Safety and Security

"With a big project comes bigger responsibility" - some scientist around the world

Abstract

A lot of iGEM projects, and synthetic biology projects in general, require a proof-of-concept using proprimary cultures or patient samples. We as well wanted to show that our diagnostic system works as we intended and thus faced the challenge to ensure responsible handling of human derived samples. As for most students, this was a task we never faced before. We found that this multifaceted topic was only accessible to us in a general and uncertain way. In addition, iGEM or other literature provides only limited possibilities to inform oneself about the risks and dangers of handling biomaterial. Based on our project and our experience with the safe handling of tumor material, we set ourselves the task of contributing a guideline for the safe and secure handling of biomaterials of all safety levels. These guidelines are based on discussions with experts , extensive literature research,the current regulations of the different nations, and what we would have liked to know beforehand when it comes to the handling of biomaterials. With these guidelines, we want to contribute a clear and simple way to iGEM (teams) and scientists of the synthetic biology community to evaluate their safety and security requirements for working with human derived samples. Additionally, this page also contains the evaluation of the biosafety and security aspects of our project ASTERISK.

Guidelines

Project-Based Wet Lab Safety

Safe-By-Design

ASTERISK consists of two parts with different safety and biosecurity requirements - integrated Nanopore Sequencing for molecular diagnosis and intraoperative mRNA treatment for tumor-cell specific therapy. Therefore, safety regulations are taken into account. Through our whole journey and the iGEM competition, the handling of responsibility with biosafety issues concerning our project and laboratory work accompanied us throughout. We emphasize that safety in science should be communicated openly and transparently and should accompany educational cations and collaborations. The discussion about biosafety is also closely connected to integrated human practices. Along with meetups and background research, resulted in our advanced project safety considerations both in and outside the wet lab including safe-by-application and safety-for-patients for our dual-strategy of intraoperative diagnosis and therapy:

Diagnostics

After interviewing stakeholders such as Dr. Matthias Simon or others associated with the work in the operating room, we concluded that our strategy for real-time intraoperative molecular profiling of the patient's tumor poses minimal health risk to patients or employes in the operation room. We take advantage of the situation of histological analysis and surgical tumor removal. Our approach does not require additional stress or interventions. Due to its integrative nature, the patient is not subjected from repeated skullcap openings. Diagnosis is performed simultaneously and in real time alongside tumor removal, saving additional resources, effort and potential complication risks for the patient and staff.
By splitting the preparation locally from the actual tumor removal, we ensure safe handling of the tumor material and at the same time adhere to pre- and post-processive procedures in the laboratory. All sample preparation and nanopore sequencing will take place in parallel with tumor removal. Our approach aims at rapid molecular profiling but may vary in duration and chance of success depending on the patient's condition and tumor status. Despite the following ethical and data safety correlated considerations, we classify the diagnostic part of our project as safe for patient and environment due to the flexibility and room for maneuver.
The comparison between tumor material and healing tissue allows better differentiation and increases diagnostic confidence. To reduce background signal, highly specific targets should be identified. The implementation of deep learning and computional inteligence facilitates the evaluation and counteracts human errors.
The more sequencing results, the more precise the molecular profiling - but this can also be misused. In recent years, data security has become an increasingly important concern for the general public. Since the sequence data defines the molecular blueprint of the human being, this information could be unintentionally misused in violation of medical confidentiality and strict regulations. Additional security measures already in place in hospitals allow us to implement the software securely.

