Please check out full PDF (with images) at Documents tab also.
Plastics are great in many ways, but the main problem with plastic is our current usage is not sustainable. Oil reservoirs are used as raw material and source of energy to produce plastic. Yet big amount of the plastic production is discarded rapidly. On the other hand, the total mass of plastic waste in landfills and in natural habitats are increasing sharply for example in ocean will be more than fish by year 2050.
The leaching of chemicals from plastic products to soil and water resources can threat wildlife and humans. Leaving plastics can make physical problems for wildlife resulting from ingestion or entanglement in plastic. Therefore, every living creature is impacted by the problem especially Sushi lovers!
Environmentalists can collect plastic bags at beaches, but what happens next? Most current PET recycle technologies (mechanically or chemically) could only down-cycle PET, because the product (e.g., pellet) will lose the original physical properties like strength, thermo-durability compared to virgin PET from crude oil.
Polyethylene terephthalate (PET) is one of the major plastics. PET can degrade into its monomers and re-polymerize into PET fibre by using enzyme called PETase and other accompanying enzyme. In molecular level, there is no difference to a Virgin PET. We want to establish a decentralized PET plastic degradation system, that serves as an anti-thesis to expensive capital expenditure (e.g., recycle factories) that only rich countries can afford.
A DIY bioreactor that uses PETase to degrade PET in a safe and easy process could be one of the optimum solutions. It can tackle plastic waste problem of waste plastic at source and reduce the shipment cost.
Biofoundries located at 3 regions in the world (Asia, Europe and Americas) will improve and deploy those enzymes (wild type or mutant) to bio enthusiasts, environmentalist, and eventually general citizens.
It will be a two-phase implementation. 1st phase that requires no genetic modification. We borrow the idea of "Bokashi" compost that houses the host bacteria (I.sakaiensis) in a closed space with the right environment, where no monomer is produced, but rather all broken down to CO2 due to metabolism. Arguably this is not a full loop. More narratives can be found here.
2nd phase is where we aim for, a closed loop of PET-monomer-PET by using a genetically modified PETase, where bio foundries are expected to clone, ship and promote in a OpenPETase ecosystem a frugal household bioreactor that uses the enzyme.
bioreactors->freeze drying->enzyme powder/tablet
Affordable/free enzyme +buffer/H2O solution->Active enzyme
monomers in the solution->OpentPETase->free enzyme, buffer
Locally, bio foundries will facilitate dispatchin biostacks to downstream bio enthusiasts, be it solo individual or community labs in their respective geographical regions. Bio enthusiasts can start the punk trend of PET degradation. Environmentalists, not necessarily knowledgable in microbiology can come in, learn from bio enthusiasts, and hopefully some of them influencers who can set the trend. The general citizen, in multitudes can catch the trend, say owning a PET compost at home in a fashionable way.
Globally, these 3 bio foundries will have an Open MTA signed by default, to streamline transfer of plasmids (containing mutant PETase) or bacteria consortium (to improve compost efficiency). They will also complement each other over legal constraints at own region. For e.g. under a frugal setup, Europe may not be able to do CRISPR easily, but US can. Then, US can perform and deliver genetic modification, while Europe can focus on cloning, or perhaps designing more efficient bio reactors.
How frugal biostacks help
increase population of bio enthusiasts (either solo individual or community labs), and eventually general citizens to degrade PET.
Provide scientific support for the procedures required to produce PETase more available and affordable globally
Introduce the team to the expert networks in different areas includes biochemical, engineering and marketing
What are those frugal biostacks
•Compost, BPA is additive for most plastic and will promote bacterial growth
•Bioreactor, incorporating BPA will promote I.sakaiensis growth
•Compost and bacteria culture protocol
•Regulation arbitrage among 3 regions (1 region can do something the others can’t)
•EU regulations on bacterial shipment(?pathogen or not?//?GMO or not?)
•Regulations on Buffer shipment
1. Foundries can genetically modify more efficient mutant PETase
2. Foundries can design better compost chamber or DIY bioreactor, and share the blueprint open-source
3. The participating Bio enthusiasts are incentivized in a Circular Bio Economy (e.g. PETcoin) to degrade PET and evangelize general citizens (starting with Environmentalists).
By 2035, need a global deployment for a global problem. Otherwise by 2050, plastics in ocean will be more than fishes.
