Team:Wageningen UR/Safety

Biosafety Considerations

Building living machines is fun. However, biological research in general, and synthetic biology in particular, are subject to inherent hazards which can affect researchers, the public and the environment. Therefore, it is crucial that we gain a thorough understanding of the possible dangers emerging from our work and take the necessary precautions to minimize the risk of hazards occurring. This page will provide an overview of the issues related to biological safety during our team’s participation in iGEM 2011.

Below, the “Track ‘n Trace” and “Synchronized Oscillatory System” BioBrick systems will both be referred to as a part of the (team) project.

1. Biological safety identification

Pathogenicity of host organisms

A. nidulans

The filamentous fungus Aspergillus nidulans (strain pyrG89 argB2 pabaB22 nkuATargB riboB2, ordered from FGSC; A1147) is capable of producing spores. Though the route of infection should be effective, according to Kim et al., disease caused by the micro-organsim is rarely seen in healthy persons (1997 Jun; Jump to references). Correspondingly, the Dutch ‘Regeling Genetisch Gemodificeerde Organismen' (Regulation of Genetically Modified Organisms) classified A. nidulans as Biosafety level (BSL) 1 and allows genetical modifications when it's performed in a ML-II lab.

Project developments may require work with Aspergillus niger in addition to A. nidulans. This micro-organism is also classified as BSL 1 and is therefore subject to the same safety considerations as A. nidulans.

Fig.1. Aspergillus nidulans is a green mould that is a common contaminant of starchy foods, such as bread.

E. coli

The Escherichia coli TOP10 strain used in this project is a derivative of the non-pathogenic K-12 laboratory strain. The likelihood of a human becoming infected is therefore low. The most probable route of transmission would occur by accidental ingestion, though the bacterium’s inability to be retained in the human gut decreases the chance of it to develop disease in humans (EPA, 2011; Jump to References). Mainly for these reasons, this organism is also classified as BSL 1.

Fig.2. A microscopic picture of E. coli cells. The length of a cell is about 2 micrometers. The colours are not real.

Probabilities of hazards

Fungal Track ‘n Trace

The main hazard in this project is the accidental release of genetically modified spores into the environment. This could potentially occur during transportation if samples are not properly contained. Because the life cycle of Aspergillus contains a sporulation phase, it is subject to a higher risk of being released into the environment than organisms which replicate through simple cell division.

In order to perform microscopy studies of genetically modified A. nidulans strains, it is necessary that the fungus be transported between two nearby buildings. The probability of release into the environment without secondary physical containment (like air filtering) is high. The usage of a sterile box container sealed with protective film will decrease the probability of this. At the actual point of the microscopy, the sample will have to be exposed in a lab space without physical containment equipment. Therefore the probability of genetically modified material being released in this way will be minimized, as described in the following. It was advised by a biosafety instructor to remove the spore forming bodies (and spores with it) from the sample –leaving the hyphae behind– and close it up with nail polish or sterilize the surface by passing it quickly (to prevent damage to the hyphae inside) through a Bunsen flame in the ML-II lab before transport. To reduce the probability of release by sporulation, samples are covered up in petri dishes as much as possible and handled carefully, according to the Dutch Good Microbial Practice and Containment standards.

Synchronized Oscillatory System

As E. coli does not produce spores, the route of infection towards practitioners is improbable via air transport. Accidental formation of aerosols, especially during the construction of the BioBrick device, could still bring workers in contact with E. coli and increase the probability of environmental contamination. The effects can be minimized by filtration of the air, as situated in the ML-II lab, and its occurrence should be reported.

Accidental release to the environment

There are several scenarios in which unintentional release of genetically modified material could take place.

Labeling of the lab equipment and glassware used is necessary to prevent loss and improper waste disposal by a fellow researcher. The following problems are not as easy to prevent.

The air filtering system will have a hard time in keeping aerosols in the lab when a window gets broken. If this happens through a thunderstorm, an electricity break-down is not unlikely and would increase the chance of release furthermore. Under Good Microbial Practice, though, the formation of aerosols is prevented as much as possible, thus the combination of these probabilities leaves a total probability that is not that high.

