Team:Groningen/Implementation

Current regulations

There have been several legal frameworks established by the EU to ensure the safe development of agricultural GMO products (European Commission, https://ec.europa.eu/food/plant/gmo/legislation_en). The primary objectives of these frameworks are:

  1. To implement the highest possible safety assessment standards at EU level before a GMO is introduced into the market.

  2. To maximize the efficiency, time-limit and transparency of the procedures for risk assessment and authorization of GMOs.

  3. To verify that all GM food products are labelled accordingly when introduced on the market.

  4. To validate that all GM food products are traceable.

Overall, this initiative has made the GM product evaluation process more elaborate and effective. Therefore, our strategy for the safe and efficient implementation of RootPatch involves the development of a straightforward application method, control over GMO biocontainment, and off-target RootPatch activities. These considerations will allow us to minimize the risk factors and achieve an easier RootPatch implementation into the market.

How to apply RootPatch utilized by farmers in the field

The application of RootPatch is simple and straightforward for the farmer. Our approach is to apply RootPatch as a granulate, a powder form similar to many fertilizers that are already utilized by farmers on the field. Thus, existing equipment can be used to allow large-scale applications of RootPatch. The advantage of a granular form is that RootPatch can be stored easily and for a long period of time (Lobo et al., 2018). This is facilitated by the spore-forming capability of the Bacillus spp. and because our bioformulation consists of talc powder, which prevents hydrogen bonds and makes our product even more suitable for long-term storage [Martinez-Álvarez et al., 2016]. The formulation consists of low-cost compounds to keep the price of large-scale production of the granulate low. Take a look at the Engineering page for more information about the composition of the formulation.

Because the bacteria will be able to maintain their own population size RootPatch only needs, in principle, to be applied once. If re-application should be necessary, our bacteria population model has pointed out that it would be best to do this at a moment of high humidity and temperature.

Safety - the use of neuropeptides in nature

Neuropeptides-like proteins (NLPs) are short peptides naturally produced by the nematode’s neurons and facilitate synaptic communication. From our discussion with Hudson River Biotechnology, we were advised to examine how common the sequence of the NLPs is. It has happened before that due to sequence similarities, pesticides had unexpected off-target effects.
To address these concerns, we BLASTed the amino-acid sequence of our candidate NLPs, and apparently, the amino acid sequence is unique for the NLPs tested. You can take a look at the results of our BLAST-analysis by clicking on the image next to this paragraph. Of course, because of the limited size, sequences similar to that of the NLP are present in larger proteins, but only as a part of a domain. While it is impossible to completely rule out off-target effects, so far we have not identified any solid evidence that they could exist. In our experimental approach, we plan to look at the off-target effects in common beneficial nematodes in the soil as they are expected to be the most susceptible.

Click on the table to see the results of the BLAST search!

Safety - the use of GM microbes in nature

As with any other genetically modified organism (GMO), there will always be safety concerns regarding their uncontrolled spreading in the environment. In situations where it is not possible to integrate a physical barrier, the best way of overcoming these is by implementing an intrinsic barrier, such as a kill switch mechanism. In brief, a kill switch is a molecular constraint that prevents the escape of the GMO from the designated area.

To prevent the spreading of RootPatch bacteria in the field, we suggest using one or a combination of two approaches. The most simple one is creating a strain that requires the amino acid tryptophan. Thus, the bacteria will be contained close to the roots where the concentration of tryptophan is high. For the second kill switch option, we plan to make it very specific for the potato plant. Therefore, we designed a system that allows the survival of the bacteria only if solanine is present in the environment. Solanine is a glycoalkaloid synthesized by the potato plant and is also part of the root exudate. Moreover, its incidence in the soil directly follows the presence and growth of the plant (Jensen et al., 2009). B. mycoides (our host organism) will constitutively express a toxin while the antitoxin will only be expressed in the presence of solanine. Hence, RootPatch will not survive when it is far away from the roots. Check out the Engineering page for more information.

Jensen, P. H., Strobel, B. W., Hansen, H. C. B., & Jacobsen, O. S. (2009). Fate of Toxic Potato Glycoalkaloids in a Potato Field. Journal of Agricultural and Food Chemistry, 57(7), 2862–2867. https://doi.org/10.1021/jf803564v

Lobo, C. B., Juárez Tomás, M. S., Viruel, E., Ferrero, M. A., & Lucca, M. E. (2019). Development of low-cost formulations of plant growth-promoting bacteria to be used as inoculants in beneficial agricultural technologies. Microbiological Research, 219(November 2018), 12–25. https://doi.org/10.1016/j.micres.2018.10.012

Martínez-Álvarez, J.C., Castro-Martínez, C., Sánchez-Peña, P., Gutiérrez-Dorado, R., Maldonado-Mendoza, I.E., 2016. Development of a powder formulation based on Bacillus cereus sensu lato strain B25 spores for biological control of Fusarium verti- cillioides in maize plants. World J. Microbiol. Biotechnol. 32, 75. https://doi.org/10. 1007/s11274-015-2000-5