Biosafety and Ethics

General Issues in Synthetic Biology

Synthetic biology is a new chapter in biological sciences which comprises advances in different fields such as molecular biology, engineering, computer sciences and organic chemistry to create new biological systems which do not exist in nature. Therefore it can be seen as the final transformation of biology from a describing science to a designing technology. Synthetic biology is expected to provide huge benefits to society, going from detecting and curing diseases, fabrication of biodegradable plastics to the promise to produce CO₂ neutral fuel. But next to expectations, synthetic biology raises ethical questions such as concerns about biosecurity or to what extend man is legitimatized to manipulate nature. Some of those aspects will be discussed in the following. However, it has to be noted that by now many applications of synthetic biology and therefore its ethical implications are more or less just plans and intellectual games so far.

One of the biggest issues in contemporary literature is the fear of bioterrorism emerging from synthetic organisms. Since the synthetic biology community is carried by an open source mentality, knowledge and techniques to create harmful organisms are widely accessible. Just recently a reporter from The Guardian could order genomic parts of the smallpox virus. The CIA raises the fear that “engineered biological agents could be worse than any disease known to man”^[1]. But even in the borders of known diseases, a certain caution is needed: for example researchers already managed to reconstruct the deadly Spanish Flu virus from 1918, which is estimated to have killed more people than the first world war, reviving this virus and setting it free would lead to a development described by Craig Venter as “the first Jurassic Park scenario"^[2]. It is even feared that the widely spread Do-it-yourself mentality in the field might lead to something called “biohacking”: Copying and assembling parts and manipulating organisms for the sake of creating something dangerous just to prove you can do it.
Those are dangers which should be neither under- nor overestimated. It took years, a whole lab of very skilled scientists and an awful lot of money for Craig Venter to copy the first organism artificially and it is not likely that bioterrorists will achieve the same even with something as simple as a virus in short time frames. Scientists working on harmful pathogens should be forced to obey high international safety standards to make sure that the risk to themeselves and the surrounding population is minimized. Most likely illnesses from uncharted regions and threats from zoonosis followed by disease invasion are a much higher risk than new assembled pathogens created using synthetic biology. Nevertheless many governments are starting to realize the promises originating from synthetic biology. For most politicians it is obvious that an emerging field with thousands of stakeholders can not be controlled anymore by a gentleman's agreement like it took place between the pioneers of genetic engineering in Asilomar 1975. Instead panels of experts were created to keep track on the field and to give advice to decision makers. In Europe the expert from the European commission releases an report on the field every few years, whereas the newly reconstituted US Presidential Commission for the Study of Bioethical Issues is the central advisory board in the US. Transnational research groups emerged in the field of humanities like SYNBIOSAFE to investigate recent developments of synthetic biology from an ethical standpoint. In addition to that the economy already tries to anticipate governmental regulations by introducing a self control like it happened for gene synthesis companies, in order to prevent unauthorized orders of harmful genes.

Another issue which is currently discussed is the unintended release of synthetically created organisms, which is similar to the discussion of genetically modified organisms in general. It is possible that synthetic organisms leave the lab, replicate, evolve and transfer genes to other organisms in the environment. However experts from the European commission point out that the problem is not different compared to classically genetic engineered organisms which found their way on some acres already. Therefore current legislation does not need to be changed for synthetic biological research, the ethical problems remain the same and were already discussed in detail elsewhere. Synthetic biological approaches might even be suitable to reduce the life span of modified organisms in the environment and genetic “watermarks”, which were implemented in the first synthetic cell by Craig Venter, could be used to identify the origin of released organisms, adding more security or at least the possibility to backtrack.

Crossing borders also is a keyword to another central question which evolves from synthetic biology. In a concrete term, one of the most discussed questions upon Venter creating his “artificial cell”, was, how far science should be allowed to go. Many articles used the term “playing god” when it comes to manmade creation of new life and if humans should be allowed to do so just because they are able to. Again, the central question is, more general this time, should scientists be allowed to do things just because they are able to. At which point is it irresponsible to create something with risks that cannot be predicted yet? What are the ethical consequences if man actually create an organism de novo?
It is hard to discuss those questions in general and it is even harder to offer something close to a general solution. Next to the philosophical question practical issues, like patent rights, evolve.
As the ethical framework to address this question is still underdeveloped, the European Union High-Level Experts Group (HLEG) emphasis the importance of a general debate about synthetic biology in society^[3]. Then the society can deliberate whether synthetic biology as a field should be supported by the public funding and which ethical limits should be set. In order to allow such a reasonable debate it is most important that the public knows about the uses and risks in synthetic biology and it is our job as scientists to provide these information.

