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Inteins as a Tool for Post-‐Transla0onal Control The tale of how a band of misfits shot for the stars, missed, and built a SynBio legacy from the ground up
Table of Contents
1. Introduc0on a) Post-‐transla0onal control b) Inteins
2. Our project
a) Project 1: Mito-‐inteins b) Project 2: Condi0onal inteins
3. The turning point
a) “Roots vs. Shoots” b) Building our founda0ons
4. Local and poli0cal presence a) Shad Valley & Science Quest b) OGI and Ted Hsu
5. Skill development
a) Synthe0c biology curriculum
6. How can we help you? 7. Acknowledgements
Why Post-‐Transla3onal Control?
Post-‐Transla3onal Control is Fast
Olsen & Tabor, 2012
Mammalian Yeast E. coli
Inteins are Self-‐Splicing Protein Elements
N-‐Extein Intein C-‐Extein
N-‐Extein C-‐Extein
Excision
Intein
Liga3on Complete Protein
Cis Intein
Trans Intein
N-‐Extein N-‐Intein C-‐Extein C-‐Intein
Table of Contents
1. Introduc0on a) Post-‐transla0onal control b) Inteins
2. Our project
a) Project 1: Mito-‐inteins b) Project 2: Condi0onal inteins
3. The turning point
a) “Roots vs. Shoots” b) Building our founda0ons
4. Local and poli0cal presence a) Shad Valley & Science Quest b) OGI and Ted Hsu
5. Skill development
a) Synthe0c biology curriculum
6. How can we help you? 7. Acknowledgements
• About 1/2000 children will develop the disease by age 10 • Similar prevalence to childhood cancer! • Maternally inherited, has its own genome • Promising treatments? • Gene therapy:
Project Part 1: Mitochondrial Disease
How can we do gene therapy on mitochondrial genes?
Added Localiza0on
Signal
Mitochondria (13 proteins)
Allotopic Expression from the Nucleus
Too hydrophobic!!
Nucleus (~1100 proteins)
Changing Hydrophobicity with Inteins
Protein-‐N N-‐Intein MTS
Protein-‐C C-‐Intein MTS *
*
Cleavage of MTS by Mitochondrial Processing Pep3de (MPP)
Excision
Func3onal Protein
1. Split up the hydrophobic regions 2. Link Splicing to Import with MTS
Project Part II: Condi3onal Inteins
N-‐Extein Intein C-‐Extein
Natural Conditions
N-‐Extein Intein C-‐Extein
Engineered Conditions
Intein Complete Protein Complete Protein Intein
pH Temperature Spontaneous
Light Ligands Other Peptides
• What other conditions could we create other than just spatial?
• How can we gain temporal control?
Crea3ng a Light-‐Inducible Intein
N-‐Extein C-‐Extein Int-‐N Int-C
PIF3
PhyB
-‐ PIF3 and PhyB associate in the presence of light
Crea3ng a Light-‐Inducible Intein
N-‐Extein C-‐Extein Int-‐N Int-C
PIF3
PhyB
660nm 660nm
N-‐Extein Int-‐N
PIF3
C-‐Extein Int-C
PhyB
Intein
Complete Protein
PIF3
PhyB
PhyB and PIF3 associate
Extein liga3on
Table of Contents
1. Introduc0on a) Post-‐transla0onal control b) Inteins
2. Our project
a) Project 1: Mito-‐inteins b) Project 2: Condi0onal inteins
3. The turning point
a) “Roots vs. Shoots” b) Building our founda0ons
4. Local and poli0cal presence a) Shad Valley & Science Quest b) OGI and Ted Hsu
5. Skill development
a) Synthe0c biology curriculum
6. How can we help you? 7. Acknowledgements
Results
We submi^ed two crucial parts of our mito-‐construct – Bba_K1435001 & Bba_K1435002 We’re proud of our work! But we recognized that there are challenges and limita0ons to being a predominantly student-‐run enterprise from a mid-‐sized ins0tu0on in a rela0vely undervalued field
Our expecta3ons…
…versus reality
What is the solu3on?
Successful enterprises in synthe0c biology
require a strong, sustainable support network
We wanted to start building our own.
