What is Biosafety?

According to the Introduction to the Cartagena Protocol [1] - an international agreement on the safe transfer, use and handling of genetically modified organisms - biosafety can be defined as:

"A range of measures, policies and procedures for minimizing potential risks that biotechnology may pose to the environment and human health. Establishing credible and effective safeguards for GMOs is critical for maximizing the benefits of biotechnology while minimizing its risks"

In our project, there are two angles we approach Biosafety from: the safety assessment of using cell-free systems in research; and considerations about the development of new plant varieties and the biosafety concerns related to agricultural use of transplastomic plants.

Biosafety Aspects of Cell-Free Systems

One of the concerns regarding lab biosafety is the possibility of living organisms “escaping” and either causing diseases in the local flora or being introduced in the ecosystem as an invasive species [2]. For plant synthetic biology research, this means that all engineered plants need to be confined to S1/S2 greenhouses, with special guidelines for their disposal after the experiments are done. Such measures entail extra costs and logistical hurdles to anyone in this field, which might be prohibitive for iGEM teams.

Since cell-free systems enable transcription and translation but are abiotic and do not reproduce, they open up new possibilities . For instance, it would be possible to rapidly implement several DBTL cycles without risking the unintentional release of an organism. In the case of plants, it would mean less transgenic pollen, seeds and biomaterial being produced, while in other cases it means a safer workplace for researchers who would otherwise work with living pathogens [3]. Thinking beyond our project's scope, this technology could replace organisms being currently used to synthesise pharmaceuticals and other molecules of value with the added benefit of improved biocontainment [4].

Biosafety Aspects of Transplastomic Plants

As we all know, chloroplasts - much like the mitochondria - were once free living bacteria. Their endosymbiont path has made these organelles somewhat independent from the rest of the cell, chloroplasts have their own genome with instructions for a transcription and translation machinery, tRNAs and other photosynthesis related proteins [5, 6]. This independence is per se one of the reasons why chloroplasts are such an interesting Synbio platform, but they have yet another trick up their sleeve: maternal inheritance [7].

In most economically significant plant species, sperm cells do not carry chloroplasts, in other species they are excluded during fertilization, this means all the chloroplast genome - plastome - is inherited from the egg cell, and not the pollen grain. Like everything in biology, there are exceptions: there is a miniscule possibility that some chloroplasts are passed on paternally [8]; transplastomics represents, however, an enormous decrease in the gene-flow probability in comparison to nuclear-engineered plants. While nuclear-transformed plants separated by a 10m corridor have a cross-pollination frequency of about 0.01, chloroplast paternal inheritance in similar conditions has a frequency of about one million times lower [9,8]. We strongly believe that a significant improvement in biosafety can pave the way to public acceptance of plant biotechnology.

Dual-Use Considerations

Another important advantage of transforming chloroplasts is their capability for high foreign protein expression [10]. For instance, this can be advantageous to reduce the chance of insects developing resistance against the Bt toxin [11], or to use plants as bioreactors to produce a high quantity of antibodies and vaccines [10]. We have a duty, however, to also think about the ways our technology can be misused by parties aiming to harm others; In that regard, the high protein expression can be used to create plants that produce toxins and allergens, and by facilitating the engineering of chloroplasts, cell-free systems like ours could also contribute to their misuse. On the other hand, cell-free systems do not significantly shift the current threat model of recombinant protein production, as the same hurdles of purifying and weaponizing the synthesized protein still apply [12].

  1. Secretariat of the Convention on Biological Diversity. (2003). Biosafety and the Environment: An introduction to the Cartagena Protocol on Biosafety. Secretariat of the Convention on Biological Diversity.
  2. Tinafar, A., Jaenes, K., Pardee, K., 2019. Synthetic Biology Goes Cell-Free. BMC Biol. 17, 1–14.
  3. Khambhati, K., Bhattacharjee, G., Gohil, N., Braddick, D., Kulkarni, V., Singh, V., 2019. Exploring the Potential of Cell-Free Protein Synthesis for Extending the Abilities of Biological Systems. Front. Bioeng. Biotechnol. 7, 248.
  4. Garenne, D., Haines, M.C., Romantseva, E.F., Freemont, P., Strychalski, E.A., Noireaux, V., 2021. Cell-free gene expression. Nat. Rev. Methods Primer 1, 1–18.
  5. Dobrogojski, J., Adamiec, M., Luciński, R., 2020. The chloroplast genome: a review. Acta Physiol. Plant. 42, 98.
  6. Sibbald, S.J., Archibald, J.M., 2020. Genomic Insights into Plastid Evolution. Genome Biol. Evol. 12, 978–990.
  7. Daniell, H., 2007. Transgene containment by maternal inheritance: Effective or elusive? Proc. Natl. Acad. Sci. 104, 6879–6880.
  8. Ruf, S., Karcher, D., Bock, R., 2007. Determining the transgene containment level provided by chloroplast transformation. Proc. Natl. Acad. Sci. U. S. A. 104, 6998–7002.
  9. Paul, E.M., Capiau, K., Jacobs, M., Dunwell, J.M., 1995. A Study of Gene Dispersal Via Pollen in Nicotiana tabacum Using Introduced Genetic Markers. J. Appl. Ecol. 32, 875–882.
  10. Daniell, H., 2006. Production of biopharmaceuticals and vaccines in plants via the chloroplast genome. Biotechnol. J. 1, 1071–1079.
  11. Kota, M., Daniell, H., Varma, S., Garczynski, S. F., Gould, F., & Moar, W. J. (1999). Overexpression of the Bacillus thuringiensis (Bt) Cry2Aa2 protein in chloroplasts confers resistance to plants against susceptible and Bt-resistant insects. Proceedings of the National Academy of Sciences of the United States of America, 96(5), 1840–1845.
  12. Ben Ouagrham-Gormley, S. (2014). Barriers to bioweapons: The challenges of expertise and organization for weapons development. Cornell University Press.