Synthetic biology also raises new issues in philosophical disciplines such as ontology: the philosophical studies of the categories of being. It is essential to know how to categorize products of synthetic biology in order to decide on how to act appropriate. Is a synthetically created organism still life or is it more a man made machine? And what are the consequences of either case? This questions aims directly to the heart of our project since the central point of our iGEM contribution is to evolve a network which can at least partly turn a cell into something which can be controlled like a computer.

Biosafety and Ethics concerning engineered networks in living organisms

In our iGEM project we attempt to create logic gates based on RNA molecules and eventually implement these in living cells. As we applied principles known from computer science to biological molecules, the idea of logic gates itself is obviously not very new and our RNA circuits will not reach the complexity of electronic devices due to difficulties in handling biomolecules.
So what is the beneficial difference in utilizing logic gates based on RNA compared to electronics? It is the fact, that we simply changed the “chassis” for the logic gates. What once was a computer is now a cell.

Therefore our network of RNA switches has one big problem compared to an electronic device: It cannot be manufactured as precisely as it is possible to make a waver using lithographic techniques, simply because the parts are not fixed in space. Although the characteristics of RNA make it easier to construct logic gates compared to biomolecular switches used so far, it is still way more complicated than using a lithographic template to precisely etch every transistor where it should be.
But even with a network which is determined to stay simpler than most electronic device it still has indisputable advantages over any sophisticated computer: It is embedded into a biological system and therefore capable of handling biological in- and outputs, process and generate information from and for a cell, a living organism.

Then the question arises whether there are ethical problems in borrowing the metabolism, the environment and the genomic background of a bacterial cell and utilizing it as the basis for our circuits. This on the other hand has been answered long ago and when dealing with genetically modified organisms in a classical way the question whether or not it is okay to use living cells as protein production machines does not appear very often. We use, manipulate and kill cells very often and as an everyday work or duty. Ethical problems concerning the elimination of life consisting of only single cells does never play a role when it comes to bacterial cells, cell culture or just cleaning up your bathroom but is highly discussed and sacrified when the single cells are human embryonic stem cells. It does not concern our topic so we will not discuss that in detail just to show how the general question of how important something "living" can be to human society arises on very different levels. Beside this highly controversial sidestep, the basic answer maybe that our network does not utilize cells beyond everdays labwork and is otherwise highly dependent on what parts are used as biological in- and output.

In coupling our network with existing Biobricks, the full potential of bacterial cells as utilized, living particles can be exploited. Therefore major ethical problems of our engineered artificial network should not be seen as the network itself but of the inputs and outputs which are used to feed information and regain information. This on the other hand is nothing we can influence. We provide the concept and the basic bricks of our network to an open source community which can use our contribution in any possible way. To speak technically, we only provide a part of the hardware but what kind of software is enabled by it, is not and cannot be our responsibility anymore. The network itself is, like mentioned above, for physical reasons alone, much less effective as any computer is today.

So ethical problems concerning artificial intelligence will never reach our computing cell since our concept relying on RNA switches can never reach a complexity comparable to normal computers today. By introducing a synthetic network we may add a tool for better control over the cell but we do not make our cells smarter in any way. The only way, a cell may get smarter with our network is by evolving it itself.

Possible influences of the cellular environment on engineered circuits in organisms

The other potential advantage of utilizing logic circuits in biological surrounding is the main force behind progress: Evolution. As computers are not a subject of replication, mutation and selection, this principle is not really contrivable with electronic circuits, so it is an interesting question what will happen to our RNA-based devices. It would be a big advantage of biological circuits if they could be optimized by directed evolution approaches. Thus it might be possible to let nature design our logic circuits by mutation and selection, and relieve the “wiring diagram” from limitation of human creativity. One could imagine that once the basic logic gates are established in cells, you just have to select for solving a certain problem in a typical directed evolution approach: either solve it, or perish! Those cells have then optimized their circuits by means of replication and evolution, a thing impossible for a classical computer.
So, what is going to happen if we lose our cells and the artificial logical network is able to develop on its own for a while? Is it possible that evolution optimizes our modular switches? This is an interesting approach and maybe a topic for future iGEM teams. Using evolution as a force to develop artificial modular networks sounds both promising and tempting. On the other hand the question than arises, where are the borders of the definition of synthetic biology? Is a network optimized by the cell based on artificial components still something artificial or is it already just copying from nature like everything else? The quintessence maybe that nature is just hard to beat when it comes to optimization and that our approach is not to top natural circuits but to allow much easier handling and to make an complex process easier by modularity.