Therapy

We implement safe-by-design strategies based on discussions with representatives of the "Dutch national institute for public health and Environment (RIVM)” and the “Robert Koch Institute (RKI)”. Their concepts help to assess risks involved, both in the laboratory, during the conception of the safe-by-design cycle, and in the possible dual-use application. Beside our mechanism, which provides a safety switch itself, the implementation of additional safety regulations on molecular level e.g., riboswitch previously used by iGEM teams, we aim to develop safe therapy. To minimize off-target effects, we optimize our sensing and kill switch to increase sensitivity and specificity. Only transcripts with additional A-to-I Editing-sites allow specific activation of our therapeutic mRNA. Our circuit is regulated by the presence of specific editing sites that mediate translational regulation. We designed our system to prevent sensing in the absence of the characteristic transcript motifs. This ensures an inhibition of unspecific translation, minimizing false positive results and off target effects. The mutation mediated control of the expression in our therapeutic circuit represents a modular regulatory element for translational expression.
We have designed our sensing and kill switch to address and resolve the potential difficulties of conventional methods. The non-systemic application reduces potential off-target effects in other tissues or organs. Our approach bypasses the blood-brain barrier limitation but works in a brain-specific and localized manner. However, due to infection and immunodeficiency in cancer, BBB permeability varies. Potential systemic effects are minimized due to the choice of only locally acting transport vehicles. In comparison to alternatives e.g. chemotherapy or antibody-mediated strategies we design our therapeutic mRNA to act non-systemic, more specific and highly efficient.
The identification of tumor-specific transcripts is crucial for the molecular differentiation of healthy autologous and unhealthy degenerated cells. The safety of our approach is based on our mechanism itself - only certain deviations from the wild type are targeted and allow tumor cell-specific translation. In addition to using cytotoxic payloads such as Apoptin or Diphtheria toxin, we have replaced intrinsically acting factors with extrinsically acting ones. Additionally, an inhibition mechanism as an extra fail-safe measure via non-toxic chemical or anti-toxins increase the safety standard of our diagnosis platform. The co-translation of interleukins in the heterotrophic tumor miniature ecosystem allows the recruitment of immune cells to naturally counteract tumor growth. In addition, cancer-related toxic substances can be recognized and degraded by the immune system.
Safety mechanism designed to inhibit cytotoxic effects of our therapeutic mRNA
- Inhibition of cell entrance
- Inhibition of translation
- Inhibition of Payload

Our point-of-care aims at providing a highly sensitive and selective approach to use of special motives and transcript-specific target sites. Semi-quantitative measurement implemented in our software generates optimal motifs.

Choice of Therapy Strategy

Potential side effects of our therapeutic can be estimated by quantifying the in vitro off-target effects. The same effects would probably occur in patients. Since not all effects and interactions of the therapeutic molecule can be modeled in vitro, additional side effects are likely to occur. There are multiple possible sources of side effects throughout the process of the RNA entering the cells, binding to target sequences, and being translated. We will give an overview of possible sources of side effects and the measures we took to minimize the risk they pose. This information might prove useful for future iGEM teams that use similar approaches.
Once the RNA is inside a healthy cell, it could be recognized as foreign and trigger an immune response. The immune response to in-vitro-transcribed RNA can be triggered by side products of the transcription such as abortive RNA fragments or double-stranded RNA. An immune response can also be triggered by the chemical properties of the nucleotides used in the transcription process.

Choice of Delivery Strategy

We used liposomes to transfect the cells with our RNA construct. Liposomes are generally considered to have high biocompatibility and to be very safe. Even so there is a risk of cytotoxic effects occurring. The severity of these effects can be estimated by comparing the viability of transfected cells to that of cells that were not transfected. The components of liposomes can have negative effects on tissues. Phosphatidylserine, a common component of liposomes, has endocrine side effects, which is a serious concern, given that our therapeutic is applied directly onto the brain. Positively charged lipids were found to have a hepatotoxic effect when injected into the bloodstream. Since our therapy is not designed to get past the blood-brain-barrier and enter the bloodstream, it might not be the biggest source of toxicity. However, some liposomes might still enter the bloodstream and cause tissue damage or other unwanted side effects. Therefore, further testing on how liposomes could move through the blood-brain-barrier is needed before liposomes can be employed in the brain. In addition to that, there is a risk of an immune response to the liposome. The risk increases with increased size or cholesterol content as well as cationic or anionic charge. Using polyethylene glycol (PEG) or PEGylated lipids can help to hide the lipid from the immune system but at the same time, they can also cause an immune response by binding to anti-PEG antibodies. All these effects make it necessary to determine the side effects of different material compositions before clinical application. Unfortunately, we didn't have the time to run cytotoxicity assays on different types of liposomes so we only have data on the performance of one transfection system.

