The tools of biotechnology can avert human civilization’s looming mass extinction caused by fossil energy. However, intellectual property, skills, and high costs of hardware and wetware create massive barriers. These barriers were profoundly felt throughout the pandemic as sparse access to biomanufacturing infrastructure severely limited medical relief, testing, and vaccination. With an internationally distributed team of 10 nationalities and 4 laboratories, we aim to break these barriers by frugally producing and purifying thermostable enzymes essential for synthetic biology solutions. We have established a pipeline for enzyme production in frugal bio-foundries that’s based on open-sourced software, hardware, material transfer agreement, and wetware constructs of B. subtilis for reducing protein extraction costs. Our genetic constructs will be freely distributed by FreeGenes. To gauge the global needs of implementing a frugal biofoundry in different parts of the world, we set up a hackathon in partnership with JOGL. Our project contributes towards democratising biotechnology.
Team:Open Science Global/Description
Abstract
Introduction: The Global Crisis
We live in a dichotomy. Half the world is getting more prosperous, healthier, better educated, more peaceful, and better connected, whereas the other half suffers from poverty, hunger, and inaccessibility to primary healthcare. Then, there are common problems that all of us are facing together, like the climate crisis, global warming, and currently the wrath of the COVID-19 pandemic. We need massive worldwide political, economic, social, and technological mobilization to transition humanity off fossil energy and materials, build a sustainable civilization and avert mass extinction. Electrification, renewable energy, and clean transportation infrastructure can supply society with the joules and Newton-meters to replace fossil fuel power plants and the internal combustion engine. But what about the atoms?
We need to eliminate and replace the fossilized carbon polymers and fossil energy subsidies at the material base of our modern civilization. We have to change how we make cement, fertilizers, protein-rich food, etc., keeping in mind all these concerns! We need to ruggedize our global and local production capacity to make agricultural, industrial, and energy systems resilient to the disruptive climate shocks we’ve already locked into.
Where do we even begin?
We need to find common ground that has limited progress in all these frontiers. But, what’s the point if the problems of the world exceed the problem solvers? And now we have one more issue to add to the pile.
After brainstorming with the general public, scientists, hobbyists throughout the world, we realized
that biotechnology and synthetic biology have tools that could be applied to approach every one of
these problems. The tools in our problem-solving toolbox are
The Friendzymes Project
This is where we come in, Team Friendzymes! Last fall, in PrakashLab’s global Frugal Science class, some of us met with the common goal of wanting to democratize biotechnology. Our idea of democratizing biotechnology meant sharing resources to anyone and everyone despite their background knowledge. Our team expanded through the year incorporating team members from 10 nationalities and including four community laboratories involving people with different backgrounds and skillsets.
Our inspirations are our partners.
But, we were not alone in this pursuit. There have been initiatives before us that have seen the need for exactly the same. The FreeGenes initiative, Open Insulin project and Open MTA were crucial projects where we sought our inspiration and relationships for and beyond iGEM. The Biobrick foundation’s FreeGenes project contributes to the democratization effort by free distribution of bio-security screened, off-patent and IP-free DNA parts to anyone in any part of the world provided they share the genetic constructs they make openly accessible to all through Open MTA. With the Open Material Transfer Agreement, a wetware analog of open-source software licenses, anyone can build an unambiguous biotechnological commons of genes & cells, with full rights to modification, re-sharing, and commercialization.
The Open Insulin project used both of these resources very well. They tackled the problem of insulin production, a hormone necessary to keep diabetes in control. They created models of insulin which can be cheaply and sustainably made in small-scale manufacturing units using the FreeGenes DNA plasmids and DNA sequences. All of their insulin models were under OpenMTA , which eliminated the expensive importing and exporting costs of insulin.
Seeing the progress and success of Open insulin, we knew that our eventual direction was to enable local production just like Open Insulin. Scott Pownall, major part of Open insulin, joined hands in our initiative. With the growing popularity of bio-foundries worldwide[1], we focused on empowering people to tackle these global crisis problems in a bio-foundry setup.
But, first of all, what is a bio-foundry?
A biofoundry is an integrated molecular biology facility that includes robotic liquid-handling equipment, high-throughput analytical equipment, and the software, personnel , and data management systems required to run the equipment and broader biofoundry capabilities. Biofoundries marry synthetic biology with automation engineering to create new high-throughput biological solutions that help build and strengthen a Design-Build-Test-Learn (DBTL) approach to biological engineering.
