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The “human” part of designing an iGEM project

Current methods of chemical synthesis from petroleum derived chemicals have led to great environmental disruption and continue to be a strong contributor to global warming. It has also become increasingly clear during the recent decades that the raw material of this process, crude oil, is not a resource that is renewable within a viable timeframe. Biosynthesis of complex molecules and chemicals can be regarded as one of the most promising technologies capable of replacing current production strategies. Large-scale biosynthesis is already a viable industry: acetic acid, citric acid and ethanol are examples of chemicals widely produced using this process. Furthermore, complex molecules such as enzymes are almost exclusively produced using industrial fermentation.

Due to the promises that this field holds, it was clear that this is what our iGEM team wanted to focus on. An important part of the project is to consider how our project would affect society. To name a few, there are ethical, social and safety aspects to consider when attempting a biosynthesis-oriented project, our integrated human practices has been a major part of the design and formulation of our project. The following text covers some of these issues and how they were considered, discussed and dealt with during the design and development of the project concept.

Integrated human practices

Few areas of science have caused such an intense public debate as synthetic biology, especially for its application in the food industry. Though the scientific community at large agree that GMOs and the methods of synthetic biology can be used safely, the public debate continues. A recent letter addressed to the UN, describing the safety and need for GMOs, was signed by over 100 Nobel laureates spread across several scientific fields [1]. However, that is not to say that the debate should cease only because some people agree. On the contrary, it should be elevated and taken seriously by all. This discussion ought to be based on the facts, while arguments based on emotion and dogma should be put aside. Furthermore, one can argue that a technology (or knowledge of it) carries with it no ethical dilemmas, but it is the application of it that has an effect we can discuss. In the same way that nuclear fission has no ethical or moral agenda, but it is the application of the knowledge that should be evaluated and discussed.

Fig 1. Arguing on the facts [10].

This topic also relates to genetic engineering of microorganisms. Genetic modification of microorganisms differs from modification of higher animals in that it does not cause any physical or psychological suffering as we currently understand and define it. Therefore, our focus will be on potential impacts that the modified organisms could have on the environment and biosphere of the planet as well as human life. The discussion regarding genetic engineering of life in general, and whether it is good or bad, is certainly still relevant but outside the scope and aim of our integrated human practices discussion. The focus will be on the application of synthetic biology and genetic engineering of microorganisms, and the considerations that were made during the design and development of our project idea.

Safety of Genetically engineered microorganisms

To be able to assess the risks of using a genetically modified organism (GMO), several factors have to be taken into consideration, especially when estimating the potential danger to human health as well as to ecosystems. As all microorganisms currently used within the field of synthetic biology originate from nature, unmodified strains of these organisms could be considered safe to the ecosystem at large (of course excluding directly pathogenic strains). To make a reasonable argument of the risks regarding the use of a GMO, the specific modifications must be considered. For a GMO to be considered as a threat to the ecosystem, the modification needs to provide a significant evolutionary advantage over the wild type (WT) of the organism. This relates to unintentional damage that GMOs might cause if it ends up in the wrong hands (intentional misuse of the technology is further discussed later in this section).

In general, the modifications made to industrially used strains of microorganisms rarely provide such an evolutionary advantage. Most often the opposite is true. For example, modifying a strain of Bacillus subtilis to produce an enzyme (a common application of biosynthesis) is highly metabolically costly for the organism and would put it at a significant evolutionary disadvantage compared to the WT strain. This would lead to the GMO quickly being outcompeted by the WT in a natural setting. In fact, contamination of the fermentation culture, by wild bacteria, in an industrial reactor is a constant issue for biotech industry. In such a case, it could be argued that the product itself (the enzyme) is where the focus of concern should be. Though most products produced by industrial bacteria may be considered safe in small amounts, large scale fermentation does produce them in large amounts and high concentration. This raises the question that maybe the products are what should be the focus of a discussion of safety GMO, rather than the organism itself.

This is not to say that there is not a safety concern regarding the use of GMOs, but the concern needs to be focused on each case and not on GMOs in general. In cases where the effects of the modifications are not well studied or when many modifications are made, the organism needs to be evaluated and safeguards put in place. Such safeguards can be put in place as part of the modifications. Examples of this are making the microorganisms reliant on a certain kind of medium (food), requiring specific signaling to proliferate or incorporating a kind of suicide switch by inserting a lethal gene that can be activated on command etc.