Human Subject Research Safety

When working in the laboratory, handling GMOs usually builds the lion's gate of attention. However, we are convinced that data protection is equally important. During our project, we conducted online and offline meetings, webinars, surveys, interviews and information events to spread awareness of safety and security especially in the context of our project. We made sure to take all relevant precautions and advice by relevant laws and regulations or institutional rules or guidance. We extensively researched and prepared data safety and informed consent to ensure safety of all people including in our project.
Based on the AREA framework from past teams, responsible data collection and protection are more accessible. In particular, the contribution of previous iGEM teams led to a basis of consent sheets collection. Additionally, we checked our informed consent sheet with the iGEM safety and security committee, supervisors, ethics committee of university bielefeld including legal representatives and data protection officers of our university.
TODO: (Verweis auf Human Practice - Science Street Day + Bilder davon)

Talks and webinars

Permission of using visual and auditory material before publishing on the team's wiki page or via social media was obtained via a consent form. All clearly identifiable individuals, including experts and audience members, signed that we may use both audio and visual material in a responsible manner.

Surveys

The surveys released by our Human Practice and Outreach team had been approved by our institution before publication. It was pointed out that only adult persons are authorized to fill in the survey independently. In the case of minors, the express consent of their legal guardians was required. The declaration of consent preceded the actual survey and explicitly informed the participants that the entire information would be treated confidentially and published in an anonymized form. It was made clear that the data was only collected and published within the framework of the project.

Choice of Organism

As part of the safety management and risk assessment, we decided to work with safety strains of model organisms compatible with laboratories of biosafety level 1 (BSL-1) standards. We continued the open source character of the iGEM community, by making our project more accessible for other teams. In terms of safety and biosecurity, we constantly discuss our conversations with our instructors, principal investigation and different experts on their respective field about biosafety, various regulations and hazardous potential of our project. The risk of release and spreading of genetically modified organisms was reduced by autoclaving waste and vessels after usage. The release of modified organism or bacteria strains were prevented to inhibit the spreading and genetic exchange of genetically engineered organisms. The work and release of GMOs is strictly regulated by different laws and regulations in Germany.
After discussion of an appropriate organism for genetic design, many considerations were taken into account. The standard model organism E. coli with BSL-1 was chosen for its outstanding properties:
rapid reproduction allows fast and cost effective amplification allows easy genetic engineering via standardized cloning strategies such as Gibson Assembly or Golden Gate Assembly established as safe for us and the environment
All E. colis derived from fitness-deficit strain K12 derivatives (e.g. commercially available and validated DH5alpha, DH10alpha), which prevents unwanted escape. All parts utilized or designed during this project are on the iGEM whitelist or the necessary applications submitted.
By using eye protection, face masks or protective visors, we prevented the entrance of Genetically Modified Organisms in our body. Due to possible changes of the darmflora, all work with bacterial cultures was performed in a laminar airflow hood using protective equipment. To minimize contamination, work was performed under semi-sterile conditions and with an active flame that reduces the amount of bacteria in the air.
In case of gene shift due to exposure in the environment, the biggest problem will be antibiotic resistance. The impact of this risk is reduced by the presence of all resistance genes in the natural environment. By implementing the safe strategies of last year's iGEM team, such as antibiotic-free selection system or self-destruction mechanism upon release, reduces risk potentials in general.
Because of the low risk potential of our project combined with the lower risk potential of the organisms used within this study, we downregulated our safety issues to a minimum.

Laboratory Safety

Both our diagnostic and therapeutic parts require strict safety implementation. Safety regulations must be carefully observed. Our goal was to increase safety regulations on different levels in the laboratory and future application of ASTERISK. Our main experiments were performed in the laboratory of Prof. Dr. Jörn Kalinowskis at the Center for Biotechnology, Bielefeld University. Some of our experiments, which require other prerequisites and need special equipment, were performed in the laboratory of Prof. Dr. Kristian Müllers at the Biotechnikum. All experiments were performed under BSL-1 standard operating procedures.

