Proposed Implementation

“Human-induced climate change is already affecting many weather and climate extremes in every region across the globe. Evidence of observed changes in extremes such as heatwaves, heavy precipitation, droughts, and tropical cyclones, and, in particular, their attribution to human influence, has strengthened since the Fifth Assessment Report (AR5).” - Sixth IPCC Report, Summary for Policymakers


The data is indisputable, climate change is already negatively impacting the lives of millions, some of these changes are irreversible, and the effects will only get more pronounced in the decades to come. Agriculture as we know it has provided the basis for human civilization to grow into what it is today. And it will have to change. Not only because it is, in part, responsible for the climate crisis we face; millions of hectares of forest are being destroyed each year [1], while it alone produces circa 11% of all anthropogenic greenhouse gases [2], but also because it won’t be able to continue feeding the growing human population in an increasingly unpredictable climate. We need to act, now.

Who Are the End Users?

By introducing a prototyping platform with the potential of significantly accelerating plant synthetic biology, OpenPlast opens up a series of new possibilities. There already are examples of plants being engineered to grow in salty [3] or dry [4] conditions. Given the chloroplast's elevated capability for protein production, it offers a great platform for synthesizing proteins such as vaccines, BAS or insulin. Additionally, through the use of more complex metabolic engineering approaches, that potential could even be harnessed to fixate nitrogen from the atmosphere [5], reducing the need for chemical fertilizers. After consulting with specialists working in the industry, our team learned that our project is contributing to this future. Current transformation methods are the main reason for year-long iterations of each DBTL cycle, and the amount of iterations needed for metabolic engineering makes transplastomics non-viable for the industry. Cell-free systems, on the other hand, make it trivially fast to test many genetic constructs in parallel - in fact, we established a workflow that goes from idea to expression data in less than 8 hours - putting more complex projects in chloroplast biotechnology within our grasp. This could be an invaluable tool for research groups (and iGEM teams) around the world wanting to use the chloroplast as a platform, but who might be dissuaded by the large time required for in vivo work or might lack the necessary means to do so.

And the potential of cell-free chloroplasts doesn't stop there. It took a lot of resources and work from very dedicated people to produce the relatively small volume of cell-free systems (CFS) we used, but we hope future upscaling will reduce costs, making them commonplace in plant synthetic biology labs. The possibility of freeze drying extracts for long term - ambient temperature - storage has the potential to make it an off the shelf solution for rapid prototyping [6], a scrapbook for jotting ideas before turning them into a work of art. But what are the masterpieces going to look like?

Applications Inside and Outside the Lab

The process of synthetically fixating nitrogen to produce the chemical fertilizers that sustain modern agriculture is one of the main emitters of greenhouse gases, being responsible for 2% of global energy usage [7]. It not only pollutes our atmosphere, but it’s overuse is a direct cause of water eutrophisation. Engineering nitrogen fixation in plants currently incapable of doing so on their own would be the Holy Grail of plant synthetic biology. We envision OpenPlast and other similar CFS approaches to be the prototyping platform that makes this dream come true. Benefits of implementing it in the chloroplasts include the possibility of leveraging their internal supply of reducing agents, such as NADPH and ATP, to drive the fixation process. Furthermore, ammonia products could be directly integrated in the glutamine synthetase/glutamate synthase pathway [8]. This is no small undertaking, but the possibility of characterizing hundreds of genetic parts in a fast and high throughput fashion can cut down the development time by decades. Moreover, we have also demonstrated the feasibility of using OpenPlast to screen for new selection markers. High throughput screening can be implemented in the future to establish transformation protocols in plants that are currently hard or even impossible to transform.

Another way of increasing crop efficiency without increasing fertilizer use is by improving photosynthesis itself. While it is a remarkable biochemical process, the vast majority of plants (including wheat and soy) fixate CO2 in a process called C3 Photosynthesis. This mechanism is highly inefficient and limits their growth in arid conditions. Plants that do thrive in regions with higher temperatures and solar intensity have evolved a more efficient carbon fixation pathway: C4 Photosynthesis. Hence, engineering it in previously C3-reliant plants has been another major goal for synthetic biologists [9]. Because most of this reaction takes place in the chloroplast, major engineering in the plastome would be indispensable to accommodate a whole new carbon fixation pathway. Other research groups are interested in completely replacing the carbon fixation pathway, essentially designing a better and more efficient one from scratch [10].

