Project Safety

Safety Declaration of BerryVax

Introduction

The iGEM_ITESO team considers biosafety, bioethics, and biosecurity as essential aspects in synthetic biology innovation. Although the project remained theoretical because we were not able to perform any experiments, we still considered biosafety, biosecurity, and national and institutional regulations in the design of our protocols, experiments, and genetic circuit. It is important to note that we executed several simulations of our experiments in different software applications, we also attended a biosafety and biosecurity workshop from the Engineering Biology Research Consortium (EBRC) called “Malice Analysis”, which helped us to identify the risks associated to laboratory work and implementation of the solution while ensuring that all the procedures comply with national laws and the guidelines of our university. It is important to note that ITESO’s Biotechnology Laboratories Policies were based on the Laboratory Biosafety Manual and Good Laboratory Practice manuals by the World Health Organization. Also, the following Mexican laws and norms were considered: General Law for the Prevention and Integral Management of Residues, NOM-087-ECOL-SSA1-2002, NOM-052-SEMARNAT-2005, Biosafety of Genetic Modified Organisms Law, and NOM-164-SEMARNAT/SAGARPA-2013. All of the previous material, activities, and research helped us to write the following declaration.

Laboratory Safety

Location

The Biotechnology Laboratories of ITESO are located on the first floor of CEGINT’s third building in ITESO’s Technology Park. There are six areas that comprise the Biotechnology Laboratories: Bioengineering, Molecular Biology, Cell Culture, Microbiology, Analysis, and Biosafety Level 2.

Equipment and reactants

The available equipment includes microscopes, photo-documentation system, incubators (with rotation and different temperature modes), microplate reader, bioreactors with different capacities, autoclaves, biosafety cabinets, visible light spectrophotometer, -20°C freezers, 4°C refrigerators, centrifuges with different temperature modes, microcentrifuge, thermocycler, extraction fume hood, carbon dioxide incubator, among others. All chemical solutions and reactants are properly stored in the adequate containers and sections in the laboratory.

Supervision

Dr. Alejandro Arana Sánchez (the biotechnology laboratory coordinator) oversees the biosecurity policies and can help if a risk is discovered. He also revises the protocols of any experimental procedure before issuing an approval statement. In our university, we have a biosecurity committee that, along with the coordinator of the biotechnology laboratories and other experts such as teachers and researchers, stipulated the biosecurity and biosafety guidelines (ITESO’s Biotechnology Laboratories Policies). The ITESO HSE board has a branch of biological hazards that can help assess and manage risks that could potentially come up while working in the laboratory. It is important to note that those guidelines are continuously updated. The laboratories coordinator oversees the risks regarding the research protocols, and the guidelines are given directly from him among the PIs to conduct the research in a safe and ethical manner.

Training and Laboratory Work

Although no experiments were conducted in the laboratory, it is essential to note that every member of team and collaborator is aware of ITESO’s Biotechnology Laboratories Policies. Before using the facilities, it is necessary to sign the documents that states that the person that will use the laboratories is aware of the security and hygiene measures, dress code, behavior, emergency procedures, and proper handling of reactants, residues, and microorganisms to ensure good laboratory practices (guideline included in ITESO’s Biotechnology Laboratories Policies). Also, approval of the protocols from Dr. Alejandro Arana Sánchez is essential to proceed with future experimentation. Additionally, the project instructor, M.S. Sarah Ratkovich González revised the designed protocols before submission and imparted safety training as well as the proper use of the previously mentioned equipment.

Residue management

All of the residues are disposed according to the Material Safety Data Sheets (MSDS) of the reactants. Additionally, the only reactants that be disposed of in the sink only if they have a neutral pH and if regulations or the MSDS permit it. Other reactants must be disposed in designated containers to avoid release into the sewage. This is essential to void damage to the facilities and the environment. Characterization of residues is done based on the Official Mexican Norm NOM-052-SEMARNAT-2005, which determines the characteristics, procedures, classification, and listing of hazardous residues. Also, the Official Mexican Norm NOM-087-ECOL-SSA1-2002 defines biological and infectious residues, and stipulates their management and disposal in safely manner, which is considered in every laboratory procedure. Finally, the General Law for the Prevention and Integral Management of Residues states the protocols and regulations that are necessary to prevent environmental harm, the information mechanisms to properly dispose of hazardous reactants, and the organizations or enterprises in charge of waste management, and potential actions for remediation.

Project Development, Analysis, and Implementation

Microorganism review

To comply with the biosafety levels of the laboratories of our university (BSL-1 and BSL-2), to reduce the environmental impact, risk of infection, and contamination of the workplace, we decided to choose microorganisms that belong to the BSL-1 (Biosafety Level 1) classification, which were Bacillus amyloliquefaciens ssp_plantarum, Bacillus subtilis DSM 5660, Escherichia coli HB101 (Reimer et al., 2022; Ta et al., 2019).

Parts Review

All the selected parts can be found in the part registry, and all of them are BSL-1. Therefore, it was not necessary to fill out a check-in form to register a new part or to use one from a higher biosafety level.

