Team:Sorbonne U Paris/Engineering

Design

Engineering



To drive our project, we were inspired by physicist Richard Feynman’s famous quote “What I cannot create, I do not understand”. We implemented this thinking through the use of a fundamental pipeline:

Research → Imagine → Design → Build → Test → Learn → Improve

Research

Our project entitled “The Chlamy Cleaner” consists in making the photosynthetic green microalgae named Chlamydomonas reinhardtii efficient for bioremediation of hazardous compounds found in the Seine river water. This model organism is well characterized and easy to cultivate. It made sense to use it since a MoClo toolkit has been adapted to Chlamy, making it suitable for synthetic biology projects. The widespread use of that microalgae in various bioremediation strategies was also an advantage.

Next, we established a list of interesting polluants to target. We learned from an exhaustive search that several classes of hazardous compounds can be found in the water environment : pesticides, antibiotics, polycyclic aromatic hydrocarbon (PAH), synthetic hormones... Since atrazine, an herbicide of the triazine class, is one of the most broadly found and a degradation pathway has been described, we chose to target it.

We also wanted to develop a kill switch system suitable for Chlamydomonas. While checking the literature about what had been done or not, we came across the system developed by the iGEM team in Munich 2013 for their PhyscoFilter project where they had developed an innovative system in Physcomitrella patens moss. We got inspiration from their concept in order to adapt it to Chlamydomonas reinhardtii.

Imagine

We imagined an engineered microalgae able to express a part of the atrazine degradation pathway found in the bacterium Pseudomonas sp. ADP strain (de Souza et al. 1996; de Souza et al. 1998). Because of the experimental difficulty to introduce a whole new pathway in a host organism, we limited ourselves to the first 3 genes: atzA, B and C which metabolize atrazine into cyanuric acid, a less toxic compound.

To prevent any environmental pollution due to an accidental spread of our engineered Chlamydomonas reinhardtii strain, we also imagined a kill switch device, based on a UV light sensible programmed cell death. This safeguard relies on the release of a nuclease anchored on the plasma membrane induced by COP1 and URV8 dimerisation. It will allow the assembly of the N-ter and C-ter of the TEV protease, addressing the nuclease to the nucleus. Thus, a UV light highpass filter would have to be implemented in our bioremediation filter.

Design

According to the MoClo guidelines, each gene was designed as level 0 plasmids containing strong promoters and implemented in level 1 transcriptional unit plasmids. Finally, multigenic level M plasmids were assembled.

For more information see the page Design.

Build

We carried out the experiments in the laboratory since August in order to :

- Synthesize our new originals parts
- Adapt them to MoClo standard for Chlamydomonas reinhardtii
- Clone them in level 0 plasmids
- Construct our transcriptional units in  level 1 plasmids
- Construct our multigenic level M plasmids for transfection to Chlamydomonas reinhardtii

Test

We could not test the efficiency of our genetic designs due to safety measures. Still, we had planned some tests to ensure the expression and functionality of our parts.

Gene experssion:

Human Influenza hemagglutinin tag (HA-tag) was introduced in the atzA, atzB and atzC C-terminal coding region in order to confirm the expression of our transgenes. With these elements, the expressed protein would be detectable by separation on a SDS PAGE coupled with immunodetection:

- Fusion protein atrazine chlorohydrolase (atzA) - HA - tag has a theoretical molecular weight of 55,99 kDa (505 amino acids) and pI of 6,05.
- Fusion protein hydroxyde-chloro-atrazine ethylaminohydrolase (atzB) - HA - tag has a theoretical molecular weight of 55,39 kDa (512 amino acids) and pI of 5,39.
- Fusion protein N-isopropylammelide isopropyl amidohydrolase (atzC) - HA - tag has a theoretical molecular weight of 48,45 kDa (512 amino acids) and pI of 4,85.

As the molecular weights of atzA and atzB are similar, bidimensional electrophoresis (using isoelectric point) followed by a western-blot is well suited to detect their expression.

