PROS by the Stony Brook University 2022 iGEM Team

Safety and Security in our Project

Our project, PROS, will address the treatment of protein S deficiency and related disorders by proposing a protocol for the in-cell production of recombinant human protein S, for administration. The long term goal is to administer the protein intravenously once the in-cell produced protein matches the qualities of the human protein S exactly. Once that is achieved, the synthetic protein S would have the capacity to improve patient survival and quality of life. We aim to make protein S available in larger amounts by biosynthesis in microorganisms (Escherichia coli and SF9 cells), in order to provide reliable supplies of the protein worldwide at affordable costs.

From the very beginning of our project, we designed our experiments under the assumption that our solution would be clinically practical. Therefore, we needed to consider the risks that our therapy could pose to producers, developers, patients, and health care workers. Our project needs to fully guarantee both safety and effectiveness of use, so we thoroughly analyzed the material relations of risk and explored the hazards behind manufacturing human protein S using gene technology.

Risk Assessment Method

Microorganisms are associated with humans, animals, plants and the environment, and their influence is often beneficial. However, scientific considerations for safe use of these microorganisms is critical, because some of them are pathogenic, and others may have a negative impact on the environment (IBC 2020).

Risk assessment is ultimately a subjective process. We followed the risk assessment framework of the NIH Guidelines, which uses the risk group of the parent organism as a starting point for determining the necessary containment level (NIH 2019).

SF9 Expression

Vector

Host

For expression of protein S specifically in insect cells (SF9), our project involved two main working cell lines.

The first cell line is the Baculovirus Expression Vector System (BEVS), where we used commercially available DH10b E. coli cells for the homologous recombination of our constructs. The second working line was the insect cell line Spodoptera frugiperda (SF9), which we were initially planning to genetically modify through infection with the innocuous baculovirus. There was a lot of consideration and research done in order to ensure proper handling of the biological material and improve our overall project safety.

The first consideration for our project was analysis of the homologous recombination carried out in the baculovirus expression system. We specifically sought to find out if handling this type of technology represented a risk for humans, and a thorough investigation was carried out to ensure the safety of the overall use of the BEVS technology. The history of BEVS technology use, from eukaryotic vector systems to recombinant protein expression, has been extensively used and studied since 1983 (iGEM Tec-Monterrey 2015). One of the advantages of this system is the fact that baculoviruses are nonpathogenic to both mammals and plants (Life Technologies). In particular, the host range of the baculovirus is restricted to specific invertebrate species, and mainly arthropods (McWilliam 2006). Additionally, there are no helper cell lines or viruses required with this system due to the fact that the baculovirus genome has all the genetic information necessary and requires minimal containment conditions for caring. There has also been extensive testing for the safety of more than 30 baculoviruses over the past 40 years, resulting in a long and complete safety record that demonstrates there is absolutely no adverse effect on human health (McWilliam 2006). Additionally, we met daily with the members of Dr. Michael Airola’s laboratory, who have extensive experience working with SF9 cells. They advised us on handling the cell line and carrying out procedures.

Secondly, the risks of liberating biological material of the baculovirus outside the laboratory facilities were considered. We determined that accidental release of baculovirus expression vectors poses negligible hazard to the environment (Stacey and Possee 1996).

Thirdly, we aimed to assess safety issues regarding the transformed SF9 cells. Specifically, we wanted to characterize the different promoters, proteins, reporters, and signal peptides that were being added. These included the polyhedrin promoter, HisTags, Ampicillin and Gentamicin resistance gene promoters, mini Tn7 elements for the SF9 vector (pFastBac) and T7 promoter, HisTag, Ampicillin resistance gene promoter for the E Coli vector (pET His6 TEV LIC cloning vector (2B-T)), and others mentioned in our project description. None of these represent a threat to the environment or human health. Since these were to be recombined in the system prior to the insect cell infection, the only organisms prone to infection were SF9 cells themselves. Therefore, the appropriate management of the insect cell line was followed in order to ensure a safe laboratory environment.

SF9 Cell Types

For expression of protein S specifically in insect cells (SF9), our project involved two main working cell lines. The first one is the Baculovirus Expression Vector System (BEVS), where we used commercially available DH10b E. coli cells for the homologous recombination of our constructs. The second working line was the insect cell line Spodoptera frugiperda SF9, which was genetically modified through infection with the innocuous baculovirus. There was a lot of consideration and research done in order to ensure proper handling of the biological material and improve our overall project safety.