General Laboratory Safety

Our lab was maintained for the competition by the intern organization for laboratory safety and certified for commissioning. This ensures that our environment meets the requirements of our project and guarantees safe working conditions for all objects and persons, both inside and outside the building. The regulations include a signposted and barrier-free escape and rescue route, preventive safety precautions against fire or chemical injuries including a hygiene plan. In addition, there is a cleaning plan for the proper disposal of harmful chemicals and for contamination-free work. Of course, decontamination has been carried out with appropriate solutions. We achieved sterility by autoclave according to established protocols. We use an online data bank in the intranet of our institution to inform us of potential hazards like UV light or magnetic radiation. Printed versions of the safety data sheets and important instructions were placed in a safety folder in the laboratory. TODO: (Bilderkarussel mit Bilder vom Labor aus der Safety Form)

Lab Guidelines

We design this 10-points-plan to guarantee the safe and secure working within our team and of future iGEM teams. These guidelines serve as orientation and summarize the basic behavior in and outside the laboratory which is mandatory for good laboratory practice and research:
0. Every participant must inform himself independently about potential risks or hazardous substances before each experiment. Hazard Statements (H-phrase-) and Precautionary Statements (P-phrases) define the handling. The team assures them to take care of each other and not to endanger anyone else including themselves.
1. Before performing any activity in the laboratory, all participants need to inform themselves about the procedure of the exposure and the potential hazards and risks associated with it. In addition, all experiments are conducted under the strict supervision of our mentors, supervisors, investigators, lab assistants and graduate students after receiving apprentice instruction. Every student must follow the code of conduct in the lab. In case of unforeseen emergency, working alone in the lab is prohibited. Minimal two persons are allowed in the lab, so that each other can be taken into consideration and in case of an emergency each other can perform first aid.
2. Wearing the personal protective gear (PPG including lab coat, safety google, nitrile gloves) is a necessary precondition for performing any experiment. The PPG serves as self-protection against potential hazards, but also protects the samples from possible contamination. In addition, closed-toe shoes, sleeve length clothing, and hair tied back even when spectating are part of the PPG.
3. All workspaces (benchtops, biosafety cabinets, PCR cabinets, etc.) are disinfected or sterilized using appropriate methods. An aseptic technique was always used before and after every microbiological procedure. All workstations were sterilized with 70 % EtOH both prior to and after each experiment. Smoking, eating and drinking is prohibited in the laboratories. Separate rooms outside the S1 area were used to store personal items.
4. All participants were familiarized with the location and use of safety showers, fire extinguishers, eye wash stations and fire alarms. Additionally, the emergency exit plan in the event of a fire and blackout protocol were trained. All safety equipment is located in the immediate and tangible vicinity.
5. Using appropriate chlorine/chloroform solution, all plates or cultures of bacterial parts or mammalian cell cultures were sterilized before being autoclaved and incinerated. This process is repeated until the plates or cultures no longer contain living organisms to prevent accidental release of genetically modified organisms.
6. Substances of chemical concern, such as ethidium bromide or phenol, are handled with care, following the instructions of our laboratory managers. We use a segregated work area to handle hazardous substances, minimize spills by using appropriate protocols, wear extra-thick gloves, and change gloves regularly.
7. Waste is appropriately segregated into various coloured bins that are stored in the laboratory inside the safety fume hood.
8. Warning signs, safety instructions and operating instructions are available and posted throughout the laboratory. In addition, solutions, chemicals and reagents are labeled with hazard pictograms.
9. All steps performed are documented in the lab notebook. In order to clarify misunderstandings and to avoid them later, every deviation from the standard, no matter how small, is documented in detail.
Our laboratories are secured through three major security levels:
Level 1 - Biosecurity
personalized electronic access system for all labs allowing fine-grained restriction of access tracking of activities by the authorities of Bielefeld University Level 2 - Biosafety
mandatory lab safety trainings continuous surveillance of measures provides safety regulation strategy Level 3 - Awareness
detailed and transparent documentation of all experimental work including all levels of accidents Implementation depends on the atmosphere created by each team member - responsible planning and exploratory work form the basis for good laboratory practice and excellent scientific work.
TODO: 3 Icons für Biosecurity, Biosafety, Awareness einfügen

Sustainable Solutions for Environment

Safety Instructions

The safety briefing was mandatory for all participants before starting the laboratory work. According to German regulations the participation of every team member had to be confirmed with a personal signature. The safety briefing was carried out by the safety officer Dr. Thomas Patschkowski and must be refreshed annually (see §12 ArbSchG):
1. General lab safety
2. Regulations concerning hazardous and toxic substances
3. Regulations concerning biological substances
4. Regulation concerning genetic engineering
We obtain special instructions for safe operation with every device. The instruction was conducted by the Safety and Security Officer within the laboratory. This person pointed out respective hazards and associated precautionary measures. This guarantees the safe and correct usage of the device, but also enables the collection of reliable data.

