Nekeisha Nogal , Gabriel Pineda, Keith Neeves, Judy Schoonmaker ,

1. NekeishaNogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway, Erika Tinoco

2. Goals and Outcomes • Unit: Engineering recombinant DNA vaccines • Goal: To understand the molecular biology sequence of antigen  vaccine • Goals for Today: Making a recombinant DNA vector • Students will understand how restriction enzymes work • Students will understand how DNA ligase works • Outcomes • Students will be able to organize the main steps in DNA vaccine development • Students will be able to recognize the specificity of ligation sites following cleavage by restriction enzymes • Students will be able to create a script that applies the concepts of gene insertion

3. Course Context Biology I or II • Freshmen • ~100 students Prior knowledge • DNA structure • Central dogma • Restriction enzymes • Vaccines and immune response

4. Have you gotten YOUR HPV Vaccine? • 20 million Americans currently infected • 6 million infected/yr • 50% sexually active people have HPV at some point DNA Vaccine Recombinant Vector HPV Vaccine http://hplusmagazine.com/2009/08/31/take-shot/ Accessed 8/3/12

5. Steps in DNA vaccine development Cut vector and DNA insert with restriction enzymes Ligate DNA insert into vector Purify recombinant vector Deliver recombinant HPV vector vaccine Determine coding DNA for insert Identify HPV immunogenic protein Choose a vector Culture transformed bacterial host cells

6. Steps in DNA vaccine development Identify HPV immunogenic protein Determine coding DNA for insert Choose a vector Cut vector and DNA insert with restriction enzymes Ligate DNA insert into vector Culture transformed bacterial host cells Purify recombinant vector Deliver recombinant HPV vector vaccine

7. Cut vector and DNA insert with restriction enzymes Campbell Essential Biology with Physiology, Simon et al., 3rd Ed.

8. The restriction enzymes RE1 and RE2 cut along the red lines: If one sample of DNA was cut with RE1 and another with RE2, and these two samples were mixed and treated with DNA ligase, what would happen? A. DNA cut with RE1 and RE2 would be ligated (joined together) with no preference. B. The RE1-cut DNA would be ligated to itself. C. The RE2-cut DNA would be ligated to itself. D. Both B and C. E. No DNAs would be ligated.

9. Ligate HPV specific DNA insert into vector • Write a script for this movie using these terms: • Restriction enzyme • Plasmid • DNA ligase • DNA insert • Sticky ends • Restriction site • Peer review script • Turn in revised script to teacher. http://www.youtube.com/watch?v=aA5fyWJh5S0

10. Have you gotten YOUR HPV Vaccine? • 20 million Americans currently infected • 6 million infected/yr • 50% sexually active people have HPV at some point DNA Vaccine Recombinant Vector HPV Vaccine http://hplusmagazine.com/2009/08/31/take-shot/ Accessed 8/3/12

11. Homework Assignment(summative assessment) • Make a concept map that shows how a scientist would proceed from a gene sequence for a protein to a vaccine using recombinant technology. • You should use the terms below as a skeleton framework. Add as many additional terms as you feel appropriate. • Restriction enzyme • Vector • DNA ligase • DNA insert • Sticky ends • Vaccine

12. If you had an HPV-specific DNA insert that has sticky ends created by digestion with BamHI, which of the three restriction enzymes below could be used to cut a vector to create a recombinant vector vaccine for HPV? Exam Question(summative assessment) • BamHI only • BamHI and BslI • BslI and EcoRI • EcoRI only • EcoRI and BamHI BamHI – 5’-G|GATCC BslI – 5’-C|GATCG EcoRI – 5’-G|AATTC

13. Alignment