The intentional misuse of genetically engineered microorganisms is a separate issue that may be more difficult to handle. Especially since the technology for performing genetic modifications is becoming more and more readily available. However, this did not have a huge impact in the formulation of our project idea, since we would consider misuse of genetic engineering to be more of a regulatory issue. Due to this it was outside the scope of our integrated human practices.

Environmental vs social sustainability

Safety issues are not the only problem that needs to be addressed if large scale biosynthesis is to replace the current petroleum based chemical production. Industrial fermentation requires large amounts of substrate, mainly in the form of sugars. These are currently derived mainly from plant-based sources such as corn, sugarcane and sugar beets [2].

The use of these crops as industrial substrate raises concerns regarding competition for these feedstocks as food or industrial substrate. In countries like Brazil, where large-scale bioethanol production has started to replace conventional fuel production, this has already been shown to have negative social consequences. The competition for farmland has led to an increase in the prices of raw feedstocks [3]. This example of biosynthesis can teach us what effects large-scale implementation of this type of production can have. The price difference in developed countries is relatively small, since the value of the raw ingredients comprise a small part of the total cost of processed food [4]. However, the reverse is true in undeveloped countries, where the diet is comprised of mainly unprocessed food, resulting in a market more sensitive to the price of these crops. Families below the poverty line spend upwards of 80 % of their income on food, leaving low margins for a price change of basic food goods.

Should environmental sustainability come before the basic needs of human beings? The future of all is of course predicated on the fact that there is a livable earth for us to inhabit. “The needs of the many outweigh the needs of the few” as the saying goes. But do the two goals need to be mutually exclusive? We certainly hope not.

Self-sustaining production system

How can the need for industrial substrate be limited and at the same time move the world onto a sustainable production of chemicals and fuels? Our answer: Create a production system that produces its own substrate. Photosynthesis was the key to this solution, as photosynthetic microorganisms have the ability to convert atmospheric carbon dioxide into sugars by converting sunlight to bioavailable energy (ATP) to fuel this process. The use of cyanobacteria addresses both the substrate issue as well as decreasing the carbon footprint of the industry. A photosynthesis-based production system would not require the arable land that food crops need, since the microorganisms would be grown in contained bioreactor systems.

However, there are some disadvantages when working with cyanobacteria. One of them is their slower growth rate than most chemotrophs, making genetic engineering of them slower. Since a future expansion of the biosynthesis field certainly will require extensive genetic engineering, working with this organism is not ideal. Leaving the product synthesis to faster growing organisms such as Saccharomyces cerevisiae and Bacillus subtilis, which already have already proven capable of viable production, could therefore be the better choice.

The approach we chose in our project was to modify the cyanobacteria to simply produce the substrate for the production organism. The genetic modifications performed in the cyanobacterium will therefore only be focused on one task - the production of substrate - and the organism will not have to be reengineered for each new product that one wishes to produce. Furthermore, dividing functions into different strains can lead to decreased metabolic load and prevent competition between the different functions within one organism [5].

How do we create a system that is self-regulating and at the same time avoid the risks discussed earlier?

To prevent one organism from outcompeting the other, there has to be some sort of communication between them. Several approaches to this has been attempted in the past. A quorum sensing (QS) system is one alternative that has proven capable of maintaining population equilibrium within a system comprised of different strains of the same species [6,7]. However, QS within the same species might be conceivable, but achieving a functioning QS system between organisms of different species that originate from completely different environments is a challenge to say the least.

Another alternative is the exchange of nutritional metabolites, e.g. amino acids. This approach has been shown to provide an additional fitness benefit due to the division of metabolic labour [8]. It has also been shown to be capable of purging cheaters from the system, i.e. cheaters do not seem to be able to outcompete the cooperating population [9]. Furthermore, in a scenario where the industrial strain escapes, an auxotrophic bacterium will have a significant fitness disadvantage without its partner and will therefore pose a very small threat to the wild flora and fauna.