Design and Implementation of a Biocontainment System

Having control over a genetically modified organism at the time of releasing it into the environment is essential, therefore, it was sought to implement a biocontainment mechanism. A mechanism previously used by another iGEM 2012 team LMU Munich, was found, which uses a σ factor and a target promoter. We decided to implement a kill switch system to regulate the lifespan of our bacteria. After a certain period of time, the kill switch will start expressing an intracellular toxin that will cause apoptosis through the degrading of mRNA. The biocontainment system is described with more details in the design section.

Selection of Preventive and Corrective Substances

Surfactin was selected as a preventive substance and chitinase was selected as a corrective substance. In the case of surfactin, it was selected because several strains of the Bacillus genus produce it and they have the capability of antagonizing antagonize phytopathogenic agents and induce systemic resistance strategies in associated plants (Valenzuela et al., 2020). In the case of chitinase, it was selected because chitin is part of the cell wall of yeasts and fungi (Stoykov, 2015). Therefore, the enzyme chitinase will be able to degrade this material in the cell wall of the mentioned microorganisms, which will inflict damage and eventual cell death. Chitinase has the ability to inhibit Fusarium oxysporum and Botryodiplodia theobromae with efficiency of up to 83% (Swain, 2008). More details of these substances and the implemented design of the genetic circuit to secrete them can be found in the design section.

Application of BerryVax Outside of Laboratory Containment

As fields and crops are situated in distinct places (which is considered outside of laboratory containment), BerryVax would be implemented in berry crops (conventional, hydroponic, and semihydroponic) of fields/farms that are located in the western region of Mexico, as any other biocontrol product, via irrigation. Once applied, the bacteria will be complying with its purpose (producing surfactin as a prevention mechanism and producing chitinase as an antifungal). Eventually, the bacteria will die through the triggering of a biocontainment system (a kill switch) that will degrade the mRNA of Bacillus subtilis through the expression of an intracellular toxin that will cause apoptosis on the cells (previously described in the preceding point).

National Laws and Regulations

In Mexico, the Biosafety and Genetic Modified Organisms Intersectoral Commission is responsible for coordinating the policies about biosafety and the production, imports, exports, release, consumption, and, in general, the use of genetically modified organisms (GMOs). There is also another regulatory entity, the Federal Commission for the Protection against Sanitary Risk for products that are meant to be consumed by humans. This Commission assesses the risk against public health, and it has the faculty of giving authorization in subjects regarding GMOs.

The institutions mentioned above operate under a legal framework that consists of the Biosafety of Genetic Modified Organisms Law, which stipulates what is allowed and what is not when it comes to research in laboratories or release of GMOs to the environment. If the product were to be released, the Official Mexican Norm NOM-002-SAG-BIO-/SEMARNAT-2017 defines the conditions and considerations that are necessary to perform risk assessment analysis. The Official Mexican Norm NOM-164-SEMARNAT/SAGARPA-2013 sets up the requirements to report the analysis of a release into an uncontrolled environment. Also, the Sustainable Rural Development Law sets up some guidelines to be considered for a GMO that is going to be used in crops, which would be the case of BerryVax.

Additionally, the Secretariat of Environment and Natural Resources, the Secretariat of Agriculture and Rural Development, and the Secretariat of Health are authorities in subjects related to biosafety and GMOs and those institutions are involved in the paperwork needed for their use outside of a controlled environment. To implement any agricultural solution, the Sustainable Rural Development Law must also be considered.

Reactants and Procedures

Even though the project remained in a theoretical phase, with experiments being simulated in different software applications, the following substances/reactants were identified as hazardous according to their respective Material Safety Data Sheet (MSDS): Ethylenediaminetetraacetic acid (EDTA), Calcium Dichloride, TE Buffer, Potassium Acetate, TAE Buffer, Sodium Dodecyl Sulphate (SDS), Ethanol, Ampicillin, SYBR Green.

To analyze the efficacy of our product, first we will be working with a plasmid (pUC19) and a strain of E. coli to verify that our genetic circuit works as we wish it does. To enhance our biocontainment strategy, we decided to use integrative vectors to avoid horizontal transfer with plasmids. Once we know the genetic circuit works in the E. coli, we will insert an integrative vector (pDG1730) in the B. subtilis and B. amyloliquefaciens and analyze which of these two give out the best results.

The protocols will be handled the same way to insert the vectors in the different bacteria with the possibility of changes once the results in the E. coli are analyzed.

To make the cells competent, the active inoculum will be added in a medium which is prepared with Spizizen salts, tryptone, yeast extract and glucose, with the goal of making the bacteria overpass the exponential growth. Once the bacteria have reached the desired growth, 0.5ml of this culture will be added in a second flask with a medium that contains Spizizen salts, CaCl2, MgCl2 and glucose, and will be incubated for 90 min. The process must be stopped with ice, and the cells will be separated from the medium with a centrifuge and resuspended with a little bit of medium and glycerol so the can be frozen (iGEM Team Brno, 2020).

