Safety Questions

According to the WHO biosafety is the prevention of unintentional exposure to pathogens and toxins, or their accidental release, whereas biosecurity is the prevention of loss, theft, misuse, diversion or intentional release of pathogens and toxins.

Key Questions

Would any of your project ideas raise safety issues in terms of researcher safety?.

Our system primary goal is hydrogen production. This gas is known to support deflagration (combustion at a subsonic velocity) when its concentration in air ranges between 4% and 70% (1), and several cases of spontaneous ignition have been reported (2). For the iGEM competition, our work will be focused solely on small containers with our bacteria (petri dishes and flasks with a capacity smaller than one liter). These containers pose a low combustion risk. However they will be handled with care, avoiding direct exposure to ignition sources.

Would any of your project ideas raise safety issues in terms of public safety?.

Yes. Our project includes the development of broad host-range plasmids, which include antibiotic resistances (e.g. gentamicin). However these plasmids are intended for use only in laboratories, under controlled conditions. This ensures that resistances won’t be horizontally transferred to any pathogenic organism.

Would any of your project ideas raise safety issues in terms of environmental safety?.

Yes. Hydrogen production could interfere with nitrogen fixation. If the strain we create lacks the nitrogen fixation system and if it becomes common in the environment it could poison the nitrogen cycle. However it is part of our objectives to maintain nitrogen fixation in our bacteria. In the case both systems are mutually exclusive, further safety evaluations and experiments would be required before considering introducing our bacteria into the environment.

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

Yes, our broad host-range plasmids with antibiotic resistances.

Did you document these issues in the Registry?.

pBBRMSC-5 is broad host-range with gentamicin resistance. While working at Lab for research purpose there is no problem, for that we think as a implentation the integrate our construction into chromosome. By the moment there are are not other risks with our bioparts.

How did you manage to handle the safety issue?.

Our bacteria will be handled exclusively in the laboratory for the iGEM competition. For further applications we have evaluated the chromosomal insertion of our system, which would make vary unlikely the transfer of any resistance gene to other bacteria.

How could other teams learn from your experience?.

A proper documentation, containing not only what safety measures were taken but also why, is crucial. This way all our team learnt from our project will be properly transmitted to future teams.

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

Yes.

What does your local biosafety group think about your project?.

They believe it to pose low or no risk at all given our bacterial strain Rhizobium etli CFN42, which has a very low fitness compared to other strains.

Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions?.

It is important to recognize which teams did a proper job considering safety issues. As well as present teams check what was done in previous winning projects, teams could check what was done regarding safety in previous competitions. Those teams whose work achieved proper considerations should be distinguished, so future teams can look back at their work.

How could parts, devices and systems be made even safer through biosafety engineering?.

Undesired and unexpected interactions among parts or among systems should be diminished to the maximum. For example, a double transcriptional terminator ensures that the transcription regulation affects only our system, avoiding the transcription of downstream genes. In this way the promoter affects only our system, ensuring that no new regulation interactions arise with the downstream genes. It is important to think that the properties of one part modiffy other parts interactions or interaction strength. For example the number of transcriptional terminators affects the promoter possible interactions.

Safety issues in our project.

Probability:

Could there be an unplanned event or series of events involving your project, resulting in death, injury, occupational illness, damage to equipment or property, or damage to the environment?.

Yes. Hydrogen production in great amounts poses a risk to the investigators and the equipment. Accidental plasmid release poses a risk to public health.

How likely is that going to happen?.

Hydrogen production will be limited to bacteria in petri dishes or flasks with one liter capacity or less. This makes the production of great amounts of hydrogen very improbable.

As of accidental plasmid release, our work is bound to the appropriate safety measures, which makes the release very improbable.

Does your project require the exposure or release of the engineered organism to people or the environment (e.g. as medicine, for bioremediation)?.

Not in this stage. It may will in further applications.

Hazard:

Could your device, when working properly, represent a hazard to people or the environment?.

No. However it will require the proper hydrogen handling devices in further applications.

Is your engineered organism infectious?.

It is known to infect neither humans nor other animals. It does infect the common bean Phaseolus vulgaris roots, forming specialized structures known as nodules, in order to enter the symbiotic stage.

Does it produce a toxic product?.

No. However hydrogen handling requires specialized techniques which, as of July 15, we are investigating.

Does it interfere with human physiology or the environment?.

Our bacteria don’t interfere with human physiology.

