Safety

In our project, we have used synthetic biology to create a ferritin container with attached cell penetrating peptides (CPPs) to transport antibacterial active substances into pathogens. The container gets degraded inside the pathogen and sets the substances free. In order to ensure that our project was in line with safety regulations, we have consulted a safety expert of University of Hamburg, Dr. Mirko Himmel, and undergone safety training for laboratories with a biosafety level 2 (BSL2) classification since our laboratory was situated within the Kolbe group at the Center for Structural Systems Biology (CSSB) in Hamburg.

All in all, we learned about the different safety aspects that come with working with Genetically Modified Organisms (GMOs). Below you can learn how we implemented various safety aspects and ethical considerations during our research and beyond!

To work safely with genetically modified organisms (GMOs) we had an elaborate safety training provided by the CSSB. This course taught and refreshed knowledge on how to properly work with GMOs.

During our research and project consolidation for the competition, we discussed the safety and security aspects of our project. To gain a comprehensive understanding of the field, Dr. Mirko Himmel, our secondary PI and bioethics specialist, provided an informative introduction. Furthermore, our team thoroughly examined the potential uses and misuses of our project by analyzing the beneficial and malevolent perspectives separately.

Content of the Introduction to Biosafety and Biosecurity

Dr. Mirko Himmel talked about biosafety and biosecurity, stressing the significance of occupational health and safety and protection against misuse in biological research.

He introduced the "spectrum of biological risk factors" (fig. 1) as a central concept, clarifying that we occupy the middle, where targeted organism modifications occur. However, Dr. Himmel cautioned against reaching the extreme ends of the spectrum, as such actions increase the likelihood of knowledge misuse by bioterrorists. The presentation included a discussion of biological and toxin weapons, highlighting the technical challenges involved in their production and proliferation.

The role of synthetic biology was also extensively examined. He emphasized the potential for genetic manipulation to optimize pathogens, and the ongoing expansion of knowledge in this field. Historical examples of the risks and misuse of biological knowledge were presented, including the pre-Cold War U.S. biological weapons program and the Japanese biological warfare program of World War II. The challenges of implementing arms control agreements such as the Biological Weapons Convention and the Chemical Weapons Convention were emphasized. Dr. Mirko Himmel also mentioned the recent developments in the Nawalni case.

He highlighted the importance of considering biosecurity measures for both protecting against dangerous organisms and preventing misuse in synthetic biology research. He stressed the significance of self-regulation and the responsibility of researchers, as demonstrated through the iGEM project.

What is meant by Biosafety and Biosecurity?

Biosafety and biosecurity involve managing and controlling dangerous biological agents to prevent harm to human, animal, plant, and environmental health, whether intentional or unintentional. While these two concepts are related, each places a unique emphasis on biological safety.

Biosafety involves implementing strategies, protocols, and containment measures to mitigate risks in laboratory and research settings that involve biological materials such as microorganisms, toxins, and genetically modified organisms (GMOs). The goal of biosafety is to protect individuals, communities, and the environment from inadvertent exposure to potentially harmful substances. The use of clear and objective language, consistent technical terms, and logical progression of information contributes to a balanced and precise discussion of the subject. Informal expressions, contractions, and unnecessary jargon are avoided in the formal register of language used in the text. This includes a range of practices, from the design of laboratories and the use of equipment to the disposal of waste and proper training. Biosafety Levels (BSLs) are commonly used to categorize conditions for confinement and necessary preventive measures when handling various types of biological agents. Four primary biosafety levels exist, ranging from BSL-1 (least dangerous) to BSL-4 (most dangerous), with each level necessitating more stringent safety measures.

Biosecurity aims to prevent intentional or malicious use of biological agents for harmful purposes such as bioterrorism and the development of biological weapons. This requires implementing measures to prevent unauthorized access, theft, loss, or release of potentially dangerous biological materials. Biosecurity measures may involve physical security, access control, personnel screening, data protection, and biological agent inventory management.

We conducted a self-assessment of our project with regard to safety under the supervision of Dr. Mirko Himmel. We divided our team into two sub-teams, with one focused on the positive applications of our project and the other on the negative ones.

Ideally, it would initially target human pathogenic bacteria in vitro and later in vivo. It is intended for human application in the future to circumvent antibiotic resistances of pathogens.
The objective is to tackle bacterial pathogens that are multidrug-resistant. The aim is to achieve high specificity through the use of a modular system.

Are we using an incorrect dosage?
Perhaps by accident?
Toxins could be packaged and not directly targeted towards human cells. Instead, human toxins can be introduced into the pathogen which will only release the toxin when reaching the eukaryotic cells. Microdosing may also mask homicides by keeping the toxin concentration below the detection limit.

