Safety
1. Prologue
Why we should be aware of Biosafety?
According to the World Health Organization (WHO), "biosafety is a strategic and integrated approach to analyzing and managing relevant risks to human, animal and plant life and health and associated risks for the environment." It addresses the safe handling and containment of infectious microorganisms and hazardous biological materials.
The lack of consciousness of biosafety and biosecurity could yield devastating consequences, such as the leakage of experiment organisms that could break ecological equilibrium or the exposure to toxins that could threaten our lives; if we have a high biosafety consciousness and lower the possibility of biological hazard as much as we can, we will be able to minimize the possibility for those horrible events to occur and protect both the health of the environment and people around the globe, and of course, protect ourselves.
In iGEM, safety covers the procedures, practices, or other measures used to manage risks from accidental exposure or release; security covers the procedures, practices, or other measures used to manage risks from deliberate exposure or release. One might contend that we, high schoolers without access to hazardous materials, don't have to be extremely serious about biosafety. However, working safely and securely is a core element of responsible research and innovation. RDFZ_CHINA team addresses safety and security issues throughout the competition lifecycle. Team members consider potential risks to themselves, their communities, and the environment, and try to manage any risks through the process of project design, laboratory work, and human practice.
2. Safety in project design-microorganism information
If E. coli is released into the environment, due to the weakening of its varieties, its survival ability has been greatly reduced, which will not cause ecological problems. Occasionally, however, probiotics may have adverse clinical effects, including septicemia. If both the microbiota and adaptive immunity are defective, translocation across the intestinal epithelium and dissemination of the probiotic E. coli strain may occur. Considering this risk, we plan to use Escherichia coli Nissle 1917. Escherichia coli strain Nissle 1917 (EcN) is a remarkable probiotic bacterium, first described by Alfred Nissle in 1916/1917. This strain of E. Coli is not a pathogen and EcN has been well-researched over decades. E. coli Nissle 1917 has been commercially available with no toxicity. Therefore, our bacteria are not harmful to the environment or the community.
3. Safety in project design-kill-switch design
3.1 Our design
To prevent the leakage of probiotics, we did research and came out with an effective kill-switch design for our project. Among many types of kill-switch systems, we adopted one for reference, which is designed by Tore Bleckweh for the iGEM13_Bielefeld-Germany team. We adopted their gene and design but did some confirmatory experiments to improve the design, which they didn't. This biosafety system is composed of L-Rhamnose, Rhamnose promoter PRha, Regulator AraC, Alanine racemase Alr, Terminator, Arabinose promoter PBAD, and RNase Ba.
Implanting the above genes into the Nissle 1917 strain results in a powerful device, allowing us to control bacterial cell division. This control of bacterial growth is possible either active or passive. Active by inducing the PBAD promoter with L-arabinose and passive by the induction of L-rhamnose. Passive control makes it possible to control bacterial cell division in a defined closed environment, such as the intestine by continuously adding L-rhamnose to the medium. As shown in the first graph, this leads to an expression of the essential alanine racemase (alr) and the AraC regulator, so that the expression of the RNase Ba is repressed.
If bacteria exit the defined environment of the intestine or L-rhamnose is not added to the medium anymore (as shown in the second graph), both the expression of the alanine racemase (Alr) and the AraC regulator decreases so that the expression of the toxic RNase Ba (Barnase) begins. The cleavage of the intracellular RNA by the Barnase and the lack of synthesized D-alanine, caused by the repressed alanine racemase inhibit the cell division and make sure that the bacteria can only grow in the defined environment or the device of choice respectively.
There are several advantages to this design.
Firstly, we choose to use the RNase Ba as a toxin because they are intracellular cutting RNA enzymes and there is little chance for them to influence our enterocytes. Also, when the system functions, the molecules involved (such as the L-Rhamnose) are not necessities for the human body or enterocytes, and the presence or absence of these molecules will have little impact on humans. They only serve to control the bacteria.