The first consideration for our project was analysis of the homologous recombination carried out in the baculovirus expression system. We specifically sought to find out if handling this type of technology represented a risk for humans, and a thorough investigation was carried out to ensure the safety of the overall use of the BEVS technology. The history of BEVS technology use, from eukaryotic vector systems to recombinant protein expression, has been extensively used and studied since 1983 (iGEM Tec-Monterrey 2015). One of the advantages of this system is the fact that baculoviruses are nonpathogenic to both mammals and plants (Life Technologies). In particular, the host range of the baculovirus is restricted to specific invertebrate species, and mainly arthropods (McWilliam 2006). Additionally, there are no helper cell lines or viruses required with this system due to the fact that the baculovirus genome has all the genetic information necessary and requires minimal containment conditions for caring. There has also been extensive testing for the safety of more than 30 baculoviruses over the past 40 years, resulting in a long and complete safety record that demonstrates there is absolutely no adverse effect on human health (McWilliam 2006). Additionally, we met daily with the members of Dr. Michael Airola’s laboratory, who have extensive experience working with SF9 cells. They advised us on handling the cell line and carrying out procedures.

Secondly, the risks of liberating biological material of the baculovirus outside the laboratory facilities were considered. We determined that accidental release of baculovirus expression vectors poses negligible hazard to the environment (Stacey and Possee 1996).

Thirdly, we aimed to assess safety issues regarding the transformed SF9 cells. Specifically, we wanted to characterize the different promoters, proteins, reporters, and signal peptides that were being added. These included the polyhedrin promoter, HisTags, Ampicillin and Gentamicin resistance gene promoters, mini Tn7 elements for the SF9 vector (pFastBac) and T7 promoter, HisTag, Ampicillin resistance gene promoter for the E. Coli vector (pET His6 TEV LIC cloning vector (2B-T)), and others mentioned in our project description. None of these represent a threat to the environment or human health. Since these were to be recombined in the system prior to the insect cell infection, the only organisms prone to infection were SF9 cells themselves. Therefore, the appropriate management of the insect cell line was followed in order to ensure a safe laboratory environment.

E.coli Expression

In the project, we also used Escherichia. Coli; BL21 for expression, and DH5 alpha for cloning, which fall under Risk Group 1, corresponding to the certification of our laboratory (Biosafety Level 2). There are minimal risks associated with these strains (iGEM Linköping University 2014).

There are various different risks that lie in both indirect and direct bacterial contamination when handling E.coli strains. Without proper safety equipment, this can lead to sickness. Furthermore, there are risks involved with transforming plasmids which code for phenotypes that the E.coli host does not naturally have. Enzymes used during restriction digests and proteases used when extracting DNA can also cause allergic reactions in individuals.

When using E.coli, There is risk for bacterial infection if the bacteria were to be accidentally released into water sources. There are also risks involved when using bacterial strains that are transformed with antibiotic resistance. Contamination with antibiotic resistant bacteria can be dangerous for individuals. Furthermore, there are risks involved with transforming plasmids which code for phenotypes which the E.coli host does not naturally have, and the risk of horizontal gene transfer from GMOs to wild-type bacterial strains. Bacteria undergo a natural process called horizontal gene transfer, which is when DNA is transferred from one bacterium to another. Therefore, it is possible that escaped, modified bacteria, such as the ones we are producing, could have unwanted and even dangerous effects in the natural environment. A few possible scenarios are the transfer of genes to native pathogenic bacteria that would give them a new evolutionary advantage, or released GMOs spreading in an ecosystem or even in human bodies, overpowering populations of natural bacteria.

However, we identified that there are no such hazards to the environment presented by our project. We did not transform anything into our bacterial strains to make them more competitive than bacteria in the native environment. Therefore, we consider it highly unlikely that our modified bacteria could compete with natural strains. Our product also doesn't have any environmental application, so if bacteria escapes the lab, then the risk of environmental contamination is very low. Additionally, our team only used degradable antibiotics, mitigating risks. Finally, the bacteria that we used are not a threat to security. In some cases, engineered bacteria that express harmful proteins could be used as bioweapons. However, our product doesn’t contain any such proteins or modifications.

Biosecurity Assessment

We conducted a biosecurity risk assessment aligned with the methodology of The International Federation of Biosafety Associations to address any potential threats of the assets we have in the laboratory. We address our assets, insider and outsider threats that may occur, and security. In addition to the results of the assessment, control measures were developed to limit any threats.

