1. Restriction Challenge
A classroom activity or exam question

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. Challenge I

4. 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

5. The source vector

6. Question #1
What enzymes would you use to cut out the gene for Miraculin?
BamHI and NotI
PstI and NotI
BamHI and EcoRI
PstI and EcoRI
PstI and CiaI

7. 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.
800, 2200, 40, 5460

8. 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?
3000bp
6300bp
2240bp
5500bp
6260bp

9. 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)
3000bp
6300bp
2240bp
5500bp
6260bp

10. 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 Miraculin- containing 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

11. The source vector

12. Question #5
What enzymes would you use to cut out the gene for Miraculin?
BamHI and SmaI
EcoRV and HaeIII
BamHI and EcoRI
CiaI and EcoRI
HaeIII and SmaI

13. 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.
800, 2200, 40, 5460

14. 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?
3000bp
6250bp
2250bp
5500bp
6300bp

15. 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)
3000bp
6250bp
2250bp
5500bp
6300bp

16. The problem of insertion
Blunt-end versus sticky-end cutters

17. Question #9
What additional ligation products might you detect inside colonies of transformed bacteria? Write size in base pairs on the back of your card.
2200, 4550

18. 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?
HaeIII
CiaI
EcoRI
HindIII
SmaI
EcoRI is the best choice because the cutting site for CiaI is nearly in the middle of the miraculin cassette and will not help at all.