Real Vegan Cheese Team SF BAY AREA DIY BIO A project by Biohackers - PowerPoint PPT Presentation

Real Vegan Cheese Team SF BAY AREA DIY BIO A project by Biohackers from Counter Culture Labs and BioCurious

The Team

S Process e a We can make cheese from *any* sequenced mammal! r c h f o r g e n e s Synthesize DNA o f No animals involved! i n t e r e s t i n g Insert casein genes into yeast e n o m e Using strawberry DNA as carrier Express & Purify casein proteins Secreted into supernatant Make vegan “milk” Casein micelles + sugars + fats Make Cheese!

Milk & Cheese Science 101 4 major cheese proteins: α-S1, α-S2, β , κ -casein Fam20C kinase responsible for casein phosphorylation

Milestones Verify protein Clone, expression and transform Transform secretion in Part Design plasmids to E. plasmids into yeast and purify coli, & confirm yeast proteins sequence

Part Design Internal Kex2 site Kex2 site BioBrick BioBrick prefix suffix α- factor SapI site SapI site

Clone & Transform into E. coli ● Synthesized 11 cow and human casein and kinase constructs as IDT gBlocks ● Used Electra pD1214 plasmid backbone (IP-free, scarless cloning) ● Transformed into E. coli ● Sequence confirmation for 10/11 constructs (kappa casein (Kex-) may be toxic in E. coli?) ● BBa_K1531004 - bovine beta casein ● BBa_K1531005 - bovine kappa casein

Transform Plasmids into Yeast Auxotrophic yeast strain (URA-) 5-FOA will kill yeast with intact uracil pathway Currently troubleshooting yeast contamination issues

Express, Secrete & Purify ● pD1214 plasmid carries a strong constitutive TEF yeast promoter ● Expression of kappa casein can be verified by cleaving/phosphorylation with chymosin ● Precipitate caseins by changing pH, [Ca] ● Work in progress!

Can we pull this off? This would be the first attempt at making a bulk food from recombinant protein! ● Target yield: 2.5g protein/L ● Cost? “5g cheese for the price of 1L of expensive beer” (compare to $250,000 for Mark Post’s in vitro hamburger) ● “Tasteable” amounts within months ● Actual product: 2016?

Not Just a Summer Project! ● This project goes well beyond iGEM - we’re into this for the long haul! ● We have set up a non-profit corporation. ● We raised $37,369 in donations on Indiegogo ○ $10,000 for a 50L bioreactor for scaleup ○ Narwhal Cheese!

Indiegogo Campaign > 51,000 visits > 700 tweets > 8,000 facebook shares & 700 contributors from 20 countries

“Ask a Biohacker” videos Answers to questions from IndieGogo supporters: Also: ”Tell Us Why We’re Wrong”!

Press We have been featured in over 100 articles & a Reddit AMA! It seems odd that anybody would have a reaction to this story other than "Cool, hope it works!" - Slate Real vegan narwhal cheese? Well, I’m sure synthetic biology can get weirder — but this is a great start. I’m looking forward to tasting some. - O’reilly Radar

Public Engagement Approach - we ARE the public “We believe that innovations in biology should be accessible, affordable, and open to everyone.” - BioCurious mission statement ● Grassroots formation from existing communities ● Open team. Many joined throughout. Anyone can still join ● Wrote articles for open access publications ● Invited the media to participate in our work ● Open, public meetings via Meetup.com - Reach of over 2300 ● Open membership on Google Groups - 84 members ● Maker Faire - Best in Class & Editor’s Choice Serving & protecting 4 groups

Public perceptions of synbio Source: Mazerik, Jessica, and David Rejeski. “A Guide for Communicating Synthetic biology.” Wilson Center Synthetic Biology Project, 2014.

