Introductory textbook

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This is the Real Vegan Cheese introductory textbook. This is a work in progress. The purpose of this textbook is to get newcomers rapidly up to speed so they can start contributing.

Contents

Introduction

Whenever this document references outside materials you can find these materials linked in the section Essential knowledge and further reading. If you only care about the science, skip to the Genetic engineering section.

Purpose

The purpose of this project is to create a vegan cheese that is as similar to normal hard and semi-hard cheese as possible using genetically engineered S. cerevisiae (baker's yeast) to produce vegan versions of the necessary cheese protein.

Origin

The Real Vegan Cheese project came to be when some people from Biocurious and Counter Culture Labs decided to enter into the iGEM competition. This was triggered by a rule change in the iGEM competition that allowed non academically associated citizen scientists to enter into the competition. While entering into the iGEM competition was the original intent and remains a goal of the project, the Real Vegan Cheese project rapidly grew beyond the scope of the iGEM competition.

Values

We have a Statement of ethics that is currently a work in progress. Our core values include but are not limited to:

  • Open access
  • Transparency
  • Educational outreach
  • Safety
  • Respect for the environment
  • Using biotechnology only for peaceful purposes
  • Community outreach

We use open source software, hardware and wetware where possible and release all information under an appropriate Free Culture license. Our patents, if any, will be explicitly abandoned, putting them into the public domain.

Organization

The project is for all intents and purposes horizontally organized, though we have a board of directors, and a set of officers because these are requirements for a non-profit corporation. To counterbalance this formally required hierarchy the Real Vegan Cheese project members have the power to vote out the board members. We are currently only a California state non-profit. The plan is to apply for federal 501(c)(3) status.

Becoming a member

Membership is not yet defined but is slated to be defined before the end of September 2014. Membership will be open to anyone who actively participates in the project.

Funding

We are currently 100% funded by donations. The Real Vegan Cheese project is 100% volunteer-run, with no team-members receiving payment for services. Team-members are not currently expected to pay anything to participate. Most of the funding so far has come from the crowdfunding campaign.

Here are some stats on the campaign:

  • We received a total of $37.369
  • The campaign ran for 60 days
  • Nearly 700 people contributed money to the campaign
  • Over 28,000 people watched the video

Some of this money is going to pay fees and perks (like stickers and t-shirts) but the majority is being used to pay for:

  • Lab equipment like incubators and bioreactors
  • Services like DNA synthesis and sequencing
  • Reagents and chemicals, e.g: enzymes, buffers, gels, agar plates, etc.
  • Documentation and presentation equipment like a projector, cameras, microphones, live streaming gear, etc.

Aspects of the project

People are working on at least the following aspects of the project:

  • Science and engineering, including hands-on training
  • Cheesemaking (learning the craft and building equipment)
  • Establishing Counter Culture Labs (finding and moving equipment, resurfacing floors, installing lab benches, etc.)
  • Ethical, Legal, Social and Environmental implications
  • Crowdfunding and fulfillment (designin, orderding and shipping T-shirs, stickers, etc.)
  • Media outreach and external communications (social media, interviews, etc.)
  • Virtual infrastructure (managing servers and such)
  • Video: Documentation, video-conferencing and live-streaming
  • Bureaucracy and finances

This document focuses on the science and engineering aspects, since that seems to be what most new people are interested in. However, we are working on many exciting sub-projects, such as prototyping a one-button live-streaming lab bench, investigating novel approaches to open patents and estimating predicted greenhouse gas emissions. We welcome people to help us out with any and all of these aspects, or others if you feel something is missing!

Science and engineering

The science and engineering aspects of this project are divided into two main components:

  1. Producing casein using genetically engineered yeast
  2. Producing "milk" and cheese using only purified casein and other vegan ingredients

The first component is likely the more challenging. Most of the work is currently focused on genetic engineering and casein production.

Genetic engineering

Background

The four main ingredients of cheese are water, milkfat, cheese protein (casein) and sugar (lactose). Briefly, the cheesemaking process is:

  • Add lactic acid bacteria (LAB) to milk (or rely on the naturally occurring LAB)
  • Heat milk. LAB consumes lactose producing lactic acid and lowering the pH.
  • Add rennet enzyme (chymosin) to milk
  • The rennet + pH drop makes caseins drop out of solution and coagulate, entrapping milkfat. This is the cheese curd.
  • Drain remaining liquid (whey) from cheese curds.
  • Press, salt and age cheese curds.

