By Chris Isaac, Marissa Sumathipala, Nannan Jiang, and Hassnain Q. Bokhari

Via Unspalsh - National Cancer Institute

Introduction

iGEM is a sandbox of innovation in multiple areas, including in biosafety and biosecurity. Even though there is a lack of legislation around the world governing biosafety and biosecurity, some teams have taken it upon themselves to make improvements and conduct science responsibly, safely, and securely. Historically, iGEMers have done a tremendous job in addressing any biosafety and biosecurity concerns in their individual iGEM projects, despite biosafety and biosecurity being challenging issues to address at national levels.

In this article, we aim to briefly and non-exhaustively highlight teams who have done exemplary work in areas related to biosafety and biosecurity. We hope that readers will be inspired by their stories and integrate some of these practices into –or perhaps design novel ones – for their own projects and make a deliberate effort not only to comply with the safety and security criteria of iGEM, but also to innovate and develop new tools, ideas, and efforts to raise the bar for how iGEMers and the rest of the world consider safety and security in their projects.

Biosecurity

In 2013, the University of Lethbridge iGEM team investigated an RNA element called a pseudoknot. Pseudoknots are secondary structural motifs that can displace the ribosome during translation causing a frameshift. The team wanted to create pseudoknot elements that induced frameshifting at predictable frequencies. The pseudoknot-carrying constructs will then have the ability to “compress” double the amount of coding potential into a single DNA fragment, along with enabling proteins to be expressed with variable localization signals, purification tags, or other functional domains.

Prompted by the increasing prevalence of gene synthesis and the growing recognition of the power of that technology, the University of Lethbridge iGEM team sought to determine the extent to which its project could impact the screening practices of DNA providers. They suspected that bad actors might be able to evade screening and acquire toxins or other dangerous proteins by using pseudoknots to distribute a controlled sequence across different frames of translation. Unsure of whether or not the increased complexity could defeat screening practices, the team engaged DNA synthesis providers to investigate and test the system.

The team designed several sequences using the pseudoknot element to adjust and distribute sequences of concern including ricin, Ebola matrix protein, open reading frames from Staphylococcus spp., and innocuous fluorescent protein. Major North American synthesis companies took the orders and subjected them to their screening practices. One of the companies was able to identify all hazardous sequences, indicating that there is capacity to detect sequences shifted by pseudoknots. This is likely because the companies use all six frames of translation to BLAST the sequence and that the length of the tested sequences were long enough to make high-scoring alignments to the reference sequences. The team concluded that the largest biosecurity risk in DNA synthesis is the limited adoption of screening best practices. Though no security vulnerability was discovered, their work was positively received by the DNA synthesis community, and ultimately led to the team receiving the Security Commendation from iGEM. The team has continued to engage with DNA screening in their work, investigating the impact of codon reassignment in their 2018 project.

Dual-Use Risks

The Bielefeld 2018 iGEM team identified a potential Dual-use research concern (DURC), while undertaking their project. They worked on developing a microbe that had the ability to extract metals from old electronics to help with recycling. Ideally, the bacterium would extract the metals and create metallic nanoparticles that could be used downstream, creating new value from the recycling process. However, the team noted the risk that their project could be misused to degrade useful electronics. They reasoned that even if the effect of the microbe was small and their original intentions were good-natured, that the public might negatively perceive the project if framed as a weapon or actually misused.

The consideration of their project as a fundamentally dual-use technology was the team’s first brush with dual-use, and they were concerned that none of the members of the team had previously encountered it before during their education or training. Following that thread, the team dug in, researching issues of science communication, the legal landscape of dual-use research, integrating dual-use training into universities, and reaching out to other iGEM teams.

Notably, their outreach efforts to other teams included distributing slides on dual-use, giving presentations, and rewarding teams that participated with a “Dual-Use Awareness Button” to embed on their wikis. The purpose of the button was to showcase the team’s engagement with the concept of dual-use, acting as a mark of recognition that teams with the badge had assessed their project for dual-use risk and took the issue seriously. The inclusion of the badge on the wiki also served to introduce additional teams to the concept, broadly raising the level of awareness within the competition and beyond.

More information about the experience of the Bielefeld teams can be found in “iGEM and the Value of Responsibility.”

The 2018 Bielefeld team meets with experts to discuss Dual-Use

Biosafety

The 2018 Delft University of Technology iGEM Team took a scalable approach in targeting the accessibility of biosafety training itself. The team produced an interactive virtual reality (VR) laboratory to conduct basic biosafety training. In doing so, the team hypothesized that using VR technology can reduce financial, temporal, and spatial constraints due to laboratory instrumentation, training time, and consumables, while providing training without exposure to real risks.