Secondly, we deal with some potential safety issues that are unable to control. For example, when plasmid loss occurs, the bacteria will die. When our patients decide not to continue the treatment anymore, which means there isn’t L-Rhamnose in the defined environment, and when the downstream gene group mutates, the bacteria would gradually die because they lack D-Alanine; if any of the upstream gene group mutates, the bacteria would also die either because of the lack of D-Alanine, or the release of RNase.
In conclusion, following our designed procedure, that is, the patients still take L-Rhamnose, and when bacteria exit the intestine into the environment, they will die because of the release of RNase; when patients want to drop the treatment and stop taking L-Rhamnose, the bacteria will also die in the intestine because of the release of RNase (most kill-switch system only have one of the above two mechanisms, but we have both). What’s more, when mutation and plasmid loss occurs, the bacteria still will be unable to survive.
3.2 Comparison to other group's designs
3.2.1 Team: Fudan
Their (Team: Fudan/Design - 2020.igem.org, n.d.) Kill Switch consists of a toxin/antitoxin system MazF/MazE and an RNA thermometer NoChill-06 to regulate it to deprive of the survivability of engineered Nissle in the environment when excreted from the human intestine. The antitoxin MazE is liable and expressed at a relatively high level. The MazF toxin is constitutively co-expressed with the antitoxin under the control of an RNA thermometer NoChill-06. When the temperature is equal to or higher than 37°C, the constitution promotor will trigger the system, NoChill-06 unfolds and exposes its ribosome binding site (RBS) to express. MazE and MazF neutralize each other by protein-protein interaction and form a stable complexity in a one-to-two ratio; when the bacteria encounter a cold shock (30℃), MazE is degraded rapidly by an ATP-dependent serine protease ClpAP and releases MazF. The toxin MazF acts as a site-specific endoribonuclease to almost all cellular mRNAs, resulting in cell growth arrest and finally cell death.
Advantages of their design:
Despite the restricted living condition of the engineered bacteria, their overall design is effective in controlling the survival of engineered bacteria that exit the designed environment following the designed procedure.
Defects of their design:
They pointed out that “once the Kill Switch was implanted, the engineered bacteria could no longer be frozen as a glycerol stock, and it must be maintained in the culture media above 30 degrees.” The engineered bacteria can only live in a small defined range of temperatures between 30 degrees Celsius and 37 degrees Celsius.
3.2.2 Team: Rice
For their design (Part: BBa K3247005 - parts.igem.org, n.d.), as shown in the graph, the promotor is constantly open. The ribosome binding site will open when the temperature equals to or is above 37°C (when it is cold, the site will not open), and the antitoxin RelE will be released, inhibiting toxin RelB, keeping the bacteria alive.
Defects of their design:
Firstly, the toxin is harmful to human cells. B.
Secondly, RelE/RelB module tends to have unexpected enrichment in bacteria that is not observed for other toxin/antitoxin modules such as MazE/MazF and ParD/ParE, which might generate resilience among intestinal probiotics.
Thirdly, there is a similar substance of RelE in the intestines, which might influence this designed system.
3.2.3 Team: St Andrews
The aim of their 2011 iGEM project (Team: St Andrews/Switch - 2011.igem.org, n.d.) is to create an E. coli kill switch using intracellular antimicrobial peptide (AMP) production (k628000). The pBAD promoter is found in nature governing the E. coli arabinose operon, responsible for the breakdown of the sugar arabinose into D-xylulose-5-phosphate. This promoter is induced by the binding of L-arabinose to the AraC promoter region. The team chose to use the pBAD strong promoter(k206000), which is a mutagenized form of pBAD that induces transcription at a lower arabinose concentration and has a higher maximum expression. pBAD strong is only induced in the presence of arabinose, which is not naturally produced by the cell, making it a very stable promoter to work with and greatly reducing the chance of accidental gene activation. In the first graph, J61101 is the ribosome binding site and B0015 is a double terminator according to the group's wiki.