Cheesemaking completely depends on casein and the interaction between casein and chymosin. Casein is actually four different proteins:

  • Alpha-s1 casein
  • Alpha-s2 casein
  • Beta casein
  • Kappa casein

These proteins all fold up into globular structures called micelles, with the kappa caseins coating the surface of the micelles. The kappa caseins have hydrophillic ends that stick out into the watery milk and hydrophobic ends that remain inside of the micelles. This arrangement allows large amounts of protein to remain in solution (~3.5% for cows, up to ~12% for whales) enabling easy delivery of high amounts of nutrition to the baby mammal in liquid form. When the milk reaches the baby mammals stomach, chymosin enzyme cleaves the hydrophillic tails of kappa caseins, making the protein rapidly drop out of solution (within ~1 hour) and entrapping both milkfat, water and some of the remaining lactose. While rennet used to be extracted from calf stomach linings, most of the rennet used in cheese production today is produced by genetically engineered microbes grown in large bioreactors. We intend to do the same for casein.

The plan is to take the genes that code for all four caseins from the known genetic sequence of an existing mammal, put those genes into yeast and grow yeast in bioreactors to produce vegan caseins. Those caseins can then hopefully be combined with water and vegan milkfat and lactose alternatives to create a kind of vegan milk containing the casein micelles we see in nature.

Overview of genetic engineering

There are two main ways to add genes to an organism. One way is to insert the new genes into the chromosomes of the target organism. This is called chromosomal integration and can be desirable since it is a very permanent and stable form of genetic modification, but it is somewhat more involved. The other way is to insert an extra circular piece of DNA (called a plasmid) into the cell of the target organism. This plasmid functions like a small artificial chromosome and apart of the genes being inserted (often referred to as the genes of interest or GOI) it contains an origin of replication (ORI) which is a sequence recognized by the cellular machinery that replicates the chromosomes during cell division, and usually contains regions that are recognized by different restriction enzymes (these are the enzymes that let you cut and paste DNA). The plasmid is a less stable form of genetic engineering. This is because the extra plasmid and genes carry with them an extra burden of work for the cell, so cells that manage to get rid of it (by random mutation or by error during cell division) have an advantage in being able to replicate faster and potentially out-compete the plasmid-burdened cells. For this reason the plasmid will usually contain a gene that confers some sort of survival advantage specific to the growth media the cells are grown on. A common example is a plasmid containing a gene that confers resistance to kanamycin (a common antibiotic) with kanamycin added to the growth media. This is referred to as selection or using a selective media. A plasmid used in this way is sometimes referred to as a vector or construct.

The Real Vegan Cheese project currently uses a plasmid vector. It uses a so-called shuttle vector which is a dual-use plasmid that can work in two different organisms. Our shuttle vector is made to work in E. coli as well as S. cerevisiae. E. coli is easy to work with and by growing more E. coli containing the plasmid one can rapidly generate large amounts of plasmid which can then by extracted from the cells using a so-called miniprep kit.

The Real Vegan Cheese shuttle vector has an antibiotic resistance gene (TODO which one) for selection in E. coli. For selection in S. cerevisiae we're using a strain of S. cerevisiae which is missing URA3, one of the genes necessary to produce uracil, while the shuttle vector contains the URA3 gene instead. The growth media is then specially prepared so it does not contain any uracil, meaning that only yeast cells containing the shuttle vector plasmid with the URA3 gene can survive.

Gene design and synthesis

The DNA sequence to be inserted into the plasmids are designed on a computer based on natural genetic sequences and ordered from a synthesis company such as IDT and their fairly cheap gBlock service. The DNA arrives and is inserted using some existing system usually relying on enzymes to paste the synthesized DNA into the linearized plasmid, creating the circular plasmid. We're using the Electra system from DNA 2.0. It's very easy but somewhat expensive. This section focuses on the work that has gone into designing our genetic sequences.