In transport from one end of the lab to the other, there is however a chance the transporter falls and cuts themself with the broken glassware containing hazardous biological material. Infection is a high probability in this case. For our project prophylaxes would be available, which reduce the chance on the development of a disease.

It should furthermore be reported to the Minister of ‘Housing Spatial Planning and the Environment’ and, involved, institutions and direct actions should be taken if it is found that an autoclave has been malfunctioning. The chance that this happens however is as large to happen in our project as it would be for other projects. Still it has to be checked as frequently as possible if the autoclave is functioning.

The usage of antibiotic resistance markers, to select for tetracycline resistance for example, increases the chance of spreading antibiotic resistance to pathogens. Conjugation and transduction is a way of DNA transfer between bacteria. There is a chance the antibiotic resistance genes end up in a pathogenic bacterium that isn’t intrinsically resistant to antibiotics. However, this hazard is present in most molecular microbiology research.

Some hazards have already been mentioned by giving the probabilities of them, but in the following paragraph a closer look to harmful effects will be taken.

Specific Hazards

Fungal Track ‘n Trace

The Track ‘n Trace project involves modifying the leucine metabolism of A. nidulans and using metabolic intermediates to regulate the expression of fluorescent proteins. We do not expect these modifications to increase the pathogenicity or fitness of the A. nidulans strain. Therefore, we do not anticipate any additional safety hazards going beyond those inherent to working with A. nidulans in a laboratory environment.

Synchronized Oscillatory System

This project involves the periodic oscillation of the extracellular concentration of the quorum sensing molecule AHL. Due to the important role quorum sensing molecules play in many bacterial infections, successful transfer of the oscillator BioBrick device to a pathogen could potentially affect the development of QS-related pathogenicity in humans.

Environmental impact

The way an organism arises in Synthetic Biology is significantly different compared to its reproduction ability or abilities. Therefore, when it enters into new surroundings its ability to thrive can be different from its wild type variant and the practitioners of Synthetic Biology should, at least partly, be responsible for what they produce. So, consideration has to be taken about possible mutations that could occur whilst it is in the environment.

A mutation in the synchronized oscillator, for example, could give the host organism an unexpected fitness advantage, and allow it to invade certain ecological niches. The Track ‘n Trace system would only have a plausible fitness advantage in environments with very particular requirements for the Leucine metabolism.

Risks and benefits

For both parts of the project, the risk to the practitioners is rather small. The risk on a negative effect to the environment due to the practice is considered to be intermediate. That is because the effects can’t have been studied yet – unforeseen hazards might turn up.

A robust biological oscillator that could be integrated into more complex genetic circuits would be of great utility for more advanced synthetic biology applications, especially related to quorum sensing. One can also envision the oscillator as a core component of a system sequentially performing specific enzymatic reactions.

The Track ‘n Trace project seeks mainly fundamental knowledge, and is an attempt to introduce a new, biotechnologically useful eukaryotic model to iGEM. A better understanding of, and control of intercellular mass transfer in hyphal fungi could also prove beneficial for engineering solid state fermentation cultures.

Based on these considerations, we have concluded that the potential benefits in successfully executing our projects outweighs the risk of serious hazards occurring.

2. Notification

The systems our team designed do not contain parts that produce toxins. Therefore, no additional biosafety indications will be given next to the other part information at the Registry of Standard Biological Parts (RSBP).

When there is (uncertainty about) relevant biosafety information, it would be good to indicate this in the Design Notes of the Part Design page of the RSBP. Furthermore it should become clear from the introduction on the Main Page whether the BioBrick part produces a biohazardous product or the system might be harmful in total and if a subpart falls out of the superpart. Because effects of release out of the containment can be unpredictable, biosafety information wouldn’t be at the right place on the page Hard Information. A team might have designed a part without producing and validating it. If this part is then used in a new system and turns out to be hazardous during the construction this should also be noticed under User Reviews at the Experience page. For this purpose, the team that used the BioBrick design should be able to edit this page.