General Lab Safety

Both our diagnostic and therapeutic parts require strict safety implementation. Safety regulations must be carefully observed. Our goal was to increase safety regulations on different levels in the laboratory and future application of ASTERISK. Our main experiments were performed in the laboratory of Prof. Dr. Jörn Kalinowski at the Center for Biotechnology, Bielefeld University. Some of our experiments, which require other prerequisites and need special equipment, were performed in the laboratory of Prof. Dr. Kristian Müllers at the Biotechnikum. All experiments were performed under BSL-1 standard operating procedures.

General Laboratory Safety

During the competition, our lab was maintained by the intern organization for laboratory safety and certified for commissioning. This ensured that our work environment met the requirements of our project and guaranteed safe working conditions for all objects and persons, both inside and outside the building. The regulations include a signposted and barrier-free escape and rescue route, preventive safety precautions against fire or chemical injuries including a hygiene plan. In addition, there is a cleaning plan for the proper disposal of harmful chemicals and for contamination-free work. Decontamination has been carried out with appropriate solutions. We achieved sterility by autoclave according to established protocols. We use an online data bank in the intranet of our institution to inform us of potential hazards like UV light or magnetic radiation. Printed versions of the safety data sheets and important instructions were placed in a safety folder in the laboratory.

Lab Guidelines

We design this 10-points-plan to guarantee the safe and secure working within our team and of future iGEM teams. These guidelines serve as orientation and summarize the basic behavior in and outside the laboratory which is mandatory for good laboratory practice and research:
0. Every participant must inform themselves about potential risks or hazardous substances before each experiment. Hazard Statements (H-phrase-) and Precautionary Statements (P-phrases) define the handling. The team vows to take care of each other and not to endanger anyone, including themselves.
1. Before performing any activity in the laboratory, all participants need to inform themselves about the procedure of the experiment and the potential hazards and risks associated with it. In addition, all experiments are conducted under the strict supervision of our mentors, supervisors, investigators, lab assistants and graduate students after receiving apprentice instruction. Every student must follow the code of conduct in the lab. In case of unforeseen emergency, working alone in the lab is prohibited. Minimal two persons are allowed in the lab, so that each other can be taken into consideration and in case of an emergency each other can perform first aid.
2. Wearing the personal protective gear (PPG including lab coat, safety google, nitrile gloves) is a necessary precondition for performing any experiment. The PPG serves as self-protection against potential hazards, but also protects the samples from possible contamination. In addition, closed-toe shoes, sleeve length clothing, and hair tied back even when spectating are part of the PPG.
3. All workspaces (benchtops, biosafety cabinets, PCR cabinets, etc.) are disinfected or sterilized using appropriate methods. An aseptic technique was always used before and after every microbiological procedure. All workstations were sterilized with 70 % EtOH both prior to and after each experiment. Smoking, eating and drinking is prohibited in the laboratories. Separate rooms outside the S1 area were used to store personal items.
4. All participants were familiarized with the location and use of safety showers, fire extinguishers, eye wash stations and fire alarms. Additionally, the emergency exit plan in the event of a fire and blackout protocol were trained. All safety equipment is located in the immediate and tangible vicinity.
5. Using appropriate chlorine/chloroform solution, all plates or cultures of bacterial parts or mammalian cell cultures were sterilized before being autoclaved and incinerated. This process is repeated until the plates or cultures no longer contain living organisms to prevent accidental release of genetically modified organisms.
6. Substances of chemical concern, such as ethidium bromide or phenol, are handled with care, following the instructions of our laboratory managers. We use a segregated work area to handle hazardous substances, minimize spills by using appropriate protocols, wear extra-thick gloves, and change gloves regularly.
7. Waste is appropriately separated, additionally marked with a color code
8. Warning signs, safety instructions and operating instructions are available and posted throughout the laboratory. In addition, solutions, chemicals and reagents are labeled with hazard pictograms.
9. All steps performed are documented in the lab notebook. In order to clarify misunderstandings and to avoid them later, every deviation from the standard, no matter how small, is documented in detail.
Our laboratories are secured through three major security levels:
Level 1 - Biosecurity

• personalized electronic access system for all labs
• allowing fine-grained restriction of access
• tracking of activities by the authorities of Bielefeld University

