Nekeisha Nogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway,...

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Nekeisha Nogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway, Erika Tinoco

Transcript of Nekeisha Nogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway,...

Page 1: Nekeisha Nogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway, Erika Tinoco.

Nekeisha Nogal, Gabriel Pineda,Keith Neeves, Judy Schoonmaker,Delphi Chatterjee, Diane Ordway,

Erika Tinoco

Page 2: Nekeisha Nogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway, Erika Tinoco.

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

Page 3: Nekeisha Nogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway, Erika Tinoco.

Course ContextBiology I or II – Freshmen– ~100 students

Prior knowledge– DNA structure – Central dogma– Restriction enzymes– Vaccines and immune response

Page 4: Nekeisha Nogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway, Erika Tinoco.

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

Page 5: Nekeisha Nogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway, Erika Tinoco.

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

Page 6: Nekeisha Nogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway, Erika Tinoco.

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

Page 7: Nekeisha Nogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway, Erika Tinoco.

Campbell Essential Biology with Physiology, Simon et al., 3rd Ed.

Cut vector and DNA insert with restriction enzymes

Page 8: Nekeisha Nogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway, Erika Tinoco.

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.

Page 9: Nekeisha Nogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway, Erika Tinoco.

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

Page 10: Nekeisha Nogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway, Erika Tinoco.

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

Page 11: Nekeisha Nogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway, Erika Tinoco.

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

Page 12: Nekeisha Nogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway, Erika Tinoco.

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

Page 13: Nekeisha Nogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway, Erika Tinoco.

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

Page 14: Nekeisha Nogal, Gabriel Pineda, Keith Neeves, Judy Schoonmaker, Delphi Chatterjee, Diane Ordway, Erika Tinoco.

DiversityDifferent learning styles:Kinesthetic – strip sequenceVisual – movieWriting – scriptAuditory – movie sounds

Different personalities:Activities that appeal to introverts and extroverts