Another major problem corresponding with evolution is not only how is the environment going to modify my artificial network but also how may the network influence the environment.
Will it ever reach the point at which it is more effective than the actual basic cell components? Are cells with an artificial network in any way better equipped for evolutionary challenges than other cells, so is there a threat to biodiversity if set free? The cells might use their ability to optimize their circuits in order to compete against other organisms. A problem which would never apply for electronics, or have you ever seen a “wild computer”? In a review article for the British Research Council it is already noted that “mutations in the genome of synthetic organism could produce unexpected interactions with the environment and other living, natural organisms”^[2].
This is very unlikely with the way our network is designed. First of all, all components are to be located on a plasmid. Plasmids are only kept by E. coli as long as they give advantage and allow survival in certain conditions like LB media supplied with antibiotics. Otherwise they are a waste of energy, cells with plasmids in an environment where they are not needed have an replication disadvantage and are likely to lose this plasmid over time. This is enhanced in our case by the potential inputs and outputs which may also be located on the same or different plasmids, if they consist of complex proteins they are likely to be a major disadvantage concerning growth rates and energy efficiency. It is rather likely that bacterial cells are excellent in replicating the way they are and our synthetic network will not help E. coli to gain an evolutionary advantage.

The same accounts for horizontal gene transfer and the consequences if our network happens to be introduced in other cells beside E. coli. As described above, synthetic biology tries to apply principals known from engineering to biological system. However even the simplest bacteria is an extremely complex organism which so far cannot be understood in every way. Therefore it might be helpful to introduce an additional abstraction level between the complex basic biology of the cell and the synthetic biologist. Thus logic networks, e.g. composed of our RNA networks, might provide a suitable “user interface” to manipulate cells. Similar to the engineer who does not need an overall knowledge on how the machine works, but can use it with an electronic interface such as a computer, a synthetic biologist might be able to use a bacteria by utilizing RNA networks. These digital circuits provide an additional abstraction level before it comes to basic biology. This might be a tremendous advance when it comes to “user-friendliness” compared to existing biological circuits, for example based on activators and repressors. Since (anti)termination is an ubiquitous principle of transcription, RNA is a more suitable principal to make cells “programmable” compared to specific genetic switches in classical approaches so it can in general also apply in other bacterial or maybe even eukaryotic cells.
Although our RNA circuits might introduce a higher abstraction level to the cells, this does not mean that we are trying to make “better” cells compared to those which are not manipulated. With more than 3 billion years of evolution, bacteria are one of the most thoroughly tested organisms. What our approach facilitates compared to unmodified cells, is to improve the control for scientists. However this does not yet make a better organism and is most likely not providing them with an evolutionary advantage.

Safety Declaration

So to sum it up, beside possible ethical controversity which does not only apply for our artificial network but for all work done with genetically modified organisms, all our parts should not represent a danger to individuals or the environment. We only used derivatives of E. coli K12 cells, which contain gene deletions to reduce the competitive capacity of the cells and avoid survival outside the laboratory. We worked under biosafety containment level 1 and all materials being in contact with living cells were autoclaved before disposal.

Key Questions

1) Would any of your project ideas raise safety issues in terms of:

• researcher safety,
• public safety, or
• environmental safety?
  • • NO

2) Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes,

• did you document these issues in the Registry?
• how did you manage to handle the safety issue?
• How could other teams learn from your experience?
  • • NO

3) Is there a local biosafety group, committee, or review board at your institution?

• If yes, what does your local biosafety group think about your project?
• If no, which specific biosafety rules or guidelines do you have to consider in your country?
  • • YES, every department needs a safety delegate, in our case Helene Budjarek: She thinks it is not dangerous.

4) Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

• • • Using special restriction sites with very rare enzymes for dangerous proteins. Prohibit the sell of those enzymes for everybody except certificated institutions. Build corresponding plasmids.
    • Using a special cell line which is deficient of nearly everything but is resistant to a toxic protein which is coded on every BioBrick part.

References

[1] Central Intelligence Agency (2003) The Darker Bioweapons Future prepared by Office of Transnational Issues available here: http://www.fas.org/irp/cia/product/bw1103.pdf [2] Balmer A., Martin P., 2008, Synthetic Biology: Social and Ethical Challenges, Institute for Science and Society, University of Nottingham. http://www.bbsrc.ac.uk/organisation/policies/reviews/scientific-areas/0806-synthetic-biology.aspx [3] EU Commission. (2005): Synthetic Biology - Applying Engineering to Biology ftp://ftp.cordis.europa.eu/pub/nest/docs/syntheticbiology_b5_eur21796_en.pdf