For the transformation, the cells must be taken to 37°C, once they have reached the desired temperature 100ul of competent cells will be mixed with 100ng of vectors and incubated for 60 min at 37°C and 150 rpm (iGEM Team Brno, 2020).

To determine the amount of surfactin that the cells are able to produce, we will resuspend the pellets in the supernatant and mix it with distilled water and NaOH 2M and filter the solution with a Centrifugal filter of 10kDa at 5000g. Afterwards, the retentate will be diluted with 50% methanol and let to dry for 24h at 80°C, finally the samples will be weighted and the amount of surfactin will be determined by the dry weight calculations (FCB-UANL, 2020).

To verify the fusaric acid sensor, we will place the GFP gene just before the surfactin gene, this way, the GFP gene will be activated when the surfactin gene detects the presence of the fusaric acid. To verify this statement, we would need to grow our modified B. subtilis in a Petri dish with fusaric acid in the media, if everything works as expected, we should be able to see the GFP indicating that the fusaric acid sensor is working in our modified bacteria.

To verify the inhibition of the fungal growth, a simple but effective way would be growing our modified bacteria together with the F. oxysporum and see if these in fact, do not let the fungi grow making the bacteria the dominant species. Finally, to verify the functionality of the kill switch we need to change the toxin MazF for a GFP and grow the bacteria in a liquid broth making constant samplings in the spectrophotometer, with this we will be able to graph the absorbance in the period of time programed for the bacteria and this way we can see if the toxin gene would have been active having a visual measurement (LMU, 2012).

COVID-19 Associated Safety Measures

Every student that enters ITESO’s Biotechnology Laboratories must wear a KN-95 mask, as well as eye-protection glasses. Before starting any laboratory work, the open-bench tables must be disinfected with ethanol 70%. Also, when laboratory is finished, the used surfaces must be also disinfected with ethanol 70% before leaving the workplace. Continuous or constant handwashing is promoted to reduce the risk of infection. Additionally, the laboratory visits are controlled, and must be previously booked to avoid the overcrowding of the laboratories in order to reduce the risk of infection.

Bioethics and Biosafety Manual

References

FCB-UANL, iGEM. (2020). SYNBIOFOAM. Retrieved from https://2020.igem.org/Team:FCB-UANL/Engineering#subtitle-sp

iGEM Team Brno. (2020). Handbook - How to handle Bacillus subtilis. https://static.igem.org/mediawiki/2020/5/50/T--Brno_Czech_Republic--Contribution_Handbook.pdf

LMU. (2012). Beadzillus. https://2012.igem.org/Team:LMU-Munich/Germination_Stop

Reimer, L. C., Sarda Carbasse, J., Koblitz, J., Ebeling, C., Podstawka, A., & Overmann, J. (2022). Bacillus subtilis (Ehrenberg 1835) Cohn 1872 (7.0) [Data set]. DSMZ. https://doi.org/10.13145/BACDIVE1174.20220920.7

Reimer, L. C., Sardà Carbasse, J., Koblitz, J., Ebeling, C., Podstawka, A., & Overmann, J. (2022). BacDive in 2022: The knowledge base for standardized bacterial and archaeal data. Nucleic Acids Research, 50(D1), D741-D746. https://doi.org/10.1093/nar/gkab961

Reimer, L. C., Sarda Carbasse, J., Koblitz, J., Ebeling, C., Podstawka, A., & Overmann, J. (2022). Escherichia coli (Migula 1895) Castellani and Chalmers 1919 (7.0) [Data set]. DSMZ. https://doi.org/10.13145/BACDIVE4434.20220920.7

Reimer, L. C., Sarda Carbasse, J., Koblitz, J., Podstawka, A., & Overmann, J. (2022). Bacillus amyloliquefaciens (ex Fukumoto 1943) Priest et al. 1987 emend. Wang et al. 2008 (7.0) [Data set]. DSMZ. https://doi.org/10.13145/BACDIVE598.20220920.7

Ta, L., Gosa, L., & Nathanson, D. A. (2019). Biosafety and Biohazards: Understanding Biosafety Levels and Meeting Safety Requirements of a Biobank. Methods in molecular biology (Clifton, N.J.), 1897, 213–225. https://doi.org/10.1007/978-1-4939-8935-5_19

Stoykov, Y. M., Pavlov, A. I., & Krastanov, A. I. (2015). Chitinase biotechnology: production, purification, and application. Engineering in Life Sciences, 15(1), 30-38.

Valenzuela Ruiz, V., Gálvez Gamboa, G. T., Villa Rodríguez, E. D., Parra Cota, F. I., Santoyo, G., & Santos-Villalobos, S. D. L. (2020). Lipopeptides produced by biological control agents of the genus Bacillus: a review of analytical tools used for their study. Revista mexicana de ciencias agrícolas, 11(2), 419-432.

Swain, M. R., Ray, R. C., & Nautiyal, C. S. (2008). Biocontrol efficacy of Bacillus subtilis strains isolated from cow dung against postharvest yam (Dioscorea rotundata L.) pathogens. Current microbiology, 57(5), 407-411.