These arguments lead us to construct our microbial consortia based on metabolite dependence rather than a quorum sensing mechanism. This solution has two main advantages. Firstly, amino acids are universal and most organisms on earth share the same set. Modularity becomes easier to achieve compared to a QS system, in which entire new synthesis pathways may need to be implemented for the QS molecules. Secondly, with respect to safety, a metabolic dependence based on auxotrophy of amino acids prevent the unwanted spread of the organism since it cannot survive without its partner organism providing it. Furthermore, in 2015 the Amsterdam iGEM team achieved a proof of concept in which they created a symbiotic culture between the cyanobacterium Synechocystis and Escherichia coli based on auxotrophy. This provided great inspiration in how to achieve our production system.

The system will be based on a strain of Synechocystis providing a carbon source in the form of acetate to the production organism. In return, it would provide the cyanobacterium with a required amino acid, as seen in Figure 2.

To expand the idea further and to make the system more universal, a level of modularity was desired, therefore the production organism should be interchangeable. Different species of microorganisms relevant to the field of industrial biotechnology was therefore be made compatible with the system. In this way, the system becomes more flexible and the organism best suited to produce the desired product can be applied.

Lab scale to large scale

Moving from a lab setting into a large-scale industrial process is a long road to say the least. To help us consider the possible issues that may arise with a future scale-up of our project idea, we consulted several experts within the field of industrial biotechnology and microalgal research. Carl Johan Franzén, associate professor at the biology and biological engineering, and industrial biotechnology departments at Chalmers university, provided insight into some of the technical issues that might arise. Dr. Eva Albers and Dr. Joshua Mayers, researchers at the biology and biological engineering, and industrial biotechnology departments at Chalmers university of technology with focus on algal biotechnology, provided great insight into some of the considerations needed for large scale cultivation of photosynthetic microorganisms.

One of the main technical issues discussed were product separation which would increase in cost due to the lower cultivation density resulting from a photosynthesis based system. The increased cost may however be offset by the absence of cost for a sugar source for the production organisms. To study this further a more complete life cycle analysis is needed to evaluate the cost offset this will result in. This should be one of the main tasks for a continuation of the project to see if the resulting system would could be commercially viable.

The issue of product separation is also related to the problem of water use, since cyanobacteria do require large amounts of water to grow. Since freshwater is also a limited resource on our planet a saltwater living strain may be the better choice for a photosynthesis based culture system. The use of seawater does however bring with it other problems such as salinity and contamination risks and might not be a good choice for a co-culture system specifically, but may be suitable for general culture of phototrophic microorganisms. Since photosynthetic organisms require sunlight for conversion of CO₂ into more complex carbon sources, the day-night cycle also becomes an issue. The flow of carbon will decrease or cease completely during nighttime unless artificial light is used. Due to the cost of energy, using artificial light for a large scale cultivation system is not viable.

To achieve a productivity level that is as high as possible increased levels of CO₂ is also needed. The co-culture system itself could provide this since the production organism is using the carbon excreted by the cyanobacteria, closer calculations are however needed to evaluate the balance in this reaction. Another possible solution is to couple a production facility such as this to a fossil carbon burning power plant and capturing the released CO₂ and feeding it into your bioreactors. Furthermore, the transparency of the culture is important to maintain lighting conditions for the cyanobacteria; this severely limits the biomass concentration achievable in the culture. A possible solution to this problem is separation of the two organisms by a permeable membrane or even into separate reactors with a shared volume of media circulating both cultures.

It is clear that a viable chemical production industry based on biosynthesis still has many hurdles to pass. However, with the rapid progress the field of synthetic biology is making we are confident these problems can be solved; and as discussed previously in this section the need for them to be solved is great, and growing quickly.

Viewing possible futures to find our own

The science of synthetic biology can arguably be described as one of the most powerful technologies that humankind has ever conceived. Granted, this technology is still in its infancy, but as many other branches of science it carries the burden of incredible destructive capacity. But in contrast to many other scientific fields, the tremendous potential for productive and life-saving uses are even more evident in the field of synthetic biology. With both of these potentials follows several ethical and philosophical questions on how to advance the frontier of this technology.