The genetic code

The genetic code consists of the four nucleotides G, A, T and C. DNA consists of these nucleotides, which are transcribed into RNA and from RNA translated in triplets into chains of amino acids otherwise known as proteins. This is known as the Central Dogma of biology. Transcription and translation are regulated by a number of factors, some static, but many which are dynamic and up and down regulate production of specific proteins based on anything from the sequence of the surrounding DNA to signals originating outside of the cell.

Each nucleotide triplet is translated into one amino acid, e.g. AGT is translated into a single Serine. Since proteins consists of only 22 different amino acids, but there are 4*4*4=64 different nucleotide triplets, multiple different triplets code for the same amino acid.

TODO talk about tRNAs and codon optimization.

Codon optimization

ToDo

For a more in-depth explanation see the questions and answers page.

There is an open source codon optimization tool available called pysplicer.

Post-translational modification

ToDo

Secretion

ToDo

Link original secretion article

Proteases

ToDo

Synthesis

ToDo

Genetic modification (cloning)

Plasmids

ToDo

Transformation

ToDo

Salmon sperm DNA replacement.

Sequencing for verification

ToDo

E. coli and shuttle vectors

ToDo

Growing yeast

ToDo

Detecting casein production

ToDo

Scaling up

ToDo

Producing vegan "milk" and cheese

Once we can produce vegan version of the four caseins, the next step is to make them fold correctly into micelles in water. If the micelles form correctly, then we should be able to make some type of cheese.

While this part of the project depends on our ability to produce vegan casein, there are still some relevant experiments that can be done beforehand.

One preperatory experiment is to ensure that we can actually produce cheese using normal milk. We have documented our attempts at cheese making including our home-made cheese press and web-enabled cheese incubator. In the long run We plan to partner with existing skilled cheesemakers.

A more advanced experiment is to make cheese from purified non-vegan casein powder combined with vegan alternatives to milkfat and lactose. If we manage to create a decent cheese using this process, then we know that our vegan caseins can be turned into a similar cheese. So far one experiment has been succesfully completed. Mixing water and casein powder does not get the casein powder into solution. It would seem that the micelles are either clumped or mis-formed in the dried casein powder. Our second experiment established that sonication and a small amount of baking powder was an effective way of getting casein to stay in solution]]. Since the casein stayed in solution, it seems likely that the micelles are formed correctly after sonication.

Selecting suitable vegan replacements for lactose and milkfat are also major challenges.

We are documenting our efforts here.

Essential knowledge and further reading

While this document should have provided you with a rough overview of the Real Vegan Cheese project, prospective team-members can benefit from reading a few of the more essential texts we have encountered along the way.

For a both in-depth and very readable introduction to cheesemaking, have a look at American Farmstead Cheese: The Complete Guide to Making and Selling Artisan Cheeses. To get a quick overview, just read chapter five "The Eigh

ToDo finish this section.

Conveniently some unknown pirate seems to have gathered these resources for download here.

We recommend the following free and open source software to download and read these books and articles:

Getting your hands dirty

If you are ready to roll up your sleeves and start doing some work, see the getting involved section on the main page of the wiki.

How to conduct research using online literature

ToDo

Access to tools

Access to tools and facilities for this type of work can be difficult to obtain. If you are working with us in the Bay Area then you can become a member of Biocurious in the South Bay and use their facilities. This requires passing some basic safety training. We are hard at work setting up Counter Culture Labs, a new community lab in Oakland.

If you are not in the bay area, then DIYbio.org has a list of local groups engaged in biohacking. Some of these groups have laboratory facilities, but many do not. If you are in a company with free or cheap college/university education, then signing up for a lab course is a great way to gain experience. If there are pre-requisites then do whatever you can to bypass or social engineer your way around them, as long as they are not safety courses. Always take courses related to safety or sterile technique first if available!

You can also set up your own home lab. In the U.S. craiglist, ebay, labx and biosurplus are good sources for used gear. University dumpsters can be excellent sources of old and working or broken but fixable gear. Most of the essential equipment can be improvised from off the shelf equipment, but the cost of reagents can still be daunting. If a company won't sell you reagents, try ordering with a fictitious corporation at a commercial address (you probably know someone who will let you use their commercial address) or set up a real corporation at a commercial address (it's neither difficult nor expensive).

