IGEM team meeting notes 5/19/14

May 19th 2014

Agenda

Introductions

Patrik: Bio Jamie: Biotech student at BCC Rebecca: Graduate student. Synthetic biology, science fiction and history of science. Shashank: Studying salt tolerance in plants Advait: Middle schooler interested in biology Mallory on Zoom Fran (not on Zoom, only this document): Nursing student learning about biology.

Brief intro to project for new people

wiki: https://wiki.counterculturelabs.org/index.php/Vegan_cheese

Updates from sub-projects

What happened in the last week? A brief overview.

Fundraising

Fran: Video script is written and Craig provided some very helpful initial comments. I will give recording another shot in the morning, and then upload a version with no music soundtrack and one with a music soundtrack to solicit further comments. I am committed to getting this recording done tomorrow. Anybody should feel free to take a crack at it, though, I don't want to hog the project.

In any case, once the next iteration of the recording is done, we will still need a lot of input about photos, stock footage, any captions/titles, etc.

Finding good stock footage has been challenging! Easiest route to good footage to layer into the video might just be to take it ourselves with cell phones or other devices.

Molecular biology

Craig presents on cloning.

Many vectors have multiple cloning sites: A series of restriction endonuclease sites.

You amplify your gene using PCR or cut it out of another plasmid, matching restriction sites and ligate it into the plasmid.

Meh, I can't pay attention and take notes. Here's the presentation: https://docs.google.com/viewer?a=v&pid=forums&srcid=MDQ3ODI0ODE4NDM3NTM4NjIyMzUBMDM5OTQ5MTk5OTAwNDIzOTE2MjcBa0Z0ZTFkd2xycGdKATQBAXYy&authuser=0

https://www.youtube.com/watch?v=WCWjJFU1be8

Cheese making

Miyoko Schinner - vegan chef and TV host, has written books on vegan cheese and has signed up on our meetup list. I'll (Ahnon) contact her directly.... just sent her em via meetup.

Break out into sub-groups

IP Issues discussion

Marc: should definitely patent, and do defensive license; willing to write up draft asap

Patrik: fastest way to get most people eating vegan cheese would be patent the heck out of it, show it can be made cheaper than cow's milk, and sell the IP to Kraft. - If that is the *only* issue we care about.

Mallory: if you open everything, then groups with most resources will get the upper hand Mallory was basically making the argument that big incumbent corps will take what we made and make lots of profits.

Fundraising

Patrik: If we have a fundraiser we really should put a lot of effort behind it. Ahnon: needs to contact Amyris again Patrik: needs to contact Bruce German to ask about companies working on breast milk nutritional supplements Advait: http://www.ncbiotech.org/research-grants/research-funding/biotechnology-research - too late to apply, maybe next year Advait: http://www.gofundme.com/ http://www.rockethub.com/ http://peerbackers.com/ Jamie and Marc will contact PETA office in Oakland (do they have contact with Patrick Brown as well?P Shashank: Viz. Amyris, saw Greg Warner at BioCurious today. Shashank - please remind Greg to join the iGEM team! I don't think he' son our mailing list yet. Do you have an email for him? Maybe the one I've been using doesn't work -- Patrik He was away on vacation..i have a netcom.com email.will forward a note to both of you...-s gwerner@ix.netcom.com - yeah, the ix.netcom one is the one I've been using

http://www.gofundme.com/ http://scistarter.com/ https://experiment.com/

Molecular biology

iGEM DNA distribution kit: http://parts.igem.org/Help:2014_DNA_Distribution

Proposed goals for upcoming week:

• Finalize construct design
• Plan initial experiments necessary to get "band on a gel" verification of casein and kinase expression
• Make list of all required reagents, tools and other consumables, including gBlocks and primers
• Reach out to companies asking for donated reagents (and plasmids from DNA 2.0)

Tasks

juul

• Look more closely at DPL. Follow up with Linda Kahl and Brewster Kahle.
• Follow up with Rusty to see if he can help with video / equipment
• Find the best plasmid for us to order from addgene (but check the biobrick dna distribution list first)

Patrik

• Follow up with Bruce German Re: milk protein companies

Jamie

• contact PETA and other vegan organization

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old meeting ----

May 12th 2014

Agenda

Introductions

Icebreaker question: How is genetic engineering going to save/destroy the world?

Updates from sub-projects

What happened in the last week? A brief overview.

Molecular biology

• Craig and Juul have construct designs

Cheese making

• Chr. Hansen wants to know exactly which products we want.

Break out into sub-groups

Sub-group "break-outs" may have to occur in serial because of overlap of members. Groups should plan next weeks work and assign tasks. We encourage people to meet one on one or in their sub-groups to work during the week.