    1. Define the situation
      • Identify the assets
        1. E. coli cell strains BL21, Origami (expression) and DH5alpha for cloning.
        2. Centrifuge, thermocycler, incubator, and other standard laboratory equipment
        3. Laboratory protocols
        4. Outreach efforts
        5. Dry Lab modeling
      • Identify the threats
        1. Outsider adversaries - competitive iGEM teams/researchers or criminal looking to sell/steal equipment
        2. Insider adversaries - disgruntled laboratory members
      • Define the physical and laboratory security environment
        1. Room security: The lab is locked and has a pin that the PI and team members only have access to.
        2. Asset Security: Laboratory equipment is located in the locked room.
        3. Building Security: Anyone can access the building.
        4. Personnel Reliability: Lab members are entrusted to keep doors of the laboratory closed when unattended. Lab members are not allowed to work alone.
    2. Define the risks
      • Risk of an authorized person stealing valuable biological material for malicious use e.g. a lab member who deliberately wants to misuse collected data and biological equipment/materials
      • Risk of an unauthorized person stealing valuable biological material for personal gain e.g. a criminal/adversary researcher intending to steal and sell biological equipment/materials
      • Risk of an authorized person stealing institution intellectual property (in the form of information) or confidential information e.g. a disgruntled lab member wanting to sabotage the project by leaking confidential information
    3. Characterize the risks

      4. E. coli strains:

      • Likelihood of targeting the asset by the threat? LOW
      • Likelihood of the adversary's desire to attempt to acquire the asset? LOW
      • Likelihood of successful acquisition of these assets? MODERATE
      • Consequence of the misuse causing harm to health and the environment? MODERATE

      Overall likelihood of this occurring? LOW

      Laboratory Equipment:

      • Likelihood of targeting the asset by the threat? LOW
      • Likelihood of the adversary's desire to attempt to acquire the asset? LOW
      • Likelihood of successful acquisition of these assets? MODERATE
      • Consequence of the misuse causing harm to health and the environment? MODERATE

      Overall likelihood of this occurring? LOW

    4. Determine if the risks are acceptable
      • Is the risk acceptable to the institution? No
      • Is the risk acceptable to those working in the institution? No
      • Is the risk acceptable to the community? No
    5. Implement risk mitigation measures
    Risk Risk Level Control Measures + Implementation
    Risk of an authorized person stealing valuable biological material for malicious use MODERATE Conducting background checks of lab members and all related personnel with access in order to ensure that there is no malicious intent. Implementing a “buddy system,” making sure no one is in the laboratory alone. Locking doors when the laboratory is not being used.
    Risk of an unauthorized person stealing valuable biological material for personal gain LOW Locking the door when leaving the laboratory unattended.
    Risk of an authorized person stealing institution intellectual property (in the form of information) or confidential information MODERATE Conducting background checks of lab members and all related personnel with access in order to ensure that there is no malicious intent. Implementing a “buddy system,” making sure no one is in the laboratory alone. Locking doors when the laboratory is not being used.

    References

    Life Technologies. (2015). Cell Culture Basics Handbook. Thermo Fisher Scientific Inc.

    Life Technologies. (n.d.) Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques. Retrieved from Thermofisher Manuals

    Linköping University iGEM. (2014). Safety form. Team:Linkoping Sweden/human practice/safety. Retrieved July 19, 2022, from https://2014.igem.org/Team:Linkoping_Sweden/Human_Practice/Safety

    McWilliam, A. (2006). Environmental Impact of Baculoviruses.

    NIH. (2019, April). NIH GUIDELINES FOR RESEARCH INVOLVING RECOMBINANT OR SYNTHETIC NUCLEIC ACID MOLECULES (NIH GUIDELINES). NIH.

    Sandia National Laboratories & International Federation of Biosafety Associations. Laboratory Biosafety and Biosecurity Risk Assessment Technical Guidance Document. Retrieved from https://www.aam.org.ar/descarga-archivos/Laboratory-Biosafety-Biosecurity-Guidance.pdf

    Stacey, G., & Possee, R. (1996). Safety aspects of insect cell culture. Cytotechnology, 20(1-3), 299–304. https://doi.org/10.1007/BF00350409

    Tec-Monterrey. (2015). Safety. Team:Tec-Monterrey/Safety. Retrieved July 19, 2022, from https://2015.igem.org/Team:Tec-Monterrey/Safety

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