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From 2011.igem.org

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Informing Design

We consulted numerous experts in various fields to ensure that the design of the AuxIn system respects all relevant social, ethical and legal issues. One module of our system, Gene Guard, is a direct result of brainstorming around the issues involved in the release of genetically modified organisms (GMOs). Although we have only reached the proof of concept stage, we have put a lot of thought into how AuxIn may be implemented as a product and the legal issues that would be involved.

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Discussion panels

In order to discuss the possible implications and consequences of our project, we held two human practices panels. These panels were extremely helpful in informing the design and implementation possibilities of our project. Many experts in synthetic biology but also social sciences kindly agreed to attend our panel meetings and advise us on the human practices aspects of the project.

First panel

The first panel consisted of Prof. Richard Kitney, Dr Tom Ellis, Dr Guy-Bart Stan, Charlotte Jarvis and Kirsten Jensen. The panel addressed many different questions that we later used to inform our design.

Could the bacteria impact the germination of the seeds?
The coat itself would not be prohibiting germination. It is possible to design the coat sufficiently well to ensure that this would not happen. In addition, seeds normally germinate in soil full of bacteria that do not prevent germination.

How can we ensure that the auxin does not kill the plants?
We will be able to vary the inoculum of bacteria in the coat. We will get an experimental estimate of the auxin production, which will help us estimate the ideal number of bacteria to be contained in the seed coat. While a weak promoter may be better for constitutive expression of auxin, it will be easier to weaken the promoter later. We have used an insulator sequence to separate the promoter from the RBS so that we will be able to replace the promoter very easily. This may also contribute to the fine-tuning of expression and thus help us make sure that auxin is expressed at ideal concentrations. We had decided to not put the sequence on the genome as this makes persistence of the genes more likely. The worst case scenario consists of the auxin-producing genes being transferred to other bacteria that become pathogenic. However, this could be tested exclusively beforehand and the infrastructure for this separate development and safety testing stage is already in place. In addition, unlike synthetic auxin, natural auxins such as IAA have a short half-life and degrade rapidly.

What is the risk-benefit relationship of our implementation?
In our implementation, we are trying to improve already existing practices. We do have to take a certain risk to combat desertification. However, is putting GM bacteria into soil worth speeding up the acacia tree planting process? How much does this really help? While GM bacteria may pose a risk, introducing foreign plant species that also show drought resistance and grow faster than acacia trees can be extremely risky and introduction of foreign species into ecosystems has already had negative consequences all over the world. This effect is likely to be worsened by the fact that we would be introducing the foreign species into an already damaged ecosystem. We may also be able to plant other fast growing plants at the same time as planting our coated seeds to hold the soil down while the seeds are growing.

Should we be using B. subtilis or E. coli as our chassis?
B. subtilis spores spread very easily over long distances and may thus be blown into different ecosystems where they may have negative effects. On the other hand, its spores would be easier to integrate into a seed coat. E. coli is not as easy to integrate into the seed coat. However, it does not form spores and is therefore very likely to stay inside the ecosystem into which we introduce the microbes. We have already shown that E. coli is able to survive in non-sterile soil for more than two weeks and that it can pass on its plasmid to other bacteria, enabling them to express GFP and antibiotic resistance. By using E. coli as our chassis, we can be sure that the bacteria will survive in the soil for a reasonably long period of time but not spread as rapidly as B. subtilis spores would. At the same time, we will be preventing plasmid transfer using our containment device (Gene Guard). We should be able to overcome the technical challenge of putting E. coli into the seed coat.

Would we be able to get rid of the bacteria once they are in the soil?
Previous projects have sometimes used kill switches that kill the cells after some time. Thus, spreading of cells and their modified DNA is limited by simply destroying the cells. Kill switches are never 100% effective and the bacteria may lose the kill switch plasmid. In addition, bacteria killed by kill switches still leave behind DNA that can be conjugated by other, naturally occurring bacteria. We were therefore looking for an alternative method to contain the modified DNA that we have created in this project. We simply may not be able to take the bacteria back out of the environment after they have been distributed into soil, at least not 100%. Instead, we will be aiming to prevent spread of the plasmid to any cells other than those we want. We will be using E. coli as our chassis, which should be out-competed naturally in the soil and our Gene Guard device will be used to prevent plasmid conjugation.

Second panel

We held a second panel two weeks after the first one. Several people from diverse backgrounds took part in our panel. Dr Stephan Güttinger and Alex Hamilton from the LSE BIOS centre, as well as Charlotte Jarvis from the Royal College of Art and Dr Janet Cotter, a scientific advisor for Greenpeace attended the panel. In addition, Prof Paul Freemont, Dr Geoff Baldwin, Dr Tom Ellis, and Dr Guy-Bart Stan from the Synthetic Biology Centre at Imperial College joined the panel.

What is the danger of unintended consequences and how can we avoid them?
To be able to legally release our organism, we have to have consider all possible scenarios of how the project could have unintended, potentially harmful consequences. In addition, we have to be able to eliminate the bacteria from the field. In typical GM plant field trials, plants are eliminated by burning but this would not be an option for eliminating our bacteria, as they will be distributed throughout the soil. We are still a far way away from being able to release the bacteria and we will first need to develop a lab-tested solution to eliminating the bacteria. However, we are only at the very start of the development process and should be able to find measures of how to eliminate the bacteria.

What measures can be taken to prevent the occurrence of detrimental "unknown unknowns"?
This is a serious issue with the project as soil is still poorly characterised. However, we have been in contact with experts on soil, plants and above/below ground interactions who have all stated that there should not be a big negative effect on the environment resulting from our project. In addition, the project will be tested thoroughly in the lab before any possible field trials will ever take place.

What is the danger of deliberate misuse - could our project be used as a bioweapon by a malicious individual or group?
IAA produces a stress response in E. coli and the bacteria would only be able to produce the auxin up to a certain threshold. In addition, the compound degrades rapidly. We think it is therefore fairly unlikely that our project in itself could be used to manufacture a bioweapon. In addition, while it is theoretically possible to use components of the AuxIn project for making a bioweapon, the same can be said of almost any synthetic biology project.

How will be make sure that we do not just treat the symptoms?
We will be integrating our approach with already existing practices and take a dual approach of providing a good scientific foundation for a solution in conjunction with improving the societal background. In the areas that we are targeting, underlying social issues in conjunction with climate change are responsible for soil degradation and desertification. There are already good programmes in place that help local communities establish sustainable farming practices that can support their livelihoods and do not lead to degradation of the environment.

How are we going to deal with the issues surrounding patenting?
Patenting is very problematic, especially when using a patented product in disadvantaged areas. Contamination of the patented material in other areas could lead to individuals getting sued for using the product without permission and people are very wary of this effect. The ethos in the synthetic biology field is based on open sourcing of information. However, patents on enabling technology are likely to become a big issue in the future. Nevertheless, using these enabling technologies for a commercially viable product can be seen as a different issue. In the UK, it is not possible to patent a system after it has been publicly disclosed. iGEM projects can therefore only be patented in other countries such as the US. For our project, we have considered how we will be able to help countries affected by soil erosion and desertification. We aim to do this by selling the product to developed countries that suffer from soil erosion to finance our efforts in the developing world. This will probably require patent protection of the concept. However, approaches where the same product is sold for profit in developed countries and given away at much cheaper rates or for free already exist in the pharmaceutical industry. We think that this approach can also be applied to agricultural products and products such as ours that aim to improve soil.