Overview:

Biosafety occupies a large part of our project. Indeed, the microorganisms that are contained in our solution Softer Shock are intended to be spread in the environment. Since it is a major concern here in France, we decided first to understand why, but mainly to find the best solution for our case.

Context

---

If engineered microorganisms are highly regulated, it is for many different reasons, as their spreading would cause a possible biodiversity imbalance and a species competition because of the organism presence in the environment, an increase of antibiotic resistance because of a genetic material transfer or even hygiene issues in the worst case. Also, this regulation primarily exists because of ethical questions, which is the main genetically engineered microorganisms issue. Although our microorganisms do not carry any real toxic compound, we chose to develop the project’s biosafety at its maximum^1,2,4.
To do so, in the biosafety aspects of Softer Shock, we chose to create a four walls fortress, which means a multi-layer strategy.

---

Our first wall is the auxotrophy, and we aim at engineering our organism so that it becomes dependent to a specific component: it is also called nutritional isolation. Here, we chose to make them depend to a 21st amino acid, which is not found in nature. Unless it has an access to this synthetic amino acid, the organism dies: it is confined in the area where the amino acid is spread.^2,3,6

---

Our second wall is composed of a killswitch, to kill our organism under certain inputs. It permits the avoidance of a microorganisms spreading. We chose to use the protegrin-1, which causes a membrane poration and so the cell death. Once its sequence is included in our organism genetic code with an arabinose operon, it would be activated in the presence of arabinose, making it easy for the farmers to kill our organism after harvesting².

We also thought of adding a DNAse coding sequence in our plasmid and an “anti-DNAse” coding sequence in the genomic DNA of our microorganism. If a DNA transfer occurs between a modified and a wild type organism, the wild type organism which does not contain any anti-DNAse would die⁵.
With the same idea, RNase/anti-RNase couple can be used. For instance the couple Barnase/Barstar (toxin/inhibitor), from B. amyloliquefaciens is a good candidate for this function¹².

For our third wall, we are actively looking for the most adapted chassis and we already have some tracks of naturally present organism on vine leaves and specific to the leaf environment. However, the perfect chassis does not exist, as if it extremely specific to the grapevines (so little present) a mass spraying could alter the biodiversity and on the contrary, if it is less specific the safety level would be lowered^9,10,11.

Our last wall is the physical containment. We decided to use the tunnel sprayer (see figure 5), in order to diffuse our product and to add some adjuvants to facilitate its use. This device is based on a “face to face” model in which each of the product dispenser face each other. It seems to be a good choice because the product that is sprayed on one side and doesn't end up on the plant is harvested by the panel on the other side. Also, it permits the deposit of the spray on both surfaces of leaves. The adjuvants would be a drift limitant, a bounce and shatter minimiser and a sticker and retention aid^7,8.

References

1. Torres, Krüger A, Csibra E, Gianni E, Pinheiro VB. Synthetic biology approaches to biological containment: pre-emptively tackling potential risks. Pinheiro VB, ed. Essays in Biochemistry. 2016;60(4):393-410. doi:10.1042/EBC20160013.
2. Oliver Wright & al, "Building-in biosafety for synthetic biology", Microbiology (2013), 159, 1221–1235
3. Lopez, G. and Anderson, J.C. (2015) Synthetic auxotrophs with ligand-dependent essential genes for a BL21(DE3) biosafety strain. ACS Synth. Biol. 4,1279–1286
4. Marliere P. The farther, the safer: a manifesto for securely navigating synthetic species away from the old living world. Systems and Synthetic Biology. 2009;3(1-4):77-84. doi:10.1007/s11693-009-9040-9.
5. Torres B. (2003). A dual lethal system to enhance containment of recombinant micro-organisms. Microbiology 149, 3595–3601 10.1099/mic.0.26618-0
6. Rovner and al., “Recoded organisms engineered to depend on synthetic amino acids”, Nature 518, 89-93, February 2015
7. Carra et al.,”Les panneaux récupérateurs:Atouts et limites”, IFV, 2017
8. Supofruit 2017, “Spray deposits from a recycling tunnel sprayer in vineyard: Effects of the forward speed and the nozzle type”, IFV 2017
9. Vacher et al., “The Phyllosphere: Microbial Jungle at the Plant–Climate Interface”, Annu. Rev. Ecol. Evol. Syst. 2016. 47:1–24
10. Bulgarelli D, Schlaeppi K, Spaepen S, Ver Loren van Themaat E, Schulze-Lefert P. 2013. Structure and functions of the bacterial microbiota of plants. Annu. Rev. Plant Biol. 64:807–38
11. Julia A. Vorholt, “Microbial life in the phyllosphere”, Nature Reviews Microbiology 10, 828-840 (December 2012)
12. Hartley, R. W. (1989). Barnase and barstar: two small proteins to fold and fit together. Trends in Biochemical Sciences, 14(11), 450–454. https://doi.org/10.1016/0968-0004(89)90104-7