Molecular biology

Proposed goals for upcoming week:

• Finalize construct design
• Plan initial experiments necessary to get "band on a gel" verification of casein and kinase expression
• Make list of all required reagents, tools and other consumables, including gBlocks and primers
• Reach out to companies asking for donated reagents (and plasmids from DNA 2.0)

= Background research

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Notes from previous meeting ----------

Various intros by lots of people present. Unfortunately no notes for introductions. 22 people present. - Registrations on http://www.meetup.com/BioCurious/events/180642612/ and http://www.meetup.com/Counter-Culture-Labs/events/174105062/ - ccl-igem@googlegroups.com - wiki: https://www.counterculturelabs.org/index.php/Vegan_cheese - sci-hub.org

• Patrik summarizes the vegan cheese project and iGEM for new people.

Agenda

Intro

Patrik What is Casomorphin Casomorphins are peptides, i.e., protein fragments, derived from the digestion of milk protein casein. What are their effects? Do they need to be considered in the formulation of cheese?

How can people sign up for the iGEM list/stay in touch with the team?

Google Groups Mailing List: ccl-igem@googlegroups.com

Sign up at https://groups.google.com/forum/#!forum/ccl-igem

What is the work that needs to be done by this team?

1. Fundraising a. Sub-team to take the lead on this? Crowdfunding? Who are we reaching out to? b. Support from corporations? (Mallory can reach out to - All tech in Ky bio ag company, New Harvest, Maria can contact Barracuda Networks) EPA? c. Fundraising goals $3500 fee for registration, materials $5000 Goal low $15k, medium $25k, high $50k d. Ask what crowdfunding we are/will do, crowdtilt has been done, going to restart e. Treasuer to keep track of funds, expenses, and goals with an estimated budget f. Are donation tax deductible? who's 501c3 would do this? 501c3 applied for though CCL g. Teach a class realted to the proejct, all funds going to the project h. speaking at events i.e. First Fridays i. go out to speaking events like first Oakland and talk about project j. Scrappy vegan biohaker mascot - farmer milking a yeast cell with udders for marketing k. organize a GMO conference!

2. Molecular Biology a. synthetic biology services: IDT http://eu.idtdna.com/pages/landing/igem - 25% off an order, up to $800 - 40% off gBlocks® Gene Fragments, MiniGenes, or Genes b. What constructs do we want to make? - triple-gene, inducible, construct with promoter-regulated transcription of cheese components. - single constructs that serve as controls +/- expression tags for purification. - kinases needed for the polymerization of Caseino-peptides. c. Kinases and glycosylation - Do we want to co-express anything? d. can we create a construct/work around yes/no flow chart. IE: if construct is created and secretes, then...

3. Cheese Making a. what is the process? Is is the same for synthetic cheese when compared to dairy-derived cheese? b. What are our equipment needs? c. Experiments? - determining the source of fat

Experiments:

   1. Cheese Track

a. Make powdered casein and make cheese from it b. powdered casein with Ca2+ and PO4 to make cheese c. Synthetic Casein + CA2+ to cheese d. synthetic milk to casein

2. Protein Track a. Express various caseings b. evaluate mixtures of synthetic and natural caseins c. optimize casein phophates d. results in syntehtic milk -- make cheese from syntehtic milk

3. Recipe Track a. Evaluate pH History of Water/Pwder Mixture b. Develop recipe for reconstituted milk c. Develop Recipe for Micelle formation d. Develop recipe for cleaving enzyme e. artificial micelles from fat emulsifications.

4. Background research a. Cost of productions b. Are human casains appropriate for adult humans? research about this

$83 in donations tonight (Patrik) - where do those funds go? Who holds them?

Fundraising; Maria Mallory Patrick

Molecular biology: Brian Dimitri Teddy Johan Marc Craig R. Nikola Students Colin Advait Blake

Cheese Making:

   Teddy
   Maria
   Brian
   Della
   

Background: Dimitri Mallory

Molecular Biology Genes

Full-length bovine κ Casein B genetic variant AAQ87923.1 Full-length bovine αs1 Casein Full-length bovine αs2 Casein Full-length bovine β Casein A2 genetic variant for health benefit B genetic variant for better coagulation

Full-length human κ Casein P07498.3 Full-length human αs1 Casein Full-length human αs2a Casein CSN1S2A Full-length human αs2b Casein CSN1S2B Full-length human β Casein human caseinomacropeptide FAM20C Kinase

Promoters 3 promoters (low, mid, high)

Selection Markers http://protocols.mmml.nl/protocols/yeast/yeast_selectable_markers.php

OriC

Purification Tags

Constructs: Full length single peptides

do promoters as separate biobricks (if needed) have other iGEM projects done protein secretions

HIGH Copy Vector Low Promoter SS+ Mid Promoter SS+ TAG High Promoter SS+

MID Copy Vector Low Promoter SS+ Mid Promoter SS+ TAG High Promoter SS+

LOW Copy Vector Low Promoter SS+ Mid Promoter SS+ TAG High Promoter SS+

Database of constructs, sequences and restricitons

HiFi Polymerase - PfuUltra