By removing the need of generating in vivo transformants for each one of the dozens (maybe even hundreds) of design cycles necessary to create these amazing new crops, OpenPlast can speed up their implementation in the real world. The process of engineering new crops is only half of the work to actually bring them to market though. In our conversations with members of the industry and regulatory agencies we confirmed, that the process of performing the necessary field studies and getting approval for commercialization significantly extends the time needed to start distributing the seeds. Not only that, but public concern and hesitancy on the topic has further prevented a wider adoption of biotechnology, we fear it might even hold back the development of a new generation of engineered traits, such a novel carbon fixation pathway.

Safety Considerations & Dual-Use

One of the main concerns on the minds of consumers and policymakers is the possible ecological effects of gene flow from genetically modified (GM) crops to native species, possibly reducing biodiversity and destabilizing the ecosystem. Evidence shows that one of the main vectors for gene-flow is the pollen of GM plants [11, 12], which can travel hundreds of kilometers and pollinate plants in non-GM fields or native - closely related - relatives [13]. The implementation of transplastomic plants can mitigate this danger: because the chloroplast is maternally inherited in most plants, they do not get passed on via pollen. This limits the spread of transgenes and makes transplastomic crops up to one million times less likely [14, 15] to transmit their transgenes than their traditionally engineered counterparts. This is by no means a perfect containment system, the plants are still subject to dispersal through improper handling of seeds, and the paternal inheritance of chloroplasts has been observed in crop plants, albeit in an insignificantly small frequency. Nonetheless, transplastomics still represent a huge step forward in the topic of biosafety, which we believe will promote wider acceptance of plant biotechnology.

When it comes to biosafety, working with cell-free systems represents an improvement in current lab practices. Because they are not able to reproduce, they eliminate the possibility of escape of genetically modified organisms from the lab

As it is the case with all new technologies, what makes them great is also what makes them dangerous. Chloroplast synthetic biology has been already put to use to produce high amounts of proteins [16] that can be delivered in dried form. Their isolation from the rest of the cell allows protein accumulation in concentrations that would be otherwise toxic to the cell. This remarkable trait can be, in theory, misused to synthesize high amounts of toxins with the intent of harming others. Cell-free systems themselves have also been conceived as platforms for high-throughput protein synthesis. The main hurdle in the development of bioweapons, however, has never been protein production, rather the manufacture of appropriate delivery systems for the biological material [17], which would still be an obstacle even if an individual or group succeeds in producing harmful compounds using either cell-free systems or transplastomic plants.

Future Challenges

Although we have an optimistic outlook for future implementations of the chloroplast cell-free system, there are hurdles that need to be overcome for them to become a reality. While chloroplast biotechnology paves the way to exciting new possibilities, the fact remains that protocols for chloroplast transformation in many of the most widely cultivated crops are inexistant. Consequently, there is still a tremendous lack of characterized genetic parts from chloroplasts of those and other plants, which makes the process of transforming them even harder to achieve. We aim to contribute in solving this problem with OpenPlast, where we have already taken the first step in characterizing novel parts and establishing new part designs, which we expect will facilitate further work in that area.

However, creating new chloroplast transformation protocols, establishing novel pathways or characterizing parts will have no effect in solving the climate crisis if the ideas don’t leave the drawing board. The main challenge ahead of OpenPlast is most likely changing the public perception from seeing genetically modified plants as something fundamentally wrong to seeing them as powerful technology that has the potential to solve many pressing issues. Education about synthetic biology needs to happen on a large scale (Biobits), only when enough people are informed will we see a significant shift from baseless discussions fueled by fear to an actual conversation that reaches a sensible conclusion, followed by action and policy change.

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