Defects of their design:
Though L-arabinose is not naturally produced by cells they are present in many fruits and coarse grain shells, which many people take in regularly to maintain a healthy body. If there is L-arabinose ingested, the protegrin-1, an antimicrobial peptide, might be triggered by the system and damage other intestine bacteria.
3.2.4 Team: Pasteur Paris
The team (Team: Pasteur Paris - 2018.igem.org, n.d.) uses a kill switch based on temperature: it enables bacteria to survive at human body temperature (37°C) but die at lower temperatures (under 22°C). They use toxin/antitoxin couple CcdB/CcdA. The toxin targets and inhibits the GyrA subunit of DNA gyrase, an essential bacterial enzyme that catalyzes the super-coiling of double-stranded closed circular DNA. At 37°C, the quantity of antitoxin CcdA is high enough to cope with the leaky low level of toxin produced. However, if the bacteria happen to be in an environment at a lower temperature, the toxin promoter is not repressed anymore, the quantity of toxin becomes too important, and the bacteria is not able to grow.
Advantages of their design:
In their designed environment, we can infer based on their design that when the temperature is between 37°C and 22°C, instead of dying immediately, the bacteria will gradually die or will not be in a very active state, making them somewhat resistant to accidents such as when the person drinks water to take drugs, the temperature will be lower than 37°C for a short time.
Defects of their design:
Their bacteria are restricted to be kept above 22°C, better above 37°C after production.
3.2.5 Team: NEU China A
They (Team: NEU China A - 2018.igem.org, n.d.) designed a cold shock kill switch based on the toxin-antitoxin system-mazEF- a natural toxin-antitoxin system found in E. coli. MazF is a stable toxin protein, and mazE is an unstable antitoxin protein. The team used a mRNA that is mediated by the cold-acting promoter CspA and can only be efficiently translated at a low temperature of, for example, 16 ° C to activate the expression of mazF at low temperatures. As seen in the graph.
3.2.6 Team: NEU CHINA
In 2019, team NEU CHINA used the same kill-switch design as team NEU China A used in 2018 but made some improvements by palliating the symptom of severe MazF leakage problem. They introduced MazE to reduce the toxicity of MazF and experimented to test it.
Advantages of their design:
Their system is well-organized, and the bacteria will be unlikely to survive once it leaves the designed environment under the ideal circumstances.
However, if the patient chooses to stop his/her treatment, there might be some engineered bacteria left behind.
4. Safety in the lab
4.1 Training before entering the lab
To equip team members who participate in wet-lab experiments with the necessary knowledge and skills that ensure biosafety and biosecurity, our supervisor offered us a lab instruction booklet and several online courses to inform basic lab operation principles, including an introduction to the use of machines in certain experiments, biosafety & biosecurity during operation, and some knowledge of synthetic biology, microbiology, and molecular biology.
All team members were required to take these courses before conducting wet-lab experiments. Also, members who did not read through the booklet and signed their names were forbidden to enter the wet lab. We learned about lab access and rules (including appropriate clothing, eating, and drinking), responsible individuals (such as lab or departmental specialists or institutional biosafety officers), differences between biosafety levels, biosafety equipment (such as biosafety cabinets), good microbial technique (such as lab practices), disinfection and sterilization, emergency procedures, transport rules, physical biosecurity, personnel biosecurity, dual-use and experiments of concern, data biosecurity, and chemicals, fire, and electrical safety. The first graph was the first page of our booklet.
