Team:IONIS-PARIS/project/description

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Description

Vineyards and climate change

Recently displaced from its leading position among global wine producers with a 12% recession last year, France suffers from climate change and its negative impacts on vineyards. The appearance of extreme temperature events threatens the agricultural economy and farmers have to deal with the current unsatisfactory solutions. Using synthetic biology, we are developing a thermo-adaptive biological product “Softer Shock” for protecting grapevines against climatic hazards.

French vineyards exposed to freezing (www.dna.fr, 2017)
Vineyards exposed to drought (www.lequotidien.lu, 2017)

A thermo-responsive microorganism

We are engineering a microorganism to make it express a specific protectant at the plant surface depending on the outside temperatures. Below 15°C, ice-binding proteins will interact with ice crystals to either inhibit their growth (antifreeze proteins) or favoring the nucleation process (ice-nucleation proteins). Despite their opposite functions, both strategies could be of great help to prevent frost damages in their own way and small-scale tests are required to make a final choice. Above 37°C, light-reflecting compounds will limit evapotranspiration by creating a reflective layer. Once applied on crops, our solution will possess a double protection: anti-drought and anti-frost.

This solution is used as a spray which contains our engineered organism. This idea raised some questions such as: which organism should we use? Which compounds are the best for foliar protection? How do we ensure biosafety in a limited area but still open environment? How can we evaluate toxicity? How should the spray be composed and how can we optimise its application on target? All these aspects have been discussed in our Applied Design part.

Experimental proof of concept

In order to show the thermo-responsiveness of our genetic system, we used two specific compounds as reporters. The blue chromoprotein amilCP attests that the cold response is activated, and the red flurorescent protein mRFP attests that the heat response is activated. This allows a simple and visual assessment method: bacteria should become blue when exposed to cold temperatures and red when exposed to hot temperatures. The perspective is then to test the function of both plasmids together in the same host cell by a co-transformation, and potentially ligate them into a single plasmid to obtain a real "2 in 1" response.

Our strategy concerning the Cold Shock response is to use the cold-induced cspA promoter system. The molecular components are the following:

  • The cold shock response sequence itself, which is composed of an UP element, CspA Promoter, 5’UTR and a DownStream box (DSB)

  • amilCP chromoprotein: reporter gene

Our strategy for the heat is to use the heat-shock response system in order to express red proteins at high temperatures. The molecular components are the following:

  • pL promoter, also called lambda left promoter, used in combination with the cI857 repressor. Below 30 degrees, cI857 is dimeric and binds the pL promoter to inhibit its action. Above 37°C, cI857 cannot dimerize neither perform its inhibitory action no the promoter, allowing the mRFP transcription.

  • cI857 regulation system

  • mRFP fluorescent protein: reporter gene

Molecular modeling

Molecular modeling is a key part of this project as our cold-shock expression system importantly relies on mRNA conformation. It was used to predictively design regulatory sequence modifications appearing in the coding sequence (the DownStream Box of the otherwise untranslated cspA 5’UTR). It was also used to characterize the structure of the cspA 5’UTR and propose an explanation for its function in the formation of the ribosomal pre-initiation complex at low temperatures.

Further applications

The interest of a thermo-responsive biological system can expand beyond the vines protection.

A novel “biocontrol” approach to global plant protection

Firstly, other crops could benefit from the Softer Shock technology according to their physiological properties and geographic location. When discussing with Mexican wine-growers, they mentioned the high temperature gaps existing between hot and cold seasons. In this context, having a this dual protective solution could be of great interest for farmers.

We can even go further and think about more “futuristic” approaches. Direct genetic modification of plants with a dual thermo-responsive plasmid could allow them to become more resistant to temperatures stresses and brutal climatic changes. Therefore, those plants may be grown in extreme environments where few crops are grown today, and even maybe on other planets!

Thermal time calculation for monitoring plant development

Plants “record” daily average temperature and store it in a total cumulated sum called “thermal time”. As soon as the needed thermal time is reached, the plant can go to the next stage of development. Thermosensible bacteria could be transformed to irreversibly express a red pigment over a precise temperature. A red “gradient” would then be visible as a function of the heat recorded over time. Alternatively, we could use the bacteria to record the average temperature every day, by comparing the color obtained to a reference scale of colors (like pH paper). Being able to visualize thermal time could be useful to predict crop development in a particular location or back-calculate the best sowing date.

The Softer Shock micro-organisms could also be useful in other fields than agriculture.

Self-regulation of solutions

Solution thermoregulation can be costly for industrials or scientific researchers. What could be interesting is to make our thermo-responsive organism respectively induce an exothermic reaction as the temperatures decrease, in order to reheat the medium, or an endothermic reaction as the temperatures increase in order to cool it down.

Thermo-regulated drug delivery

The interesting thermo-reactiveness activity can find valuable functions in controlled drug delivery. Tumoral or inflammatory microenvironments are characterized by an higher temperature compared to healthy tissues.

Encapsulated inside vectors, the micro-organisms could react to temperature increases by releasing a compound to destroy the surface and liberate the active principle locally.

Cold chain control

As you may know, all food products subjected to a freezing process should never be frozen again after thawing. It is particularly hard for individuals, but also industrials, to guarantee that this “cold chain” has not been broken. Thanks to the Softer Shock technology, we could elaborate a system allowing an irreversible color shift as soon as

the product has been thawed. The French company Cryolog developed a thermo-sensitive microbiological sticker for the same objective. Improving this technology by optimizing the temperature changes detection could be an interesting research axis for Softer Shock.