Team:Groningen/Safety

Safety

Introduction

The final goal of Bye-Monia is to have a genetically modified organism (GMO) that can be used safely in the agricultural industry. Therefore, it is of high importance that safety regulations are taken into account. Several aspects concerning safety were taken into account designing the constructs, during our laboratory handlings and for the future application of Bye-Monia.

Laboratory safety

When working in a microbiology laboratory, one of the first things you encounter and take into account are biological risks. For our project, we work with a variety of chemicals and organisms, all with their characteristics and possible hazards. Therefore, all our team members were required to have a Safe Microbial Techniques (SMT) certificate. Those who did not have this certificate at the beginning stages of our project followed a course to obtain the SMT certificate issued by the biological safety officer from the University of Groningen. Furthermore, the main wet lab members participated in a safety course in which they learned the basic principles of laboratory safety as well as a fire fighting course. All participating members passed the course with a certificate issued by the fire safety officer from the University of Groningen. Lastly, together with the members of the Human Practices team, we participated in the Safe by Design case study from the National Institute for Public Health and the Environment (Rijksinstituut voor Volksgezondheid en Milieu; RIVM). This case study, along with other meetups and background research, resulted in our team to design an extensive Safe-by-Design approach that accounts for the safety of our project both in and outside the wet lab.

During our project, we worked with Golden Gate cloning using the MoClo-YTK kit [1]. The following microorganisms will be part of the project; Saccharomyces cerevisiae (BY4741, ySB76, ySB77 & ySB78), Saccharomyces paradoxus (ySB85), and Escherichia coli (DH5α). All organisms are classified as biological safety level (BSL) 1, which implies that they are of no direct danger to people and the environment [2]-[5]. Therefore, all the wet lab work we perform can take place in a BSL-1 laboratory provided by the institute of Groningen Biomolecular Science and Biotechnology of the University of Groningen. Since the produced alpha-amylase does not cause any harm to humans and nature, no extra precautions are needed [6].

Biocontainment

Our project aims to create a genetically modified Saccharomyces spp.. We are aware of the relevant risks and the measures that need to be taken. The uncertainties and potential risks associated with GMOs affecting the ecosystems should be minimized. In nature, Saccharomyces spp. do not produce amylase. Therefore, our genetically engineered  Saccharomyces strains have some underlying risks if they escape from the lab. These include suppression of the existing strains in nature, disrupting food chains, and altering biodiversity. We have set up an appropriate system to keep our chassis organism from being released into the environment [7],[8].

Conditional essentiality-based biocontainment system

Every organism depends on certain essential processes inside the cell to maintain its vital activities. When lacking one of the basic elements in the processes, the cellular activities will not function properly and eventually lead to cell death. Removing critical genes that ensure the cell synthesis which maintain the cell alive is a typical approach to govern the GMO release. In this containment system, GMOs are compelled to depend on external supply of essential nutrients[9].

Circuit

For our yeast strain, we have designed a genetic circuit based on histone regulation in the cell. Histones are essential proteins that interact with DNA in the nucleus, promote the assembly of nucleosomes and the condensation of chromatin. There are several types of histones, of which the histone 3 (H3) and histone 4 (H4) are critical parts for the organization of the nucleosome core. H3 and H4 are encoded by two genes hht1-2 and hhf1-2, respectively, on the chromosome. These genes are selected for developing our biocontainment system[10], [11].

Cassette construction

Our biocontainment system aims to replace the intrinsic hht1-2 and hhf1-2 genes with synthetic counterparts HHTS and HHFS. The HHTS and HHFS are placed under the pGAL1 and pGAL7 promoter, respectively ( Fig.1). Both can be regulated by galactose, a commonly used promoter in molecular work with Saccharomyces spp.. To ensure fitness and decrease discrepancy in copy number of the plasmid inside the cell, a centromere-based vector pRS415 is opted for the cloning. The gene construct is flanked by the HO gene (right HO and left HO) for the integration. The HO locus is commonly used for gene integration and studies have proved that integration at this site does not affect cell growth[12]. To delete the endogenous hht1-2 and hhf1-2, genomic targeting vector pFA6a-natMX4 and pFA6a-kanMX6 are engineered to being flanked by two homologous sequences of hht1-2 or hhf1-2 (Fig.2).

Regulation of HHTS and HHFS by pGAL1 and pGAL7 promoters.