4.2 Lab setting
The labs in which all our wet-lab experiments were conducted were built under the supervision of experts and were approved by the Chinese government. Risk management tools included accident reporting (measures to record any accidents), personal protective equipment (lab coats, gloves, eye protection, etc.), inventory control system (measures to track who has what materials and where they are), access controls (measures to control who can access your workspaces, or where materials are kept), waste management system (measures to make sure waste is not hazardous before it leaves your institution) covered our work. The officers provided all assistance in experiments requested by our members, such as the usage of PCR machines, the cleaning of used instruments, and the shutdown of electricity or flames before we left. Our supervisor researched a lot about E. coli Nissle 1917, he was well-experienced and familiar with experimental procedures and practices.
4.3 Safety during operation
Procedures: When conducting wet-lab experiments, all team members are supervised by experienced lab experts, and the protocols used are seriously examined to ensure biosafety and biosecurity.
Biohazard disposal: All team members are required to dispose of waste appropriately. We strictly follow lab protocols and make every effort to prevent leaking.
Hygiene: no food or drinks were allowed in the lab; we washed our hands when finished and used 75% alcohol to disinfect surfaces, etc.
PPE: we wore lab coats, latex gloves, masks, trousers, sneakers, etc., while operating reactions and machines.
Biohazard handling: During the operation, to prevent cross-contamination of laboratory surfaces and biological safety cabinets, the proper use of various disinfecting sprays and aseptic techniques were employed.
Storage: we labeled the reagents properly, including the name of the chemical, information about its composition, sensitivity to environmental conditions, physical properties, chemical compatibility, disposal method, expiration, etc., and after understanding the purpose of different freezers, we store experimental materials accordingly.
Emergency: we learned how to handle it in case of spill or contamination, etc.
5. Safety in potentially harmful reagents and procedures
5.1 UV lights
UV lights are essential for the synthetize of Vitamin D, which in turn helps the human body to synthesize Ca+ ions. The lack of Ca+, or UV light, could lead to diseases lick Celiac rickets; but UV lights could also harm the human body. Too much UV light could lead to skin cancer.
In our experiment, Ultraviolet Light (UV) was used to sterilize biological safety cabinets and the equipment contained in them, as well as for DNA imaging in agarose gel needs. We wore Personal Protection Equipment (PPE) during these procedures to avoid exceeding UV radiation and protect our skin.
5.2 Antibiotics
We used a few antibiotics, such as ampicillin, for the selection procedure. According to the material safety data sheet (MSDS) for ampicillin, chloramphenicol, and kanamycin, these compounds are known to have hazardous effects at high concentrations and both acute and chronic exposure to humans via inhalation, ingestion, or contact with eyes. Therefore, our team wore PPE when handling the compound, studied the safety protocols, was equipped with the necessary information to handle spills, and acute exposures, and disposed of the product following the disposal protocols as well as the state safety regulation.
6. Safety in human practice
6.1 Biosafety knowledge and awareness dissemination
6.1.1 Bio-camp
In order to spread and let more people spot the fascinating knowledge of Synthetic Biology (including biosafety), we established a synthetic biology camp in our campus's laboratory, organized and told by RDFZ_CHINA team members. The main source of participants is our alumni, including high school students of different grades, middle school students, and students from the early development program. We took our participants to the school laboratory and lecture hall to inform them of Synthetic biology and safety regulations in a lab; familiarize experimental equipment, practice basic operations and procedures, and get hands-on experience completing Polymerase Chain Reaction (PCR), etc.
6.1.2 Pre-camp quiz
In order to better arrange our content and get to know participants better, we designed a pre-camp pop quiz. The quiz included basic biology knowledge such as the Central Dogma, DNA replication, DNA transcription, DNA translation, etc.; biosafety awareness in laboratory knowledge about the operations of machines and common sense of experiments.
Due to the photographer's phone's poor pixel, the picture of the quiz is not very clear. We listed the context of the picture, or quiz, below (the expressions will be slightly different from the real quiz because we translated it from Chinese):
Pre-camp Quiz
Name:
Class:
Section 1: basic biology knowledge.
