Team:Sorbonne U Paris/Results

Results

Results



Overview:

- Analysed the pollutant diversity and abundance in the Seine river
- Synthesized 10 level 0 and level 1 plasmids (only URV8 part missing)
- Assembled the atrazine degradation pathway multigenic plasmid
- Assessed the effect of atrazine and cyanuric acid on wild type Chlamydomonas reinhardtii by plates and photobioreactor toxicity assays
- Demonstrated the lesser toxicity of cyanuric acid on wild type Chlamydomonas reinhardtii

Chemical analysis of Seine water:

Our IGEM project, The ChlamyCleaner, aims to provide a new biological filter to reduce the environmental exposure to micropollutants in the Seine, the river that crosses the city of Paris. To gain insight into the pollutant diversity and abundance in the Seine, we sent collected samples of seine water from four different places. One in Herblay-sur-Seine (1) near the “Seine-Aval” wastewater treatment plant, the largest in the Ile-de-France region, one at the Seine-Marne confluence in Vitry-sur-Seine (2), another on the quay Saint-Bernard (3) bordering the campus of Sorbonne Université in the center of Paris and the last sample was provided to us by the METIS laboratory (CNRS UMR 7619) and was sampled from the Orgeval river (4) near a heavily agricultural area in the Seine et Marne department, in the east of Paris.

Satellite view of the Paris region with the different sampling locations indicated by a red cross.

The samples were analysed in the METIS laboratory (Organic contaminant departement) where they performed a chemical analysis by liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS). Here is the list of the pollutants detected:

LOQ stands for limits of quantification, the lowest concentration of pollutant detectable with LC MS/MS. 17 different pollutants were detected. Our targeted pollutant, atrazine, was detected in all the samples. Two products of atrazine degradation, DEA (diethyl-atrazine) and DIA (desisopropyl-atrazine) were also detected in the samples, with an even larger concentration for DEA. These findings validate the significant impact that our project can achieve.

Parts cloning:

As of october 16th 2020, by using the Moclo tool kit for Chlamydomonas Reinhardtii developed by Pierre Crozet and Stephane Lemaire, we were able to clone most of the Level 0 parts of the degradation genes of atrazine and for our kill-switch system, except for the UVR8 part. We believe we will be able to complete the UVR8 cloning, to create our Level 1 plasmids as well as the level M in the near future. We systematically checked the sequence for each Level 0 plasmid we cloned, by sending samples to Eurofin Genomics for Sanger sequencing.

We were able to finalize our genetic construct related to the atrazine degradation pathway into cyanuric acid. All the level 1 plasmids were completed and cloned into a blasticidin and spectinomycin resistant level M multigenic plasmid. 

Unfortunately, due to new covid-related safety norms, we were not able to transform our Chlamydomonas reinhardtii strain with our level M plasmid. We could not test neither its efficiency nor the increased resistance of our engineered microalgae to atrazine exposure.

Measure of the resistance of Chlamydomonas to atrazine and cyanuric acid:

In order to assess the atrazine degradation ability of our engineered Chlamydomonas strain, we needed to characterize the basal toxicity of atrazine on the Chlamydomonas reinhardtii D66 strain. We also needed to demonstrate the lesser toxicity of the final product coming from the degradation of atrazine, the cyanuric acid, on our microalgae. We performed two types of toxicity tests: exposure to atrazine and cyanuric acid on plate (TAP medium, 96H) and monitored exposure to test a wide range of pollutant concentration. The Algem® photobioreactor (Algenuity®) allowed us to record more parameters (pH, Temperature) and perform the toxicity test with a larger volume (from 1mL to 1L).

The first toxicity test performed was the exposure to atrazine. We tested a range of concentrations from 0 to 1500 µg/L in 1mL TAPmedium 24 wells plates. At T=0, the Chlamydomonas cells were in exponential phase (5 millions cells/mL). Simultaneously, we ran the experiment with cyanuric acid and the same concentration range of polluant.

These experiments show that exposure to DMSO did not alter the growth of Chlamydomonas reinhardtii strain D66. Thus, any observed effect will be caused by the pollutant in the other wells. Exposure to atrazine alters the growth of Chlamydomonas reinhardtii strain D66 at the minimal concentration of 250µg/L in TAP medium.

Cyanuric acid did not seem to alter the growth of Chlamydomonas reinhardtii strain D66 in TAP medium, even at the maximum exposure levels.

These results provide two keypoints for the future of our project:

- It strengthens our strategy : the atrazine degradation into cyanuric acid is sufficient to hamper its toxic effect. 

- We were able to determine a threshold value to test the efficiency of our genetically engineered microalgae. Growing on high atrazine concentrations will be required.

The third toxicity test performed was the exposure to atrazine in the Algem® photobioreactor system. We tested a range of concentration from 0 to 500 µg/L in 400mL TAP medium in a 1L erlenmeyer flask. At t=0, the Chlamydomonas concentration was 1 million cells/mL. Environmental parameters were set : pH=7 and T=28°C and were stable during the experiment.

Growth curve of Chlamydomonas reinhardtii D66 strain D66 at various atrazine concentrations:
Green: 0 µg/L ; yellow: 77 µg/L ; orange: 250 µg/L ; red: 500 µg/L.

These results do not fit completely with the previous ones. All the atrazine condition hampers the microalgae growth. However, the polluant starts to hinder it at 77µg/L, below the threshold of 250 established earlier. It was expected since the Algem has more precise data. 

All Chlamydomonas reinhardtii cultures were made in a rich medium (TAP), the growth of the microalgae was not nutrient-limited. This medium is not representative of the final environment of our filter: the Seine river, a nutrient low environment. Therefore, we wanted to run our experiment in a minimum medium (HSM) or even better, in sterilized Seine water. It was not possible due to the new covid-related safety norms which cut our lab access.