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From 2011.igem.org

Human Practices

Our team has decided to go down a novel route in tackling the human practices issues surrounding not only our project but also iGEM in general. Instead of sticking to the established routes of either proposing complete containment or relying on "kill switches" to prevent spread of GM bacteria, we have decided to engineer a containment switch that will not kill our AuxIn bacteria but all other microorganisms that take up the auxin-producing plasmid. In addition, we have consulted many experts and will conduct experiments that demonstrate the safety our device. The true scope of many iGEM projects can only be fulfilled if release is possible and we will be attempting to take a first step towards making this possible for our project.

Historically, iGEM teams have tried to control GM release via two mechanisms: complete containment and "suicide" mechanisms in which the bacteria kill themselves in the absence or presence of specific stimuli. However, when considering the eventual use of many projects – be it bioremediation (e.g. Peking 2010’s project), crop-enhancing projects (e.g. Bristol’s 2010 project) or other applications – full use of synthetic biology organisms will only be achieved by release and full containment is often not a realistic option. In addition, kill switches may be effective to an extent but they are easily selected against by evolution as they present a strong selective disadvantage. Stress defence mechanisms such as the SOS response in E coli add to this effect. In addition, transgenes can be transferred to other bacteria in the environment using naturally occurring mechanisms such as conjugation of plasmids. Finally, while it may be argued that engineered lab strains will quickly be outcompeted, bacteria with GM markers have been found in the environment more than a decade after they were released REF. In some cases, endurance of the bacteria in specific environments may even be desirable.

In light of these issues, we have decided to engineer Gene Guard, a containment switch that will lead to the lysis of natural soil bacteria that take up plasmid DNA from our engineered bacteria. We have consulted experts and the literature about the implications of our project and used this information to design an effective containment switch. However, we also tried to address all possible problems and complications arising from the impossibility of absolute control. Accordingly, we used the information we gathered to influence our release strategy and design.

However, we also acknowledge that this containment switch is never going to be completely effective. Accordingly, we have consulted ecologists and other experts on auxin, plants and soil to ensure that our device is as safe as possible and we can justify release. We researched other organisms such as soil microbes and earthworms that may be affected by the AuxIn bacteria and, with the help of the experts we consulted, devised experiments to test the safety and impact of many aspects of our project.

References:

Human practices panel discussion

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 possibilites 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.

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. 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 we introduce the microbes into. 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 the BacTrap. 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?
The kill switch is never 100% effective and the bacteria will lose the plasmid. In addition, bacteria killed by kill switches still leave behind DNA that can be conjugated by other, naturally occurring bacteria. We may not be able to take the bacteria back out of the environment after they have been distributed into soil. Instead, we will be aiming to prevent spread of the plasmid. We will be using E. coli as our chassis, which should be outcompeted in the soil and our BacTrap 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 joined the panel.