Rhizobial organisms participate in the nitrogen cycle performing nitrogen fixation. Experiments evaluating the release to the environment of genetically modified Rhizobia inoculants have been carried out. Most studies revealed that the number of Rhizobial organisms dropped rapidly after application to soil or seeds but then numbers stabilized for months or years (3).

What would happen if one or several bioparts change their function or stop working as intended (e.g. through mutation)?.

The hydrogen production pathway is composed of a hydrogenase, two hydrogenase maturases and a pyruvate-Ferredoxin OxidoReductase (PFOR). None of these proteins are expected to change their function through mutation. If any of them stops working at all (either by a missense or a nonsense mutation) they will only shut down hydrogen production. Proteins would still be produced, consuming resources and decreasing the organism’s fitness. Competition with other bacteria would either kill the mutated organism or lower its reproduction rate.

Other parts in our system include two promoters and transcriptional terminators. The first promoter controls the expression of the hydrogenase and PFOR in hypoxic conditions. The latter regulates the expression of both maturases in a constitutive fashion. If any promoter loses its function all the system is shut down because both the maturases and the hydrogenase are required for hydrogen production. If the first promoter loses its capacity to detect hypoxia, and becomes constitutive, a slight decrease in fitness is expected, since hydrogenase would be produced in an aerobic environment that renders it useless. Any defect in a promoters leads to a reduced fitness phenotype since proteins regulated by the other promoter would be expressed, spending metabolic energy.

As of transcriptional terminators, each construction will contain two of them. If the first one fails or is deactivated by mutations, the second one stops the transcription of our operons. This is very important to ensure that our system doesn’t interfere with adjacent ORF’s and their transcription, maintaining its modularity.

How would the whole device or system change its properties and what unintended effects would result thereof?.

We have analyzed two cases that seem plausible with our system. The first one is our system deactivation. Under this scenario the organism would have a reduced fitness both producing unnecessary proteins that fail to complete the system and consuming metabolites in the produced proteins reactions.

The second scenario is the excessive consumption of metabolites by our system. This would probably kill our bacteria since we will be utilizing reduction potentials, which play a central role in the bacterial metabolism.

What unintended effects could you foresee after your engineered organism is released to the environment?.

It is possible that our strain infects other plant species, harming or poisoning them. However it seems improbable because the interactions between Rhizobium etli and Phaseolus vulgaris are very speciffic and have been affected by co-evolution processes.

Try to think outside the box. What is the absolute worst case scenario for human health or the environment that you could imagine?.

The absolute worst case scenario requires our strain to be very efficient in the bean root infection process. It also requires our strain to be bad at or lack at all nitrogen fixation, and to acquire a great viability in free life. With these assumptions, the infection of a bean root with our strain would kill or exclude other strains from the root and deprive the bean from the nitrogen fixation process. The free life viability would allow our strain to remain in the soil and infect more plants in its surroundings. The constant competition for roots would always exclude nitrogen fixating bacteria, stopping the fixation process at all in the release environment, affecting all life forms given that nitrogen is required by all organisms.

We believe this scenario to be completely out of the question for the following reasons:

• The strain that we are working on (CFN42) presents a naturally very low fitness when competing with other Rhizobium etli strains.

• The hydrogen production system consumes reductive metabolites, which carry energy. This consumption is expected to reduce the modified strain fitness compared to its unmodified counterpart.

• None of the hydrogen pathway components are expected to interfere with the bacterium-plant communication, or the infection system. So our strain is not expected to increase its infection efficiency.

• It is possible that hydrogen production interferes with nitrogen fixation, either reducing its efficiency or inhibiting it at all. Yet, Phaseolus vulgaris has a system that senses nitrogen fixation and has the ability to kill bacteria lacking the system. If nitrogen fixation is lost the strain infection efficiency is expected to be diminished by this system.

References

(1) Carcassi, M.N.; Fineschi, F. (2005). "[http://www.sciencedirect.com/science/article/pii/S0360544204000532 Deflagrations of H2–air and CH4–air lean mixtures in a vented multi-compartment environment]". Energy 30 (8): 1439–1451. doi:10.1016/j.energy.2004.02.012.

(2) Mr J Gummer &Dr S Hawksworth “[http://www.hse.gov.uk/research/rrpdf/rr615.pdf Spontaneous ignition of hydrogen]” Literature Review, Research ReportRR615. Health and Safety Executive, 2008.

(3) Penny R. Hirsch, “[http://www.springerlink.com/content/m25jp22p431h2k17/ Release of transgenic bacterial inoculants – rhizobia as a case study]”, Plant and Soil 266: 1–10, 2004.

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