Ultimately, the goal of every iGEM project is to provide some sort of real-world benefit, and in some cases, that envisioned benefit can only be realized if the engineered organism is released into the wild. iGEM has an extremely strict Do Not Release policy forbidding teams from testing their organisms outside the lab. However, some teams are preparing for a future in which policies would allow for the environmental release of engineered organisms under strict safety guidelines and clear, predictable consequences. In regards to environmental protection through containment, the 2012 Paris Bettencourt team designed an entire system called the bWARE containment module to protect natural organisms from horizontal gene transfer from GMOs. Their system would hopefully allow for the introduction of engineered organisms into the environment without contaminating the existing genetic biodiversity.

The team began by engineering a programmed delay in gene expression that disables an antitoxin gene, thus compromising viability of their genetically modified host through a colicin kill-switch. Using this same restriction enzyme approach, the team not only regulated the cleavage of key genes, but also of whole plasmids. Next, the 2012 team introduced a semantic containment system to include a premature stop codon in specific proteins, which can be overcome by a suppressor system engineered within the host organisms. Lastly, the team used physical encapsulation of their genetically engineered hosts in gel beads to prevent further spread into the environment. This system is one of many small steps that may take us closer to safe and controlled environmental applications.

Though most microbes are harmless, pathogenic bacteria pose a considerable threat to human health. With this in mind, the 2014 Aachen iGEM Team developed a recombinant biosensor system centered around their measuring device “WatsOn.” The instrument identifies the contents of a chip, specifically targeting Pseudomonas aeruginosa, by using a genetically modified reporter strain that responds by quorum sensing. Realizing that they were dealing with GMOs and opportunistic human pathogens, the team went to great lengths to address their biosafety concerns upfront.

The WatsOn device was designed as an overall closed system, which prevents unintentional contamination outside the measurement device. Single-use sensor chips further reduce risk of contamination and are sterilized following use. The team conducted a risk assessment and determined a low risk assessment even in the event of improper handling of the chip or measuring device.

iGEM Teams also serve as catalysts to develop knowledge and create resources for new frontiers. Last year, the iGEM São-Carlos team undertook a project to facilitate the production of ethanol and fermented food on Mars. They used synthetic biology techniques to display a protein anchoring system on yeast, which uses melanin as an armor to protect the cell from UV radiation. They proposed testing their system in the stratosphere–which has radiation signatures similar to ones recorded on Mars–within a stratospheric probe. The probe would consist of a high-altitude helium balloon attached to a box that contains the engineered system to be tested in the stratosphere.

Given the nature of their test, it was difficult for them to evaluate whether the launch system comes under the scope of environmental release or not, and what kind of safety regulations they have to be wary of for a stratospheric experiment. They reached out to a diverse set of experts both locally and internationally to help them understand containment regimens, and regulations for conducting testing of genetically engineered organisms in the stratosphere. Their effort helped identify the current gaps in literature relevant to their project, and the lack of both national and international publicly available guidelines. You can see a detailed documentation of their effort on the team’s wiki.

Other collaborative efforts took a bird’s eye view from a policy perspective and consolidated the current regulatory landscape of GMOs. Realizing that biosafety would become an increasing issue as the world moves from industrial factories to cellular ones, the 2017 South China University of Technology and Northwestern Polytechnical University iGEM teams looked at GMO regulations from an emissions standpoint across China, the EU, and the US. The teams detailed their findings in their report available here. Along similar lines, the 2017 University of Manchester iGEM Team instigated a 10-team collaborative effort to expand their analysis of GMO regulations across 10 nations. Using their results, the team was able to identify and prioritize locations for bioremediation efforts.

Conclusion

It is clear that safety and security are playing larger roles as projects become more sophisticated. It is oftentimes easier to focus on the experimental results of a project, but the teams that address biosafety and biosecurity issues with intent increase the sustainability and regulatory compliance of their projects in the long run. Other teams that have made contributions to the safety and security ecosystem of iGEM are mentioned in Table 1, which is adapted from Whitford et al. 2018. Moving forward, we applaud past teams and hope that additional current and future teams expand on the ideas above and bring their own unique perspectives and expertise to the thoughtful design of future projects. By ensuring that we can do synthetic biology more safely, we can expand the range

Citation:

Whitford CM, Dymek S, Kerkhoff D, März C, Schmidt O, Edich M, Droste J, Pucker B, Rückert C, Kalinowski J. Auxotrophy to Xeno-DNA: an exploration of combinatorial mechanisms for a high-fidelity biosafety system for synthetic biology applications. Journal of biological engineering. 2018 Dec 1;12(1):13.

Table 1 along with the rest of the articles of Issue 5 of the Digest can be found here: Issue 5 of the After iGEM Digest