Access to knowledge

Unfortunately many textbooks and scientific articles are not available under free and open licenses. While a more in-depth guide is currently being written, here are some ways to get access:

Scientific Articles

University access

If you have a University login already, you should be able to use your University's proxy server to access scientific articles. If you don't, someone you know may be willing to lend you their login. It may be worth it to sign up for a class at a community college to get a login (that may not expire for a long time after the end of the class) but all university access is not created equal so you may still be denied access to some journals.

If you are willing to take some amount of risk then you can trade or buy logins from some of the shadier forums online (ezproxy is a good keyword), or you could simply set up something like the WiFi Pineapple at a place where University Students hang out, and phish for login information. Many students never change their passwords unless forced and if you only use it for article access it is unlikely to be detected, but use Tor to be safe.

Library Genesis

The main Library Genesis Search Engine also has scientific articles. They have a surprising amount of articles for direct download.

sci-hub.org

If you can't find your article anywhere else, try sci-hub. This site is awesome. It has an astounding amount of articles and if it doesn't have the article you need it will use one of a set of hacked paid accounts to download the article and serve it up.

As of this writing it is in hibernation attempting to get donations to continue operations.

Request on mailing lists

Asking on one of the following mailing lists is a good last resort for getting access to an article:

or you can try the mailing list of your local hackerspace which can always be found via Hackerspaces.org or for bio-hackerspaces try the diybio.org list of groups.

Books

Library Genesis

The Russian pirate library Library Genesis is awesome. They have a large amount sof all english language textbooks for free direct download. They cap their download speeds at a few KB per second, but books are usually small downloads anyway. Sometimes the downloads will fail part way through. Use something that's capable of restarting a failed download just in case (e.g. Firefox).

There are several mirrors of Library genesis, and not all of them are complete. I recommend trying Bookfi.org first (it's faster) and if that doesn't work then try The main Library Genesis Search Engine.

Pirate from open torrent trackers

If you are lucky you can find the book via one of the open torrent trackers. These are good places to start:

Pirate from private torrent trackers

There are many private torrent trackers. Most of them are of much higher quality than the open trackers and several are dedicated to ebooks.

Some of them have an interview process. Some of them are invite only. All of them require the user to upload at least as much as they download and follow a set of strict rules. This can be somewhat technically challenging as it can require a bit of strategy in selecting what to download (downloading only unpopular things means no-one downloads from you), or it may mean that you need to set up port forwarding or even a dedicated seed box to live up to the seeding requirements. This may seem daunting, but once you know how it works and have access it can be a great resource and community!

The private trackers generally do not like to be mentioned on other websites and we don't want to attract their ire, so if you want to go this route, talk to us in person.

Libraries

Go to OpenLibrary.org and search for the book. If you're lucky there is a free download available. If you're less lucky you may be able to borrow a digital edition via your local library. If you're eve

Even if OpenLibrary doesn't list the ability to borrow a digital edition, your local library may still have a digital edition. Check with them directly.

Even if no copies are available at your local library you may be able to borrow it from a remote library, though it may take some time. Many libraries allow you to request that they buy a certain book and some will let you sign up to be the first to borrow it once it has been bought.

Liberate from Amazon

If you really can't find the book anywhere, check if there is a digital edition available for sale on Amazon. If there is, simply download and install the free Kindle application for your operating system. Then purchase the digital edition from Amazon. It should download into your Kindle application. Now close the Kindle application and download and install Calibre (the free and open ebook management and reading application)/. Follow this guide to install the the DeDRM plugin for Calibre. This plugin will allow you to remove Digital Restrictions Management from the book you bought from Amazon. Now open the book you bought from Amazon. You may have to search for the file for the book to figure out where the Kindle application saved it. Once it is in Calibre, use the DeDRM plugin to remove the DRM. Make a copy of the book with the DRM stripped somewhere safe just in case. Now you can return the digital book to Amazon for a full refund. You may have to do a bit of searching to actually find out how to do this, but it _is_ possible. Once it is returned you have your free ebook that you can share with your friends without restrictions! Hurry up and put it on your favorite torrent site!

It almost goes without saying that you don't want to do this too often from the same Amazon account.

Buy used

For many books you can find a used copy cheap from a third-party reseller via Aamazon. If you have to resort to this, then please ensure that the book is scanned and made available for others. Talk to us about access to a book scanner at info@realvegancheese.org

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