The “Roots before Shoots” Approach
Local Presence
Political Support
Expertise & Advice
Skill Development
Table of Contents
1. Introduc0on a) Post-‐transla0onal control b) Inteins
2. Our project
a) Project 1: Mito-‐inteins b) Project 2: Condi0onal inteins
3. The turning point
a) “Roots vs. Shoots” b) Building our founda0ons
4. Local and poli0cal presence a) Shad Valley & Science Quest b) OGI and Ted Hsu
5. Skill development
a) Synthe0c biology curriculum
6. How can we help you? 7. Acknowledgements
Local Presence Community Partnerships
Projects inspired by community defined needs.
Grade 4-‐8
Grade 9-‐12
Local Presence Innova3ve Educa3on Modules
Local Presence By the Numbers
700+ students
“Awesome workshop, great instructors! Kids loved it, perfect intro to lab se]ng”
Almost 100% of par3cipants enjoyed and recommended the workshop
Poli3cal Support Ontario Genomics Ins3tute
-‐ Collabora0on with industry professionals -‐ Long term funding for Ontario iGEM teams
Exper3se & Advice Ontario Genomics Ins3tute
Poli3cal Support Ted Hsu, Member of Federal Parliament
-‐ Engaged the science, technology and post-‐secondary educa0on cri0c of the Liberal Party
-‐ Reached 10,000+ Kingstonians
-‐ Created connec3on with Federal Government
Table of Contents
1. Introduc0on a) Post-‐transla0onal control b) Inteins
2. Our project
a) Project 1: Mito-‐inteins b) Project 2: Condi0onal inteins
3. The turning point
a) “Roots vs. Shoots” b) Building our founda0ons
4. Local and poli0cal presence a) Shad Valley & Science Quest b) OGI and Ted Hsu
5. Skill development
a) Synthe0c biology curriculum
6. How can we help you? 7. Acknowledgements
Skill Development Educa3onal Innova3on in Synthe3c Biology
Three-‐pronged Approach 1. 4 Problem Based
Learning (PBL) Tutorials
2. 8 Spotlight Lectures
3. Interac0ve E-‐resource
Spotlight Lectures Exposing students to the cu]ng edge
-‐ First undergraduate students to lecture in a major science course at Queen’s university
-‐ Bringing cujng edge synthe0c biology to 400+ students
-‐ A resource that can be used at all iGEM ins3tu3ons
Problem Based Learning Tutorials Providing tools to explore synthe3c biology
-‐ Providing the tools and mindset to 400+ students -‐ A resource that can be used at all iGEM ins3tu3ons
Sharing the Wealth A Synthe3c Biology Curriculum For All
Right now, we are pooling resources with Waterloo
iGEM Teams around the world!
The Future of Synthe3c Biology at Queen’s University
Bachelor’s of Science Honors in Synthe3c
Biology
Queen’s University
-‐ Start of something brand new at Queen’s University
-‐ Poten0al of an en0re course built around synthe0c biology
-‐ Years down the line: Synthe0c biology program!
Developments from Course Redesign
-‐ Crea0ng materials that will be a part of the curriculum for next 5-‐10 years
-‐ Part of an Ontario-‐wide, provincial online biochemistry course created by Queen’s University
-‐ The first 0me at Queen’s University that student created materials will be used in class and online
Table of Contents
1. Introduc0on a) Post-‐transla0onal control b) Inteins
2. Our project
a) Project 1: Mito-‐inteins b) Project 2: Condi0onal inteins
3. The turning point
a) “Roots vs. Shoots” b) Building our founda0ons
4. Local and poli0cal presence a) Shad Valley & Science Quest b) OGI and Ted Hsu
5. Skill development
a) Synthe0c biology curriculum
6. How can we help you? 7. Acknowledgements
Serving the community
Government contact
Corporate partnerships and project ideas
Knowledgeable and enthusiastic
students
Thanks to our sponsors
Thanks to our advisors
Dr. John Allingham Dr. Mar0n Petkovich Dr. Ken Ko
Dr. Paul Young Dr. Madhuri Ko0
director@qgemteam.com
Appendix 1: Intein mechanism
Appendix 2: Mitochondrial Transport
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