• mandatory lab safety trainings
• continuous surveillance of measures provides safety regulation strategy

• detailed and transparent documentation of all experimental work including all levels of accidents

Safety Instructions

The safety briefing was mandatory for all participants before starting the laboratory work. According to German regulations the participation of every team member had to be confirmed with a personal signature. The safety briefing was carried out by the safety officer Dr. Thomas Patschkowski and must be refreshed annually (see §12 ArbSchG):
General lab safety
Regulations concerning hazardous and toxic substances
Regulations concerning biological substances
Regulation concerning genetic engineering
We obtain special instructions for safe operation with every device. The instruction was conducted by the Safety and Security Officer within the laboratory. This guarantees the safe and correct usage of the device, but also enables the collection of reliable data.

Safety Measures

General lab safety covers mandatory measures to obtain a safe working environment and ensure safety operation strategies. Our lab safety includes the correct behavior in case of emergency and contains strategies to prevent unsafe work. Our Safety and Security Officer checked the safety regulations and performed safety and security evaluation annually.
Our team advisors were responsible for full compliance of biological safety in our labs. Therefore, all responsibilities are intimately involved in biosafety considerations of our project. We discussed all safety and security considerations with our primary PI, Prof. Dr. Jörn Kalinowski and our secondary PI, Prof. Dr. Kristian Müller as well as the iGEM Safety & Security Committee and representatives at University Bielefeld.
The safety instruction covers general laboratory safety including the prescribed controls for achieving a safe working environment and behavior in emergencies. In addition, regulations on hazardous and toxic substances, biological substances and genetic engineering were also covered. For working with equipment, we received special instruction on the safe operation of, for example, the autoclave or the ultracentrifuge. Learning how to handle laboratory equipment properly prevents injuries and enables us to collect reliable data. Responsible persons instructed us in proper handling, pointed out appropriate hazards or precautions, and are always available to answer questions and provide support. However, all regulations are accessible in the intranet and in paper format within the laboratory.
In order to work in a microbiology laboratory, every team member should be aware of biological risks accompanied with performing certain experiments. By obtaining the “Safe Microbial Techniques (SMT)” certificate, we learn how to behave properly in the lab and how to handle various organisms or chemicals. The course was supervised by our personal biological Safety and Security Officer Dr. Thomas Patschkowski. Our mentor gives us a detailed safety and security workshop including health and safety awareness, laboratory safety awareness and waste management systems. We ensure that all members attend through safety and security training including handling bacterial cultures or disposal of contaminated materials before allowed to enter the laboratory. Furthermore, we discussed several scenarios and got practical training to prevent risk and learn how to act in case of emergencies.
The safety briefing itself focused on potential hazards posed by carcinogenic, mutagenic, inflammable chemicals, acids or bases, corrosive chemicals, cytotoxic reagents, neurotoxic and irritating reagents. The workshop also dealt with disinfection and sterilization procedures. This was followed by important information on health risks caused by poor posture and constant standing. Our team provided the biosafety course for future iGEM teams, that help guide them during their laboratory practice.

Safety Management

The observance of various factors allows safe working in an environment protected for all. The laboratory is located in an area physically inaccessible by security doors and electronic locking. Only authorized persons are allowed to enter or leave the premises. Each team member has received a personal key after the security briefing and laboratory introduction. In addition, uncontrolled or unauthorized access or removal of biomaterial is ensured. These security precautions allow controlled work and protect against misuse. During the entire creation process we followed the general safety requirements and biology department safety guidelines. In Particular, the iGEM Safety Rules and Policies were also followed during research activity in our lab and were conducted via our Safety and Security Officer within the team.
During the entire work, the obligation to wear personal protective clothing and equipment (short PPE) was observed and monitored. Lab Coat, safety goggles and protective gloves are the standard protective equipment of each team member. Work with hot or cold materials was performed with protective gloves and, if necessary, under a safety fume hood.
The use of a high-speed ultracentrifuge was carried out by trained team members. All relevant protocols and operating instructions as well as safety data sheets and notes were recorded in the digital laboratory book as well as deposited in the safety folder in the laboratory. Contingency plans are also in place in case of property or equipment damage. Decontamination protocols followed at the end of laboratory work include proper waste separation, cleaning of equipment, and autoclaving of bottles/vessels were used.
In case of an emergency, a telephone with direct connection to authorities is available. In addition, the rescue plan in case of fire or evacuation was rehearsed. Emergency showers, eye showers and different types of detergents are distributed in the laboratory. Emergency switches regulate the gas and electricity supply, which can be switched off immediately in case of an emergency. In addition to the first aid kit, a defibrillator is also available. Work in the laboratory was only carried out under the supervision of the safety officer and in the presence of a first aider. All standard methods of our wet lab experiments were performed in the BSL-1 certified laboratory provided by the Center of Biotechnology (CeBiTec) of Bielefeld University.

