Goal: Move the Gene for Miraculin into an Expression Vector

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2 LEARNING OBJECTIVES Proper use of restriction endonucleases Tying restriction fragmentation to electrophoresis Critical Thinking Use of internet resources Students will need to develop strategies using only restriction endonucleases and electrophoresis to solve plasmid cloning problems.

3 DESIGN OF ACTIVITY The Problem: Students continue to have difficulties visualizing and representing the use of enzymes that cut and ligate circular DNA Previous Activities: Kreuzer and Massey activities (limited) We wished to create a new challenge that: is more similar to actual plasmid architecture requires students to think critically about enzyme selection has built-in wrong choices

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5 Goal: Move the Gene for Miraculin into an Expression Vector Miraculin a glycoprotein produced by the berry Synsepalum dulcificum Alters the perception of flavors acid! sweet Binds strongly to sweet taste receptors When exposed to acid, changes configuration Bound saccharides interact with receptors to induce taste Effects last for over an hour

6 THE SOURCE VECTOR

7 QUESTION #1 What enzymes would you use to cut out the gene for Miraculin? A. BamHI and NotI B. PstI and NotI C. BamHI and EcoRI D. PstI and EcoRI E. PstI and CiaI

8 QUESTION #2 What size fragments would you create if you cut psource I and ptarget with PstI and NotI? Write your answer on the back of a card with marker provided.

9 QUESTION #3 Assuming that only plasmids with a single origin of replication are possible, which of the following products will not be made when ligase is added to the mixture? A. 3000bp B. 6300bp C. 2240bp D. 5500bp E. 6260bp

10 QUESTION #4 If the growth medium contains ampicillin, which of the following ligation products could you detect inside colonies of transformed bacteria? (more than one answer may be correct) A. 3000bp B. 6300bp C. 2240bp D. 5500bp E. 6260bp

11 STUDENT HOMEWORK, PART I Students would be directed to create a scheme for distinguishing between the two potential isolates, the regenerated ptarget vector and the recombinant Miraculincontaining vector. Students would then draw gels showing the products from their restriction analysis and describe how they would distinguish the recombinant vector. And now, Challenge II

12 THE SOURCE VECTOR PstI

13 QUESTION #5 What enzymes would you use to cut out the gene for Miraculin? PstI A. BamHI and SmaI B. PstI and SmaI C. BamHI and EcoRI D. CiaI and EcoRI E. HaeIII and SmaI

14 QUESTION #6 What size fragments would you create if you cut psource II and ptarget with HaeIII and SmaI? Write your answer on the back of a card with marker provided.

15 QUESTION #7 Assuming that only plasmids with a single origin of replication are possible, which of the following products will not be made when ligase is added to the mixture? A. 3000bp B. 6250bp C. 2250bp D. 5500bp E. 6300bp

16 QUESTION #8 If the growth medium contains ampicillin, which of the following ligation products could you detect inside colonies of transformed bacteria? (more than one answer may be correct) A. 3000bp B. 6250bp C. 2250bp D. 5500bp E. 6300bp

17 THE PROBLEM OF INSERTION Blunt-end versus sticky-end cutters

18 QUESTION #9 What additional ligation products might you detect inside colonies of transformed bacteria? Write size in basepairs on the back of your card.

19 STUDENT HOMEWORK, PART II Students would be directed to create a scheme for distinguishing between the two potential isolates, the regenerated ptarget vector and the recombinant Miraculin-containing vector. For you, Question #10: What restriction enzyme would you use to create unique fragments that would help distinguish between ptarget and the recombinant plasmid? A. HaeIII B. CiaI C. EcoRI D. HindIII E. SmaI

20 FINAL THOUGHTS What parts of the activity were the most challenging? If visualization is the problem, how could you help your students? If you gave this problem as a test question, what preparation would you give ahead of time?