1. human resource:
-biohackers from all over the world:
to make better PETase enzyme; to find better host organism; to make cheaper better purification processes.
to make machines for Composting via Ideonella sakaiensis for Composting with the enzyme only. To make software for said machines connect software with information from devices.
to establish PETcoin in 2025-2030
Check the legality of regions
In some areas of Eu, there are strict rules against GMO’s and in some parts, enzymes are treated/categorized as reagents so the only possible way for in home or mobile engineer group degradation is using the Ideonella sakaiensis itself. But there are strict rules about bacteria. Ideonella sakaiensis might be treated as a pathogen so our goal can’t be achieved that easily.
Plasmid shipment during 2nd phase (as example of frugality)
Shipping and Receiving Plasmids on Filter Paper:
1) Mark a circled area with a pencil (not a marker pen) on a clean Whatman #1 filter paper (or equivalent).
2) Spot about 2 µg of plasmid DNA into the circle. Allow the filter paper dry at room temperature.
3) Insert spotted filter paper inside a plastic bag and seal it.
4) Send by regular (air) mail.
1) To recover the DNA, use clean gloves and cut the marked circle area that contains the dried plasmid DNA.
2) Using clean forceps, insert the filter paper into a 1.5 ml micro centrifuge tube. Add 100 µl of TE buffer (Or molecular biology water), tap on the tube to mix, and incubate at room temperature for 10 minutes. Tap again and centrifuge the tube for a few seconds. (Do Not Vortex).
3) Remove about 3-5 µl of supernatant for use in transfecting E. coli by electroporation or chemical means. Please do not try to use the DNA directly for any application other than to transform bacteria and prepare a plasmid stock.
4) Store the remainder of the filter paper/TE mix at -20 or -80 C as a permanent archive in case that your plasmid stock ever gets lost or if something turns out to be wrong with it
- Whatman #1 paper is cheaper alternative
- plasmid shipped this way hardly ever degrade
-New technique (trouble shooting)
Paperless Plasmid Pouch (PPP) Method V1.0 1):
1, Add a few uLs of purified plasmid into vacuum sealing pouch. Anywhere between 500ng and 2ugs seems like plenty.
2) Open the pouch so the water can evaporate, the fewer uL you put in the pouch the better. Dry in a dessicator or a centrivap if you have one.
3) Vacuum seal the pouch for the sake of fanciness
DNA plasmid shipping in TE buffer
TE Buffer cold package (blue or dry ice) for shipment degradation is low but ice packages are needed
1. Ideonella sakaiensis:
- bacteria that degrades PET (A bacterium that degrades and assimilates poly(ethylene terephthalate), Science, Yoshida et al)
-enhanced degrading of PET(Low modification of PETase enhances its activity toward degrading PET: Effect of conjugate monomer property)
-heat denaturation resistant PETase (An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films)
3.BPA toxicity/growth promoting:
-Growth Promotion(The rapid degradation of bisphenol A induced by the response of indigenous bacterial communities in sediment)
4.Freeze drying PETase:
-(It is also noteworthy that in this study, PETase was freeze-dried and shipped between continents, and that it retained similar performance profiles after freeze-drying, which is a positive feature for its potential use in applications that require enzyme production and use be distinct, as it would potentially be the case for most biobased recycling options.)
5. Thompson, R. C., Moore, C. J., Saal, F. S. V. & Swan, S. H. Plastics, the environment and human health: Current consensus and future trends. Philos. Trans. R. Soc. B Biol. Sci. 364, 2153–2166 (2009).
6. Kawai, F., Kawabata, T. & Oda, M. Current knowledge on enzymatic PET degradation and its possible application to waste stream management and other fields. Appl. Microbiol. Biotechnol. 103, 4253–4268 (2019).
7. Zimmermann, L. et al. Plastic Products Leach Chemicals That Induce In Vitro Toxicity under Realistic Use Conditions . Environ. Sci. Technol. 55, 11814–11823 (2021).
8. Yang, C. Z., Yaniger, S. I., Jordan, V. C., Klein, D. J. & Bittner, G. D. Most plastic products release estrogenic chemicals: a potential health problem that can be solved. Environ. Health Perspect. 119, 989–996 (2011).
- Short Name: #OpenPETase
- Created on: October 2, 2021
- Last update: November 4, 2021