Functionality of our parts:

Toxicity assays like those we performed on the wild type microalgae to test the efficiency of the implemented degradation pathway would have measured the effect of atrazine exposure on the growth of the engineered Chlamydomonas reinhardtii strain. If an increase of the tolerated concentration of atrazine compared to the wild type Chlamydomonas reinhardtii D66 strain is observed with our modified strain, we could conclude that the enzymes are well expressed and functional.

Liquid Chromatography coupled to tandem Mass Spectrometry (LC-MS/MS) could have been used to assess precisely the potential decrease of atrazine levels in the medium due to our modified microalgae.

UV light exposure of our tinkered strains would have been an easy way to determine the functionality of our kill-switch. A simple 740nm OD lecture would confirm it.

Learn

The new covid-related safety measures limited our access to the lab. We were not able to test the expression, functionality and efficiency of our parts, still we imagined what we could have learned from various scenarii.

1) Protein detection: since the molecular weight of parts atzA and atzB are very close, we may not be able to confirm their respective expression by SDS-page electrophoresis and immodetecion of the HA-tags. Separation by two-dimensional electrophoresis would allow us to separate atzA and atzB according to their isoelectric point. We could also change the design of our part and use distincts tags for immunodetection.

2) Efficiency of atrazine degradation: growth of our engineered microalgae on high atrazine concentrations over 250µg/L may not be observed. Underexpression of our transgene could be an explanation.

3) Kill-switch efficiency: growth of some microalgae clones may still be observed. A default in plasma membrane anchoring or an absence of interaction between URV8 and COP1 could be an explanation.

Improve

We faced problems during the synthesis of the UVR8 sequence by our supplier. This difficulty may be linked with the coding sequence high GC content adapted to Chlamydomonas reinhardtii (which has a high GC content genome).

To face this problem, we wanted to extract UVR8 cDNA from Chlamydomonas reinhardtii wild type strain by conducting RT-PCR experiments on total RNA extracts. Sequence UVR8 insertion into the level 0 plasmid requires insertion of enzyme BbsI fusion and restriction sites into this plasmid. In order to do that, we planned to use specific primers containing flanking nucleotides at both 3’ and 5’ ends. Gene domestication is possible if the Cr-UVR8 nucleotide sequence contains BsaI and BbsI restriction sites. It could be done via site-directed mutagenesis on the amplicon that would make silent mutations insertion. We first considered using the At-UVR8 coding sequence of Arabidopsis thaliana to express the UVR8-CterTEV fusion protein. Finally, using the Cr-UVR8 sequence would not be a problem insofar as Cr-UVR8 and At-COP1 interaction has been demonstrated (Tilbrook et al., 2016).

The huge malleability potential provided by the modular cloning allows us to consider new transcriptional units construction including other exogenous expression reporter parts.

Transgene quantification and localization could be carried out by fluorescence and confocal microscopy experiments. It would have been useful to use a fluorescent C-terminal tag to mark our proteins like GFP (Green Fluorescent protein), YFP (Yellow Fluorescent Protein) or mCherry (Red Fluorescent Protein) to conduct these microscopy experiments. It would be interesting to check if the NucA-TEVsite part is well expressed at the plasma membrane.

References:

- de Souza ML, Sadowsky MJ, Wackett LP. Atrazine chlorohydrolase from Pseudomonas sp. strain ADP: gene sequence, enzyme purification, and protein characterization. J Bacteriol. (1996).
- de Souza ML, Wackett LP, Sadowsky MJ. The atzABC genes encoding atrazine catabolism are located on a self-transmissible plasmid in Pseudomonas sp. strain ADP. Appl Environ Microbiol. (1998).
- Tilbrook K, Dubois M, Crocco CD, Yin R, Chappuis R, Allorent G, Schmid-Siegert E, Goldschmidt-Clermont M, Ulm R. UV-B Perception and Acclimation in Chlamydomonas reinhardtii. Plant Cell. (2016).