The indications of biosafety could even be more generalized when at the BioBrick part entry page of the RSBP there would be requested to enter these indications. This, by filling in two extra text entry box(es) headed with, the notions on safety indicated by iGEM:

• Foreseen BioBrick Part or System/Device risk(s) and

• ‘Chassis enhancement’

or by marking a ‘No relevant biosafety issue’ checkbox (2011; Jump to References). They could also come out better if the Main Page of the BioBrick part had a Biosafety category, that would add relevant biosafety information from the entry page automatically.

3. Biosafety regulation

In the Netherlands, any person or institution intending to perform genetic modifications needs to have licenses of the Royal Order on Genetic Modified Organisms (which is called ‘Besluit GGO’ in Dutch), Regulation Genetic Modified Organisms (‘Regeling GGO’) and environmental legislation as the Law on Environmental Management (‘Wet Milieubeheer’). Food related genetic modifications and border crossing transport of genetic modified (micro-)organisms are also regulated by European legislation.

The Royal Order on Genetic Modified Organisms is mainly about the classification of the organisms, in which consideration is taken about modification of the genomes.

In accordance with the requirements of the Regulation Genetic Modified Organisms, the institution that houses our team has ascribed a safety officer. This person has introduced every team member to the lab by giving us a “safety tour.” Therein, we came across all the rules and information we should regard: biological waste, disinfection of the lab bench, how to dress in the lab, the way of acting in case of an emergency and more. Wageningen UR also has ascribed a biosafety officer which, next to performing other tasks, sees to any genetic modification related research is being executed according to the rules involved.

The Law on Environmental Management notes especially the actions that should be taken in case the environment gets into contact with genetic modified organisms. These actions are stated generally. If this occasion would appear, it is expected from us we are responsible to do everything in our ability to solve the problem. This means we should inform the biosafety officer, which then again will inform the license holder, that informs our Minister and environmental agencies.

4. Improvements on safety

Embracing the public

Understanding should come with knowledge. It might be beneficial for the development of Synthetic Biology and the society if the public will be allowed to follow projects on close watch. Probably even more if this is as close as joining a day in the lab, well maybe by viewing it via a video monitoring. In this way the biosafety regarding human health and the spread of disease won’t be at stake and might still be as fun to do. Our team has set this up and it can be found on the Social Media page. In combination with the, freely available, Wiki information pages of iGEM it could be possible for the public to test if any concerns about ‘Synthetic Biology’ were right or exaggerated. In order to provide the possibility of noting any – in this case: nuanced – concerns people should be able to add these on a Wiki page especially arranged for it. This page should give a clear overview of comments about projects. Synthetic biologists could give answers to questions on this site. This direct possibility of interaction should reduce the feeling of missing hold on Synthetic Biology and, with it, fear about it.

With outreach our team furthermore tried to contribute to make the public more aware of the actual content of iGEM and partly Synthetic Biology.

With understanding comes involvement, this might give rise to a growing improvement on iGEM and Synthetic Biology in general.

References

Kim M, Shin JH, Suh SP, Ryang DW, Park CS, Kim C, Kook H, Kim J. Aspergillus nidulans infection in a patient with chronic granulomatous disease. J Korean Med Sci. 1997 Jun;12(3):244-248. URL: http://synapse.koreamed.org/DOIx.php?id=10.3346%2Fjkms.1997.12.3.244. A small fragment was used. Copyright © 1997 The Korean Academy of Medical Sciences.

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EPA(U.S. Environmental Protection Agency). (2011, January Monday). Escherichia coli K-12 TSCA Section 5(h)(4) Exemption: Final Decision Document. Retrieved July 2011, from Biotechnology Program under the Toxic Substances Control Act (TSCA): http://www.epa.gov/biotech_rule/pubs/fra/fd004.htm

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iGEM. (2011). Safety. Retrieved July 2011, from iGEM 2011: https://2011.igem.org/Safety

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