Quality Management

“Organization and cleanliness delivers quality and safety” - this mantra was supported through adjustments and improvements over time of laboratory work. We implemented an effective system for high quality control and management based on literature and consultation with the Quality Protection Officer and Head of Molecular Pathology Dr. Almut Mentz. By using the high Quality Management Standards of diagnostic pathology, our scientific basic research receives a similarly high level of safety and efficiency. Our system includes the documentation of changing temperature profiles of thermocyclers or cooling units, the documentation of batch protocols or the localization plan for every storage. Our Quality Management System includes regular calibrations, function testing and maintenance checks to guarantee function for safe and reliable practice.

Laws And Regulations

The national legal regulations of Germany and the European Union define the project framework. The Instruction Manuals GenTSV for BSL1 and GefStoffV of the University Bielefeld represent laws including safety requirements, laboratory guidelines and responsibilities.

1. Gentechnikgesetz (GenTG) - Genetic Engineering Law
• to protect persons against the harmful effects of genetic engineering processes and products
• to prevent the occurrence of hazards
• ensures the possibility of producing and placing products on the market using genetically modified mechanisms
• creates the legal framework for research, development, use and promotion of genetic engineering
2. Gentechnik-Sicherheitsverordnung (GenTSV) - Genetic Engineering Safety Regulation
• defines requirements for the safety standard for the construction and commissioning of genetic engineering facilities
• defines biosafety level (BSL) of native organisms and genetically modified organisms according to §7 GenTSV
• ensures safety level adapted to hazard potential subdivided into technical measures
• against uncontrolled escape of genetically modified organisms:
• to reduce the risk when handling GMOs at the workplace organizational measures
• to ensure the proper operation of genetic engineering of a genetic engineering facility biological measures
• to reduce the genetic engineering hazard potential
• Biostoffverordnung
• Technische Regel für Biologische Arbeitsstoffe TRBA 468
3. Gefahrstoffverordnung GefStoffV - Ordinance on Hazardous Substances
• regulates the protective measures for employees during activities involving hazardous substances
4. Arbeitsschutzgesetz ArbSchG - Occupational health and safety law
• ensures the safety and health of employees at work through occupational health and safety measures
• provides preventive measures for the prevention of occupational accidents and work-related health hazards

Chemical Safety

The use of chemical substances is coupled to strict control and requires separate responsibility. To protect human health and the environment, used chemicals are disposed of in accordance with the guidelines of the Center for Biotechnology. Chemical safety encompasses several scientific and technical sub-areas, including extraction, synthesis, industrial production, transportation, use, disposal of naturally occurring and self-made substances or the entire spectrum of exposure situations. Chemicals are safely stored as obligated in separated locations. Solvents are stored in explosion protected lockers and toxic substances are locked in safety cabinets. We tried to use non-toxic and non-hazardous substances and avoid potential risks accompanied. In any case, the safety data sheets were followed strictly and respective safety precautions were taken.
Working with ethidium bromide (EtBr) requires special handling and strict safety measures. Handling of a carcinogenic substance is only permitted in designated work areas. The wearing of protective equipment in these areas is extended by the wearing of double gloves, a protective visor against UV radiation and a laboratory coat with long sleeves. Radiation exposure should be kept to a minimum in order to protect the biomaterial and, above all, to protect the operator. Direct eye contact with the radiation source is prohibited. In addition, no objects may be touched with EtBr-contaminated gloves. All materials used are disposed of separately in a special waste container. The regulated handling of EtBr is documented in a separate laboratory book and monitored by instructors. In addition, working instructions are available to inform about potential risks management.