We restrict the amount of toxins or similar substances that can be introduced to a limited range of volumes.
Targeted pathogen elimination achieves a reduction in the formation of resistance.
By bringing attention to the matter, we acknowledge that toxins can also prove fatal at levels below the threshold. Methods with lower detection limits may be developed as a result. For detection, the indirect detection of toxins through the half-life of nanobodies, for example, can be considered.

We apply cell-penetrating peptides (CPPs) for bacterial transfer. To achieve specificity, nanobodies and other methods can be utilized.

What are the potential consequences of exposure to CPPs while working in the lab?
CPPs are known to cause harm to eukaryotic cells, and even with the use of nanobodies, there is no guarantee of avoiding such damage.
We must ensure that the nanobodies are also present on the surface of ferritin. Otherwise, there will be no more specificity.
We apply cell-penetrating peptides (CPPs) for bacterial transfer. To achieve specificity, nanobodies and other methods can be utilized.

Nanobodies enhance specificity, particularly when not accompanied by linkers. This leads to reduced chances of misuse and more precise results.

The suitability of ferritin as a carrier stems from its abundance of good structural data. Ferritin serves as the carrier protein, utilizing the naturally occurring human ferritin heavy chain, and modifying it through synthetic biology techniques to enable mass production via bacterial recombinant overexpression. Furthermore, it should be noted that ferritin derived from Escherichia coli lacks glycosylation which is present in human ferritin. There are several open questions to consider, such as the extent to which loading and modification affect affinity, as well as the implications on immune response.
To lock drugs within the ferritin, modification is necessary to adjust specificity for drugs within the ferritin. The aim of our transport system is to specifically target the bacteria present in the bloodstream that cause bacteremia, systemic failure, or multi-organ failure.

Since our system is highly scalable, cost-effective, and efficient, it can be used for toxin transportation.
It is possible that the body may mistake the transporting human heavy chain ferritin for the body's own iron.
Open question: Are there any concerns regarding bioaccumulation in certain organs or the stability of ferritin? What occurs with the transported substance after the bacterium has perished? How are these substances metabolized? Can ferritin precipitate and aggregate in the human body/blood, resulting in heart failure?

The human-origin ferritin we utilize will not disassemble in the bloodstream, ensuring its effectiveness. Metabolism of ferritin by the human immune response occurs when it accumulates excessively in the system.
The benefit of our work is the reduction of the risk of toxin encapsulation. Our goal is to produce ferritin that exclusively encapsulates antibacterial agents. Encapsulation of other substances requires extensive efforts due to the pH value alteration that is not feasible for non-pH stable applications.

Conclusion

Most of the hazards we evaluated are only applicable in real-life medical applications. As we are in the initial stages of the project and will not proceed to real-life applications, researching on E. coli is deemed safe. The standard research procedure comprises multiple stages, starting with conducting the project on a small model organism and progressing to a larger model organism once deemed safe. In case of significant concerns, the project requires alterations and reevaluation. Nonetheless, it is crucial to evaluate a research project beforehand, particularly its potential for dual use.

A more detailed description of all safety aspects of our project can be found in the Safety Form and Check In form.

Our laboratory, situated within the Kolbe group at the Center for Structural Systems Biology (CSSB) in Hamburg, has been designated with a biosafety level 2 (BSL2) classification. To ensure a secure working environment, we adhered to all directives and stipulations outlined in the German legislative framework (GenTG). Prior to initiating experiments within our wet lab, we underwent safety training that imparted essential knowledge for safeguarding both ourselves and our surroundings. This training included proficient handling of genetically modified organisms (GM-organisms), protocols for chemical safety, proper methods for waste disposal, and the correct utilization of personal protective equipment. Furthermore, we were educated on appropriate actions to take during emergencies, and were well-informed about the locations of emergency exits, eyewash stations, safety showers, fire extinguishers, and first aid kits. Our efforts are committed to full compliance with the safety and security regulations of iGEM, as well as The German Genetic Engineering Act (Deutsches Gentechnikgesetz, Article 1 G 2121-60-1 of 20 June 1990 I 1080 (GenTG)).

We took correct waste disposal very seriously during our lab work. When one works with GMOs an appropriate waste disposal is essential. Biological agent contaminated waste had to be disposed of in extra trash cans, which were then autoclaved. Same with genetically modified materials, they were collected in a closed designated bin and autoclaved before final disposal. Solid waste was separated into carcinogenic, toxic and other waste.