1. How genetic information is delivered based on the Central Dogma?
2. State the three processes of gene expression.
3. How many kinds of bases does DNA have? Please list them all.
4. DNA has which three components?
5. The direction of DNA replication is from which end to which end?
6. What is the difference between DNA and RNA?
7. What is semiconservative replication of DNA?
8. What is reverse transcription?
9. What is the Okazaki fragment?
10. What is a replication fork?
Section 2: bio-lab experience.
1. 1ml equals how many μl?
2. The abbreviations of DNA base pairs are?
3. From 0ml to 1ml, and among pipettes most commonly seen in a lab, how many kinds of the pipette (pipettes that have different ranges) are there?
4. How many steps are there for a complete PCR experiment?
5. Normally, the voltage of Nucleic Acid Electrophoresis is?
6. At the start of the experiment, there is 1 DNA molecule, after 30 PCR cycles, the number of DNA molecules is how many times compared to the initial number?
7. gel trays have how many sizes?
8. What does the "20×" on the label of a reagent mean?
9. What is the difference between -80°C and -20°C fridges?
10. What should we be aware of during centrifuge operations? (please state one)
6.1.3 Lectures
We arranged two lectures at RDFZ Sanya School and for The High School Affiliated to Renmin University of China's Early Development Program, contributing to the community’s awareness of synthetic biology knowledge and promoting more people to learn synthetic biology. The audiences of our lectures are from different grades and different ages, from primary school to high school.
Taking participants' varied backgrounds and familiarity of synthetic biology into consideration, we presented the lectures from shallow to deep, introduced basic biological concepts and theories at first (such as DNA and RNA structures and how those structures contributed to their functions or properties; the process of DNA replication, transcription, and translation, along with identification of structures and enzymes involved in the process; what is central dogma and how is it important for biology development, etc.), then the amazing field of synthetic biology, and dived deeper into the subject's core of biological parts, genetic circuit, metabolic engineering, and genetic engineering. In addition, we taught our audience procedures to use common machines such as the PCR, and at the end of each lecture, gave opportunities for audiences to ask questions.
More details for the above activities and their result analysis can be viewed on our “Education & Communication” page.
6.2 Research & thoughts on Biosecurity Law
Before we designed our project, we look carefully through the Biosecurity Law of the People's Republic of China and raised several thoughts for its improvement. Compared to other developed countries in the world, law for biosafety and biosecurity developed late in China, especially of synthetic biology. Nowadays, synthetic biology is advancing rapidly, new technologies and innovative products emerge all the times, some flaw might exist in the law for test subject, technology approach, or product monitoring. From this point, we gave advice to the government on its official website to improve biosafety and biosecurity law. Although we haven't recieved any reply yet.
7. Conclusion
Our team made efforts and proposed practical advices in three fields: the management of engineered bacteria, the improvement of biosafety system, and education of synthetic biology and biosafety knowledge. We hope these efforts could lay a positive effect upon biosafety and biosecurity law, and make more synthetic biology teams able to conduct their experiments in a safer environment by safer means.
8. Reference
- Team:Fudan/Design - 2020.igem.org. (2020). Retrieved September 29, 2022, from https://2020.igem.org/Team:Fudan/Design
- Part:BBa K3247005 - parts.igem.org. (n.d.). Retrieved September 29, 2022, from https://parts.igem.org/Part:BBa_K3247005
- Team: St Andrews/switch - 2011.igem.org. (2011). Retrieved September 29, 2022, from https://2011.igem.org/Team:St_Andrews/switch
- Team: Pasteur Paris - 2018.igem.org. (2018). Retrieved September 29, 2022, from https://2018.igem.org/Team:Pasteur_Paris
- Team: NEU China A - 2018.igem.org. (2018). Retrieved September 29, 2022, from https://2018.igem.org/Team:NEU_China_A
- Team: NEU CHINA/homepage.html - 2019.igem.org. (2019). Retrieved September 29, 2022, from https://2019.igem.org/Team:NEU_CHINA/homepage.html.