Chemical Usage Guidelines

1. Location - All chemicals are located in a separate work area. If possible, the substances do not leave these premises. The type of substance and the hazard classification define the handling.
2. Responsibilities - Everyone follows the Safety Data Sheet (SDS) information independently. All team members have been trained in handling through spill-proof handing protocols.
3. Authorities - Corrosives or flammable liquids are stored in special safety cabinets and secured by key. Only authorized and trained members have access to these substances.
4. Usage - Often used chemicals that produce dangerous and breathable fumes are handled inside the separate fume hood.

Waste Segregation System And Management

We take proper waste disposal very seriously in order to protect our environment and to counteract the associated pollution. Working with GMOs requires an appropriate waste disposal system. Waste contaminated with biological agents and genetically modified materials must be disposed of in a special way. Our laboratory has a sophisticated and simple coloring and sorting system. This prevents contamination and saves resources during disposal. Both the environment and people benefit from this system. Consumables and substances are disposed of by trained personnel in designated disposal facilities. Particularly contaminated consumables or chemical substances require special attention. In addition, the waste is documented in detail per charge so that the contents can be traced back. Bielefeld University has an efficient waste management system for all small and large equipment as well as chemical-containing or hazardous waste. In order to protect the environment, all harmful or polluting chemicals are collected separately and disposed by our university's chemical disposal department. This regulation has been implemented nationwide, pioneering modern waste disposal systems in Nordrhein Westphalia, with Bielefeld University optimizing this in greater depth and in a scientific context.

Biomedical Waste Segregation

at our working space

white bag - used tips, reaction vessels with S1 related contamination
yellow container - used glass waste, broken material, microscopy slides
white container - metal waste
transparent container - needles of syringes, blades, scalpels (contaminated material)

in the fume hood

white bucket - consumables contaminated with hazardous substances for e.g. phenol-contaminated reaction vessels
yellow bucket - cytotoxic contaminated consumables for e.g. ethidium bromide contaminated waste (carcinogenic, mutagenic) such as agarose gel, acrylamide gel, pathological waste, solid culture media, body fluid/ cytotoxic contaminated paper, cotton, swabs, tissue or cloths

inside the laboratory

orange waste bag: gloves, reaction vessels, syringes without needles, culture plates without media, pipette tips plastic waste (double walled bags to prevent break through of sharp objects)
red waste bag: EtBr

General Waste Segregation

inside or outside the laboratory (for non-contaminated material only):
blue bucket - paper waster
black bucket - plastic waste for e.g. packaging material of serological pipettes or reagent kit package
brown bucket - aluminum or metal waste
white container - korsolex contaminated liquid

Substainable Solutions For Environment

The implementation of fermentative production in vivo and biotechnological manufacturing on a large scale improves mRNA synthesis. The reduction of waste, the use of reusable tip systems and glassware instead of disposable systems, and the establishment of cost- and resource-efficient processes reduce the safety risk by improving sustainability of the environment.

Covid-19 Considerations

In comparison to last year, we are less challenged with the COVID-19 pandemic. At the beginning of 2023 competition, safety and health regulations were loosened through government and institutions. However, we try to adapt safety concepts of previous iGEM teams to prevent potential infections. The general protection between our team members and outsiders was ensured by wearing a medical face mask. In some cases, online interviews were necessary. Although our government and the University of Bielefeld have put down all measures, we have nevertheless created and implemented a self-made concept. When symptoms appear, we practice social distancing, conduct regular covid tests and implement safety guidelines in the lab rooms. We have made it possible to attend all meetings online to further reduce the risk of infection with other communicable diseases. We follow the European hygiene guidelines as well as the guidelines of the Federal Republic of Germany, the state of North Rhine-Westphalia and the city of Bielefeld - this year with non-strict compliance. We have closely followed changes in the situation and guidelines and have been flexible to implement full encapsulation. At the start of the project, all team members were fully vaccinated to further reduce the possibility of infection.

Conclusion

We take special care to the biosafety and biosecurity aspects of our project. We evaluated the safety standard of our project on different levels to design regulatable and safe sensing and killing switches. In addition, we presented further alternatives to add safety layers to the circuit in the form of Riboswitches. By creating a novel and universal mRNA-based platform for regulating possible out-of-control scenarios. We make sure to work stringent on various safety modalities and testing them to enhance our engineering circle, because safety first.