Its destructive capacity is, in our opinion, undeniable, but equally so is its potential for good. Both of these are of course dependent on how the technology is applied and used and just as with most of our modern technology, it can be abused. The question is, can we allow tremendous good to be held back by an imaginary situation where the technology is misused? How do we balance its creative power with its destructive ability?

Like many questions it comes down to people. Do we trust the character of ourselves as people to handle this powerful tool? One could also look to history: Have we done so in the past with other scientific discoveries? One could argue that our track record is questionable to say the least.

But what about the future? Of course learning from the future is hard since we do not know it, but imagining it can help us find out where we want to go.

To try to encourage reflection and debate around these questions, our team has applied speculative design into our integrated human practices. In contrast to science, which aims to describe how things are, the purpose of speculative design is to show how things could be. This can help us to discover how we think things should be and what actions to take to reach this future. Speculative design aims to provoke thought and discussion by postulating a future situation or society in which the subject matter is portrayed. This could be in the form of stories or simple descriptions of how life is in relation to the discussed subject. It could be how transportation is envisioned, how society is organised or, as in our case, how synthetic biology is applied and how it has affected the world.

Since the future consequences of new technologies may be difficult to imagine and even harder to predict accurately, the stories are intentionally fictional. The reason for this is to remove the restrictions one commonly places on imagination when trying to come up with “realistic” ideas. This problem does not arise when it comes to fiction since these restrictions are lifted when you know the story is not real. What may seem “unrealistic” today can often be commonplace tomorrow, fiction can help us embrace unconventional ideas. The purpose of speculative design is therefore to liberate our creativity and open our minds to what might seem impossible.

Consider the idea: If all industrial production of chemicals across the world is based on biosynthesis, what could the world look like in 50 or 100 years? Below, three short stories are presented, inspired by our own project idea.

Some areas that are worth considering when discussing the future of synthetic biology are: Safety, regulation(law), education and general ethical aspects.

A blue-green home

She looked out over the barren landscape, sand and rock as far as the eye could see, nothing lived there. Beep, Beep! She walked along the cliffs, felt the poisoned air burn her throat. Beep, Beep! The wind was howling, the ground started shaking, an instinct caused her to turn around, then she saw it, a wall of water was rushing towards her. Beep, Beep! She wanted to run but the air burning in her throat seemed to drain her of her strength. Beep, Beep! Just as the water was going to crush her…

Eve woke up with a jolt, a beeping sound came from the other side of her room. The DNA-synthesizer on her desk had completed another cycle, she sighed in relief, her dream had been so unpleasant. The machine humming on her desk she had bought for her high school biology project. It was old and loud, almost as big as the vintage toaster her mother kept in the kitchen. Moving over to the desk, she sat down and read the display on the device. It still had 3 more cycles to complete before the genetic constructs she had chosen the night before were complete. The constructs were going to change the “photoplastic” growing in small tubes on the roof of all the buildings in her neighborhood. They would shine in different colors depending on the season. She thought of the small life forms growing on the roof and how they provided the material for the 3D-printers everyone had in their homes.

She wondered how people had made things without them.

The idea she had suggested to her mother, not two weeks ago, played in her mind; if the small lifeforms make everything we need, then they might as well look pretty doing so! She had learned the basics of manipulating DNA in primary school and bought everything she needed online, from the DNA-synthesizer to an old TIRC machine (Transformation Integration Replication and Cultivation). That night, her mother had talked about how genetic modification of the tiny life forms had once been difficult and even forbidden.

Eve could not see how anything could be produced without them. In her world they produced everything from food to energy, even the material of the sheets she had been curling into a moment ago was produced from these tiny things. She remembered how they used the energy of sunlight and the gases in the air, freely available everywhere and to everyone, to replicate themselves. Of course, they need the small bag of minerals every few weeks or so but other than that they were completely self-sustaining.

Her teacher had once talked about how people used to pull ancient black and toxic goo from deep pockets in the ground to make things and burn for fuel. She had described how it had almost made the planet uninhabitable, how the oceans had threatened to swallow the lands and the air becoming toxic. Eve wondered how people could allow such wasteful and destructive use of the scarce resources available on the little world they all shared.

Eve thought about the dream she had, was it real? Was it a memory, no she had never seen the dead landscape before, her world was green and full of life. She returned to her bed and looked up at the early morning sky through the transparent domed ceiling of her bedroom. Some of the stars had not yet yielded to the sunrise. As she drifted back into sleep she thought of the blue-green world that was her home, and knew, it was well taken care of.