Changing the Conversation People care about: ● context ○ dedicated to open science in the public benefit ○ our IP strategy: patent and abandon

Changing the Conversation People care about: ● the environment ○ 2.7% global greenhouse gas emissions from dairy production ○ we estimate we could reduce GHG by 40- 90% per gram casein

Changing the Conversation People care about: ● health and safety ○ host organism and technique already in use ○ containment of yeast ○ in consultation with the FDA and public interest groups

Education → Participation

A Special Thanks To: CORPORATE SUPPORTERS Clontech, Teknova, IDT, DNA2.0 COMMUNITY SUPPORT - THANK YOU! Indiegogo Campaign. The campaign had 696 funders, and we raised $37,369. in support DIYBIO COMMUNITY BioCurious, Counter Culture Labs

Thanks for Watching!

Questions?

INTRO (4 minutes) - Matt 1. 15 sec - One line introduction to project [We are a project from BioCurious & CCL] 2. 15 sec - One line intro to people 3. 3 min - Here’s a short introduction to our project [play video - up to 3 minutes] “we’re confident we’ll make the world’s first real vegan cheese” SCIENCE (5 minutes) - Advait 4. 15 sec segue - So, how do we do this. Let’s dig in a bit more 5. 2 min - (2 slides) Process & Science 6. .5 min - Milestones 7. .5 min - Part Design 8. .5 min - Clone & Transform 9. .5 min - Transform Plasmids into yeast 10. .5 min - Express, Secrete & Purify SCOPE OF PROJECT (1-2 minutes) - Patrik 11. segue - Can we pull it off? + Timeline for getting a product to you 12. This is part of a much larger project that goes beyond iGEM. Not just a summer project COMMUNITY OUTREACH & public engagement (4 minutes) - Patrik & Eri 13. Indiegogo campaign [Patrik] 14. Press coverage [Patrik] 15. Engagement model [Eri] Public perception & ELSEI (5 minutes) - Rebecca 16. Public perceptions (take concerns seriously) 17. Context 18. Environment 19. Health and Safety 20. From Education to Participation a. We are working toward changing the conversation and synthetic biology - DONE! ● Eri will buy a dongle and download the presentation ● Meeting at 8:15 in sheraton starbucks lobby

OLD SLIDES HERE ON…..

Team Members in the Lab

Benefits Why Real Vegan Cheese? ● We believe that using animals as large-scale food production machines is ethically and environmentally irresponsible. ● We believe that our process is more ethically responsible and environmentally sustainable than the status quo. ● We believe that all humans, vegans included, should have access to delicious and healthy cheese!

Risks/Challenges As a health conscious and ecologically responsible collective, we have our own concerns about our process. - We do not want to produce food in a way that replicates the shortcomings of some existing Genetic Engineering companies. - One of our major challenges will be to evaluate whether the technology we work with is safe. - We will need to continually re-evaluate risks and design safeguards that prevent or limit potential dangers to individuals, humanity or the planet as a whole.

Vegan Cheese. Real Vegan Cheese. Why? ● Environmental impact of dairy production ● Animal welfare ● Cheese is major reason for not switching to a fully plant- based diet ● Health issues: lactose intolerance, cow’s milk allergies Solution: Make cheese proteins in baker’s yeast!

Health Effects Not only can we make Real Vegan Cheese that is better for the environment, we can also design it to be better for you: ● For each of the four main proteins found in cheese, there are dozens of known genetic variants found in humans and in different breeds of cows. Some of these versions of the proteins are associated with various health benefits, or increased risks of allergic response. We have the opportunity to pick and choose the most desirable variants of these naturally occurring proteins, increasing health benefits and removing allergens.

Health Effects (cont) There are clear health benefits to consuming milk proteins derived from humans versus cow milk proteins. This is made possible by expressing the genes that code for human milk proteins in yeast. Using human genes to make vegan milk may not be appetizing to everyone, so for you folks that love good old-fashioned cow cheeses, we’re also making a vegan cow cheese. By creating an alternative pathway to cheese, we help the environment, increase access to tasty and healthy food, and support people who have already committed themselves to a smaller personal carbon footprint.