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Nekeisha Nogal, Gabriel Pineda,Keith Neeves, Judy Schoonmaker,Delphi Chatterjee, Diane Ordway,
Erika Tinoco
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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
• Outcomes1. Students will be able to organize the main steps in DNA
vaccine development2. Students will be able to recognize the specificity of ligation
sites following cleavage by restriction enzymes3. Students will be able to create a script that applies the
concepts of gene insertion
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Course ContextBiology I or II – Freshmen– ~100 students
Prior knowledge– DNA structure – Central dogma– Restriction enzymes– Vaccines and immune response
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DNA Vaccine
Recombinant Vector
Have you gotten YOUR HPV Vaccine?
HPV Vaccine
• 20 million Americans currently infected
• 6 million infected/yr• 50% sexually active
people have HPV at some point
http://hplusmagazine.com/2009/08/31/take-shot/Accessed 8/3/12
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Steps in DNA vaccine development
Identify HPV immunogenic protein
Choose a vector
Culture transformed bacterial host cells
Purify recombinant vector
Deliver recombinant HPV vector vaccine Determine coding DNA for insert
Cut vector and DNA insert with restriction enzymes
Ligate DNA insert into vector
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Steps in DNA vaccine developmentIdentify HPV immunogenic protein
Choose a vector
Culture transformed bacterial host cells
Purify recombinant vector
Deliver recombinant HPV vector vaccine
Determine coding DNA for insert
Cut vector and DNA insert with restriction enzymes
Ligate DNA insert into vector
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Campbell Essential Biology with Physiology, Simon et al., 3rd Ed.
Cut vector and DNA insert with restriction enzymes
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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.
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1. Write a script for this movie using these terms:A. Restriction enzymeB. PlasmidC. DNA ligaseD. DNA insertE. Sticky endsF. Restriction site
2. Peer review script3. Turn in revised script to teacher.http://www.youtube.com/watch?v=aA5fyWJh5S0
Ligate HPV specific DNA insert into vector
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DNA Vaccine
Recombinant Vector
Have you gotten YOUR HPV Vaccine?
HPV Vaccine
• 20 million Americans currently infected
• 6 million infected/yr• 50% sexually active
people have HPV at some point
http://hplusmagazine.com/2009/08/31/take-shot/Accessed 8/3/12
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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
1. Vector2. DNA ligase3. DNA insert4. Sticky ends5. Vaccine
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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?
A. BamHI onlyB. BamHI and BslI C. BslI and EcoRI D. EcoRI onlyE. EcoRI and BamHI
Exam Question(summative assessment)
BamHI – 5’-G|GATCCBslI – 5’-C|GATCGEcoRI – 5’-G|AATTC
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AlignmentLearning Objective Assessment Active
learningLow Order/High Order
Students will be able to organize the main steps in DNA vaccine development
Formative: Self-check based on key
Strip sequence
Low OrderBloom 3
Students will be able to recognize the specificity of ligation sites following cleavage by restriction enzymes
Formative: Clicker question-two polls
Clicker question + peer instruction
High OrderBloom 3-4
Students will be able to create a script that synthesizes the key concepts of gene insertion
Formative: Peer evaluation
Summative: Grade the script
Writing script of movie
High OrderBloom 5
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DiversityDifferent learning styles:Kinesthetic – strip sequenceVisual – movieWriting – scriptAuditory – movie sounds
Different personalities:Activities that appeal to introverts and extroverts