The Sporenet Protocol


If we look back on the emergence and rise of the internet, there was and still is a fundamental conflict: Information is valuable, and information wants to be free. Education is the great equalizer, and the internet has enabled people without access to traditional educational systems to educate themselves.

Information wants to be free.

In biotechnology, this idea still applies, but in a slightly different way. The Human Genome Project can easily be summed up in 6 words - "Bought the book; hard to read" (Eric Lander). In the DNA synthesis world, our current efforts can be summed up with "Easy to write; nothing to say" (Drew Endy). We are in an interesting point in history where our ability to build with biology is becoming greater than our ability to design with biology. The DNA information itself is valuable, but what is even more valuable is meta-information of how to use DNA technology and what to use it for.

So - how do we get better at using DNA and the information surrounding it? Wikipedia offers a decent model with many writers contributing to a pool of information, with this model being adopted in biotechnology with the iGEM Registry of biological parts. One of the essays which was influential in the development of Wikipedia, "The Use of Knowledge in Society", contains the core concept that I believe is most important for understanding the success of Wikipedia - the idea that knowledge is decentralized and distributed throughout society. The knowledge, context, and skills of what to use biotechnology for is also distributed throughout society, but the opportunity to engineer life, so far, is concentrated in a few places.

iGEM Registry

The Use of Knowledge in Society

Information is valuable but wants to be free, we are becoming increasingly skilled with building with biology, and knowledge, context, and skills for the uses of biotechnology are distributed throughout the world. If we want to get better at engineering life and using engineered life, we need to give more people the opportunity to engineer life. The tools to engineer life are contained in plasmids throughout the world, but they aren't widely distributed beyond academic institutions because of a few prohibitive problems. I have a potential solution to the problem of plasmid distribution, which I call the "Sporenet" protocol. I hope this protocol becomes a foundation for a more equitable biotechnological future.

Some of those prohibitive problems

The Sporenet Protocol

The basic idea of the Sporenet protocol is to store and ship plasmid DNA in Bacillus subtilis spores. Bacillus subtilis spores have the following advantages:

To distribute a plasmid using the Sporenet protocol, you'd first have to transform it into Bacillus subtilis. After transformation, you would grow a deep-well plate of Bacillus subtilis and force them to sporulate. This can easily be done by leaving the deep-well plate an incubator for a week. After this, color the liquid in the wells with some kind of food coloring, and stamp onto a sheet of normal paper with a 96-well pinner (the food coloring makes the dots of spores visible).

To recover a plasmid using the Sporenet protocol, you would punch out a food-colored dot and resuspend it in liquid media. After ~8-12 hours of shaking incubation, the liquid media should be turbid with living Bacillus subtilis cells.

Storage at room temperature means that no expensive freezers or cold chain capacities are needed to ship and use the spores. In my testing, I've seen that spores can be sent on paper and are recoverable over a year after shipping them through the post office in a normal letter. Combine room temperature storage and letter-based shipping with a low production cost (simply add spores to paper) and you have a combination that can be far more accessible than the status quo of plasmid distribution.

Challenges to Sporenet

There are 2 current alternatives to the Sporenet protocol when it comes to plasmid distribution - agar stabs and raw DNA. Agar stabs have the disadvantages of having a short shelf life and low-density while raw DNA has the disadvantages of high production cost and recovery cost. However, they both require little upfront work to deploy, since typically plasmids are in Escherichia coli already. Only time will tell if the long shelf life and low production costs of spores will outweigh the hefty upfront costs of conversion from Escherichia coli vectors to Escherichia coli - Bacillus subtilis shuttle vectors.

In addition to the upfront costs of conversion, several new protocols also need to be developed. Should PCR be the primary recovery method of plasmids from spores, or would minipreps work? How efficient are Bacillus subtilis minipreps in comparison to Escherichia coli minipreps, and what modifications have to be done to the normal miniprep protocols to increase their efficiency for Bacillus subtilis? Which origin of replications work best in Bacillus subtilis in terms of high copy number and stability?

Without coordinated and concentrated effort, it is unlikely that the Sporenet protocol will come into being. It'll be challenging to use at first, and there will be hiccups, but I believe that in the long term, we can solve the problems around plasmid distribution and give the opportunity of genetic engineering to thousands of people who otherwise would have been ignored by the status quo.

Keoni Gandall

Other historical notes

I was first interested in Bacillus subtilis at 14 years old. I was inspired by Cathal Garvey's spore germination transformation protocol to really start looking into Bacillus subtilis. What isn't written there explicitly is that one of the big reasons I was attracted to Bacillus subtilis was its ability to spore. I didn't have a -80c freezer at the time, so that was super important to me. However, my primary driver to this organism was its natural competence system.

A DIYbio post from that time

Cathal Garvey's spore germination protocol.

I wrote a series of memos for the FreeGenes Project, asking for DNA synthesis to pursue the idea of Sporenet. Though none of them got synthesized in the end, they reflect a few different ideas that are interesting. In particular, #3 gives a possible method to do zero in-vitro work for plasmid conversion.

Sporenet #1: Synthesize vectors

Sporenet #2: Local Enzyme Production

Sporenet #3: Universal Landing Pads

The idea of shipping spores on paper isn't new. I discovered this technique when I got a shipment of Bacillus subtilis from the Bacillus Genetic Stock Center, which is the best genetic stock center I've ever interacted with. A personal thanks to Dr. Daniel R. Zeigler, who was kind enough to provide me with Bacillus subtilis stocks when I first began genetic engineering.

Bacillus Genetic Stock Center

I haven't have any problems with Bacillus subtilis spore contamination of other cultures. I think this is mainly a worry because of how mold spores spread throughout labs, but I have not found this to be an issue with Bacillus subtilis.