note: LB is the nutrient mixture that is added to the plate agar to feed the bacteria. Experimental Design Explain the experimental design: 1. What is/are the independent variable(s) in this experiment?

15 2. What is/are the dependent variable(s)? 3. Which plates will serve as control plates? Do you expect cells to grow on these plates? Why or why not? What is the purpose of these controls? 4. Define plasmid. II. LAB: Your instructor will provide you will the lab protocol. Create a flowchart below of the steps you will take in this lab.

16 III. Analyzing the Results Results 1. In the table below, fill in your observations after examining your plates under both normal and UV light. Plate Plasmid? Number of Other Properties? Colonies? LB/amp LB/amp/ara LB/amp LB +DNA +DNA -DNA -DNA 2. Was your genetic transformation successful? How do you know? 3. Are your results consistent with the predictions you made in the table on the previous page? If not, why? 4. Consider the following two pairs of plates. What do the results obtained from these plates tell you about your experiment? a. -DNA LB and -DNA LB/amp b. +DNA LB/amp and -DNA LB/amp 5. After examining your results, would you revise your hypothesis? If so, restate your hypothesis below.

17 IV. Applications Discuss the various uses of GFP as a reporter protein. Students can look up scientific articles about scientists that use GFP in their experiments. Research other bioluminescent organisms, and describe how they have been incorporated into biological research. V. Extension: How could you confirm which bacterial cells contained the GFP gene? Using your results plates, design an experiment that could help you answer this question? Briefly describe what you will do. Do it. Record your results below.

18 Transformation Lab Report Requirements I. Title of Experiment Determine the lab s independent & dependent variables, then generate an appropriate title! (+2) II. Introduction Introductory paragraph describing key terms associated with experiment: plasmid, transformation, competent (+3) III. Hypothesis total) Formulate and make predictions(+2) Discuss your experimental design (+3) (5 pts IV. Methods/Procedure total) (15 pts Briefly describe/summarize experimental procedure and include the following information: (+5) o Explain importance/role of pglo tubes in experimental design o Explain importance/role of CaCl 2 in experimental method o Explain importance of and parameters for successful heat shock For each plate, identify purpose/role in experimental design and provide an hypothesis for expected results: -pglo: LB plate, -pglo: LB/amp, +pglo: LB/amp, +pglo: LB/amp/ara (+8) Explain how transformation efficiency is calculated (+2) V. Results All data tables must have a title AND all columns must be labeled at the top (20 pts total) Table 1: Observations (quantitative and qualitative) for E. coli starter plate (+5) Table 2: Transformation experiment results (+10) Do NOT use the pre-lab table you were given! Transformation efficiency calculation for your group s results for +pglo/amp plate: (+5) o Present calculations in logical, step-by-step manner. o Explain what you are calculating in each step. o Include units for all numbers within calculations & units for final answer! VI. Conclusion total) Was your transformation experiment successful? How do you know? (+5) Are your results consistent with the predicted results? If not, why? (+2) Would you revise your hypothesis? (+3) (10 pts