Figure 1: Regulation of HHTS and HHFS by pGAL1 and pGAL7 promoters.

Genomic targeting by pFA6a-natMX4 and pFA6a-kanMX6.

Figure 2: Genomic targeting by pFA6a-natMX4 and pFA6a-kanMX6.

Assembly of the biocontainment system

The vector pRS415-HO-pGAL1-HHTS-pGAL7-HHFS-HO is transformed to the yeast strain firstly. This ensures the cell viability when knocking out the intrinsic H3 and H4 of the genome in the following step. The vectors pFA6a-natMX4 and pFA6a-kanMX6 are then transformed into the selected strain. With this step, hht1-2 and hhf1-2 are replaced by natMX4 and kanMX6 that display resistance to nourseothricin and kanamycin, respectively. This generates the construct hht1-hhf1Δ::natMX4 hht2-hhf2Δ::hygMX4 HO::HHTS-HHFS in our yeast strain which is only viable when supplied with galactose[13](Fig. 3).

Visual representation of the biocontainment system

Figure 3: Visual representation of the biocontainment system

Unfortunately, there was not enough time during the project to test the biocontainment system design, but it can be implemented in future applications of Bye-Monia. The design has been considered carefully; however, for real-world application, the performance of our biocontainment system and the possibility of our GMOs escaping the lab needs to be evaluated. The designed system relies upon the availability of external supply of galactose to keep the organism alive. Therefore, the GMO would not be able to survive outside the lab.

References

  1. A Highly-characterized Yeast Toolkit for Modular, Multi-part Assembly. Lee ME, DeLoache, WC A, Cervantes B, Dueber, JE. ACS Synthetic Biology 2015. DOI: 10.1021/sb500366v.
  2. RISK ASSESSMENT EXAMPLE E. COLI K-12 DERIVATIVE EXPRESSING HUMAN GROWTH HORMONE. (2015). http://www.chem.ed.ac.uk/sites/default/files/safety/documents/GMOform_example.PDF
  3. ATTACHMENT I--FINAL RISK ASSESSMENT OF SACCHAROMYCES CEREVISIAE .(1997). https://www.epa.gov/sites/default/files/2015-09/documents/fra002.pdf
  4. ATTACHMENT I--FINAL RISK ASSESSMENT OF BACILLUS LICHENIFORMIS .(1997). https://www.epa.gov/sites/default/files/2015-09/documents/fra005.pdf
  5. ATTACHMENT I--FINAL RISK ASSESSMENT OF BACILLUS SUBTILIS. (1997). https://www.epa.gov/sites/default/files/2015-09/documents/fra009.pdf
  6. Silano, V., et al., (2018). Safety evaluation of the food enzyme α‐amylase from a genetically modified Bacillus licheniformis (strain NZYM‐AV). EFSA Journal, 16(7), e05318-n/a. 10.2903/j.efsa.2018.5318
  7. Simon, A. J. & Ellington, A. D. Recent advances in synthetic biosafety. F1000Research (2016). doi:10.12688/f1000research.8365.1
  8. Epstein, M. M. & Vermeire, T. Scientific Opinion on Risk Assessment of Synthetic Biology. Trends in Biotechnology (2016). doi:10.1016/j.tibtech.2016.04.013
  9. Kim, D. & Lee, J. W. Genetic Biocontainment Systems for the Safe Use of Engineered Microorganisms. Biotechnology and Bioprocess Engineering (2020). doi:10.1007/s12257-020-0070-1
  10. Eriksson, P. R., Ganguli, D., Nagarajavel, V. & Clark, D. J. Regulation of histone gene expression in budding yeast. Genetics (2012). doi:10.1534/genetics.112.140145
  11. Cai, Y. et al. Intrinsic biocontainment: Multiplex genome safeguards combine transcriptional and recombinational control of essential yeast genes. Proc. Natl. Acad. Sci. U. S. A. (2015). doi:10.1073/pnas.1424704112
  12. Voth, W. P., Richards, J. D., Shaw, J. M. & Stillman, D. J. Yeast vectors for integration at the HO locus. Nucleic Acids Res. (2001). doi:10.1093/nar/29.12.e59
  13. Fan, L. & Xiao, W. Study essential gene functions by plasmid shuffling. in Methods in Molecular Biology (2021). doi:10.1007/978-1-0716-0868-5_5