The end of microbe-based production of chemicals?

With general tools for genetic modification becoming commercially available, how do we tackle the arising problem of microorganisms being modified for destructive ends? Certainly, many commercial goods available today can be used for destructive means, after all a regular automobile is a 2000-pound projectile in the wrong hands. But, it could be argued that no product available today have the potential destructive capacity of a highly contagious bioweapon in the form of a genetically engineered bacterium or virus. How should questions of regulation be handled to prevent such a future?

The year is 2116. The world is threatened by a plague! Not a human plague, but a highly resistant bacteriophage threatening to eliminate the microbe-based production of food and medications that the sustainable world has become reliant on.

How could this happen?

50 years ago, there was barely any hope left for the planet Earth. The majority of the population of the Earth was shuttled to Mars, which then was more habitable than our planet. Due to this, the carbon dioxide emissions slowly began to decrease. However, this decrease was nowhere near the pace that was needed in order to save our planet. People were scared. Any solution would do. During the last three decades, a microbe-based production of chemicals using atmospheric carbon dioxide had drawn a lot of interest. Even though it showed great promise, the fear of using genetically modified organisms for production of any everyday product was even greater. But now, even the most skeptical people have agreed. Anything to save our planet.

Today, in the year of 2116, the levels of atmospheric carbon dioxide are back to the normal level. It’s too good to be true. Or is it?

Due to the availability of using this production system, anyone today is able to use the system for production of everyday products. Limiting legislations were made since it was implemented during such chaotic circumstances. The public was not well-informed of the potential risks of using this system and the importance of using it as intended.

Due to misuse of the system, a highly resistant bacteriophage has arisen. The solution that saved our planet also became a weapon that now threatens to end all human life on Earth. Is this the end of the Earth?

In a Coffee Shop...

In a galaxy far, far…. Well, not that far, it’s actually very close, it’s actually here, just not now.

Sofus: ”Do you want something to drink?”

John: ”Yes, thanks. I’ll have a latte, with the extra caffeine. I need a pick me up!”

Sofus: “Here’s your coffee – why do you need that extra kick?”

John: “I stayed up all night working. I just started a new project in the lab!”

Sofus: “Oh, what are you working on now?”

John: “Well, it’s a new form of life. I know you’re thinking ‘oh, now again’, but ever since Bohrian made that new one, I have been wanting to do it too!”

Sofus: “What kind of lifeform?”

John: “It’s sort of a little creature that can help with all the things that you back in the days needed paid workers to do – you know; laundry, shopping for food, cleaning. I hope I can get it to watch the kids when they’re forming in the womb as well!”

Sofus: “Oh yeah, you’re having one hatched. How is that coming?”

John: “I just visited it yesterday. Seems like it’s coming along fine!”

Sofus: “When do you think it’s waking up?”

John: “About a week or two – so my creature should be done by then.”

Sofus: “That should be plenty of time. Remember, we're in 2500 now.”

John: “Yeah, I know. It’s been 500 years since the dark ages… It is so weird to think about how we used to live.”

Sofus: “It’s weird to think of them as our ancestors. We have evolved since, I believe.”

John: “That we have, yes. I actually read something about their ideas of creating new life.”

Sofus: “Why would you want to read something from the dark ages?”

John: “I was drunk and did some research on how the perfect new life form should look. I stumbled on something called ‘Google’ that they used.”

Sofus: “I think I have heard of that – they had that on ‘the Internet’, right?”

John: “Yes. But, there was actually a lot of articles about life. But they all said that you shouldn’t create it yourself!”

Sofus: “… Shouldn’t?”

John: “Yes. Remember, they had that idea about not just doing things because you could. It must have been so boring!”

Sofus: “So weird. Why shouldn’t you do the things you are able to? How would you otherwise evolve?”

John: “I really don’t know but they seemed to fear the unknown, believing that science moved too fast to follow.”

Sofus: “I feel for them – if they only knew what they missed when they didn’t explore the limits of their knowledge.”

By: Rikke Friis Bentzon - iGEM team SDU-Denmark

References