Safety
Risk assessment is essential to the decision-making process for commercial release of GMOs into the environment. When GMOs end up in the environment, they can have unintended consequences for it. Genetically altered micro-organisms are able to reproduce and establish themselves as permanent populations and there can be subtle and long-term effects on the biological communities and ecosystems. Results of DNA modifications can’t be limited to the replacement of the specific characteristics of its gene. Therefore, it is important that when these organisms are released into nature, they don’t harm the environment or form any risk to human health. Such problems have led to a broader interest in the theme of risk assessment in the release of GMOs.
Synthetic biology is a new field, which is different from engineering genetics and working in the lab. We have to assume that our target consumers are probably not all familiar with biology. All in all, a cautious approach is needed. We need to assess the environmental risks of our iGEM project that might arise from the introduction of recombinant organisms.
Safety in our lab
In our lab, where we build our secret message into our bacteria, we work on a Biosafety level 1. All team members are familiar with the safe microbiological techniques to work safely with microbiology and GMOs. The according rules apply in in our lab.
- All GMO related work can only be performed by those people that have permission from the Biological Safety Officer (BVF). Work according to the rules, even if you believe there is no apparent risk.
- During GMO related work all doors and windows have to be closed. Verify that insects and other pests are not present in the lab.
- Wear a closed laboratory coat. Do not take this lab coat outside the VMT area unless it is absolutely necessary for the experiment. In case of a contamination of the lab cat, sterilize it first, with bleach or by autoclaving, before washing.
- Clean and sterilize spills immediately. Report serious contamination immediately to the BVF.
- It is absolutely prohibited to eat, drink or smoke, or to have cups, plates, mugs or silverware in the GMO area.
- Pipetting by mouth is prohibited. Used pipettes are collected in a disinfecting solution.
- Prevent aerosols. These may be created by -splashing drops; -pouring of liquids; - discharging pipettes; -opening of moist plugs; - using inoculating loops that are too hot. Use of needles only if there is absolutely no alternative.
- Glassware and instruments that have been in contact with GMO's (Genetically Modified Organisms, GGO’s in Dutch) have to be sterilized or disinfected before being washed, reused or discarded. Biological waste has to be collected in autoclavable plastic bags, which are autoclaved before discarding (use indicator tape to demonstrate that the bag was autoclaved).
- Wash hands with soap and water after work and before leaving the room. Bench surface areas have to be cleaned and disinfected daily. Keep the area clean and organized.
- Record the general nature of the work clearly in a lab journal.
Safety and security in our chassis B. subtilis
If our chassis B. subtilis is released into nature, there should be no harm to humans or the environment. In our project we work with the non-pathogenic chassis B. subtilis for which we designed a kill switch for safety and for security of our message. Nevertheless, we came up with idea for an application of CRISPR/CAS 9 for our chassis, which can be found here
The design of our safety and security kill-switch.
“We’re moving from reading the genetic code to writing it” – Craig Venter
Synthetic biology is one of the most developing and emerging technologies to date. [1] The field of synthetic biology is continuously developing. As in iGEM, real-world applications are constantly shifting ideas, more research becomes available. There is only a pressing concern, especially with the microbial systems. Genetically modified bacteria may produce undesired consequences if they are released into the environment
In order to provide an idea of the risks possible of releasing GMO in the environment, risk should be assessed. In this special case of our project, we should not only assess the safety risks but also the security risks
In general, there are two potential hazards considered generally; successful competition against other natural strains and horizontal gene transfer from engineered bacteria to natural strains. [2] Natural strains have less changes compared with GMO’s considered their fitness. For example, most designed bacteria contain antibiotic resistance cassettes in their plasmid, which are used for the cell forcing to keep an insert plasmid. In absence of selection a plasmid can be discarded by its host. [3] Therefore, due to horizontal gene transfer, some engineered genes might could have been taken up by other bacteria, which also could lead to competition with other strains. Both situations end up creating a natural imbalance between species.
There are several regulations regarding genetically modified bacteria. The one that applies to us are the ones about working safely with GMO’s and the sending of GMO’s. Following these rules should be taken seriously.
Over time, the lack of evolutionary stability of synthetic DNA might serve as safety itself. A synthetic bacteria could change to a near wild-type state, thank to pseudo-restoration. [4] But there is current consensus that multiple safety mechanisms are needed to ensure that risks are lowered in case of component inactivation. [5]
We designed a kill-switch to prevent against the two potential hazards. This kill-switch in under a constructive promotor which is always on, unless suppressed. If the spores are released in nature they will not germinate, because of tetracycline deficiency. Under the control of PtetR, the nuclease is translated, which is capable of digesting extracellular genetic material. Meaning that horizontal gene transfer isn’t a possibility.
Looking at the security aspects, both applications in the kill-switch are also to prevent that unauthorized parties gain our spores with the message or key. If the unauthorized receiver tries to grow the spores without the right compounds, the kill-switch will be activated and the message or key will be digested by the NucA. This makes our system should be safe and secure.
Safe by Design: RIVM
The RIVM, The National Institute for Public Health and Environment, challenged us to go beyond the safety forms of iGEM. By inviting iGEM teams from the Netherlands to give an implementation of safe-by-design, they wish to improve safety. At the first meeting with the RIVM, we presented our plan for safety in our project, by which we implemented by the Safe-by-Design concept.
On Tuesday 21th of September we had a second gathering entitled “Safely continue with synthetic biology” with all Dutch iGEM Teams, the RIVM and the Rathenau Institute. Here we presented our project and what our final implementations were of “Safe-by-design”. The purpose of Safe-by-design is to make sure that all the safety aspects have been considered during the design stage of your product. This made Safe-by-design the topic of the day.
The afternoon was initiated by prof. Dr. Erik Lebret, Risk Assessment researcher at the RIVM and Dr. ir. Melanie Peters, director of the Rathenau Institute. Erik Lebret gave the kick-off by stressing the importance of incorporating risk management from the beginning of the manufacturing process development. Whether Melanie Peters emphasized the responsibilities after fabricating, because safety isn’t only a matter of engineering.
This day we also spoke about the ethics side of safe by design in the application of biotechnology. According to Sabine Roeser, ethics professor at TU Delft, emotions and art are the sources of ethical in Safe-by-Design.
At the end of the day we had workshops about previous iGEM projects and discussed several important safety aspects of these projects. Perhaps we should sometimes consider whether biotechnology solutions are the best solutions.
This day highlighted a lot of safety aspects iGEM, and compelled us to consider safety and security while working on our project.
References
- [1] Synthetic biology has been described as “arguably the world’s hottest and most poorly defined scientific discipline.” Paul Voosen, Synthetic Biology Comes Down to Earth, CHRON. HIGHER
- [2] Genya V Dana et al. Four steps to avoid a synthetic biology disaster. 2012. Nature vol 483
- [3] Silva-Rocha & other authors (2013). The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. Nucleic Acids Res 41 (Database issue), D666–D675.
- [4] Oliver Wright, Guy-Bart Stan and Tom Ellis. Building-in biosafety for synthetic biology. Microbiology (2013), 159, 1221–1235
- [5] Presidential Commission for the Study of Bioethical Issues, 2010.