Establishing a biofoundry is a significant investment and requires more than simply setting up a well-equipped physical space. The emphasis on high-throughput methods requires simultaneous attention to software, protocols, and the integration of physical and digital infrastructures to efficiently prepare and track samples. In this respect, biofoundries are at the forefront of a paradigm shift in biological engineering toward a more automated, design-focused venture.
So, what are the workhorses for any synthetic biology solution that needs to be enabled in these biofoundries?
Enzymes!
Basic enzymes like polymerases,... are used in almost every experiment and design in synthetic biology and beyond. That was where we wanted to focus our attention for this iGEM 2021.
We want to democratize biotechnology by setting up a frugal pipeline for enzyme production and purification that can be adapted in a biofoundry. As we are based on five different continents, we could essentially build our own distributed mini-biofoundries where we perform different parts of enzyme production at multiple places simultaneously, and share it within this distributed network. Essentially, setting up a ‘bionet’.
There are wetware, software, hardware, IP and policy barriers that we’ve tried to tackle using this year’s project.
Barriers in Synthetic Biology
Challenge: E. coli requires expensive equipment to extract recombinant protein. Expensive sonicators or french presses needs to be used to get the enzyme out of the bacteria.
Our Approach We decided to secrete enzymes using non-standard model organisms like Bacillus subtillis and Pichia pastoris. The main advantage we found was that it allowed us to bypass expensive hardware required for popping open E. coli cells and scalable protein extraction.
Additionally, we also decided to make some clever wetware designs that will also let us perform the DBTL cycle faster and easier.
We created B. subtillis Genetic toolkit that has promoters, secretion tags, selection markers, terminators and homology arms for genomic integration and protein expression It is a plasmid library we designed that helps us (and everyone) to deal with the ~150 secretion tags of B. subtillis. We also made strategies for both replicating vectors and multi-copy recombinant gene integration in the genome, while avoiding genetic instability. If there are identical DNA sequence repeats nearby, it causes genetic instability and we addressed this through our software. Further details about our wetware strategies and how we dealt with strain selection, DNA propagatio can be found in our Wetware pages.
Challenge: High-Throughput Strain Engineering for High-Yield Protein Secretion
Our Approach Frugal biofoundry for high-capacity, semi-automated biological DBTL cycles on a low-cost liquid handling, DNA sequencing nad plate reading instruments. We’ve used OpenTrons OT-2 Liquid Handler, Oxford Nanopore MiniION DNA sequencer and a frugal plate reader. See more about how we made this happen in our Hardware subpage
Challenge: Frugal Protein Production. Standard method is to use Baffled Glass Shaker Flasks or Bioreactors all of which are very expensive ~$1000-$10000 of dollars.
Our Approach Frugal Shaker Flask: This can be made from a 2L pepsi bottle. The bottom of the Pepsi bottle has the same functional design as that of a shaker flask! Frugal Bioreactor: We modified design from Sebastian Cocioba’s bioreactor to suit our needs. It can be frugally produced under $100. The build can be found in Hardware subpage
Challenge: Protein purification for specific needs. Standard method is to use expensive Immobilized Metal Ion Affinity Chromatography column (IMAC). It costs ~$100 for Nickel IMAC resin and ten times that for the chromatography instrument!
Our Approach
- For thermostable enzymes - pursue secretion and heat purification. Phusion polymerase is one such
- For reagent grade enzymes ~90% purity, pursue secretion and dialysis using food wrap and potato chip bag clips. It would be similar to the dialysis set-up generally performed.
- For high purity enzymes ~99% purity, pursue frugal sand chromatography with silica binding tags
Addressing all of these challenges we want to manufacture a distributed way to manufacture enzymes in different bio-foundries around the world.
With an internationally distributed team, we felt it more compelling to address that this democratization should happen with an international vision, tackling barriers within each local community in different countries. Our human practices effort was seamlessly integrated into each aspect of our project and we learnt a lot from the synthetic biology community to be able to give back to the community! Check out our Human Practices
References:
[1] https://academic.oup.com/synbio/article/6/1/ysaa026/6039187
[2] https://www.nature.com/articles/nbt.4263 - paper for OpenMTA by Linda Kahl et. al (Jenny Molloy, etc) - our inspirations