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PhD student (M/F) in molecular biology for the development of depollution enzymes by directed evolution and functional screening in microfluidic systems

Send your Application before :
June 15, 2022
Offer published on :
May 23, 2022
Offer published until :
June 15, 2022

Human industrial activities of the last decades have been accompanied by the production and accumulation of a large number of fluorinated residues in the environment. Some of these compounds, sometimes extremely persistent such as PFAS (PerFluorinated Alkyl Substances), represent today a real threat not only for the environment but also, more generally, for public health. Consequently, there is an urgent need to identify and develop rapid bioremediation methods for the removal of these compounds. Fluorinated compounds are not specific to human activities but are also quite widespread in nature. Therefore, it is highly likely that many microorganisms are capable of degrading them and using them as an energy source. However, as conventional microbiological methods do not allow for an exhaustive exploration of the microbiosphere, very few such organisms have been identified to date, leaving the question of bioremediation of fluorinated wastes a largely open and essentially unsolved problem. This thesis project will be funded for 36 months by the ANR “Microfluor” program and will aim at identifying/developing new molecular tools in the form of PFAS degradation enzymes. The thesis work will be carried out in the “Digital RNA Biology” team (Institute of Molecular and Cellular Biology, Strasbourg) under the supervision of Michael RYCKELYNCK and will have as main objective to develop and exploit a new ultrahigh-throughput functional screening strategy using droplet microfluidics in order to isolate new enzymatic activities of defluorination. To do so, two complementary strategies will be exploited in parallel, in close collaboration with the team of Prof. Stéphane Vuilleumier (University of Strasbourg). First, bacteria from samples taken from sites contaminated by fluorinated compounds will be individualized in water-in-oil droplets of a few picoliters containing a minimum synthetic culture medium supplemented with the fluorinated compound to be degraded and used as a carbon source. Thus, the degradation capacity of the target compound by the organism present in the droplet will result not only in its growth, but also in the release of fluoride. The latter will be detected by an RNA molecular probe recently developed by the team and which allows to convert the presence of fluoride ions into fluorescence emission. A second approach based on directed evolution will also be used. For this, the gene coding for a dehalogenating enzyme will be used as a starting point and subjected to a mutagenesis step (random and/or partially targeted). The large collection of mutant genes thus obtained will then be analyzed by the microfluidic screening technology routinely used by the team. For this purpose, each gene of the library will be individualized in small droplets of PCR mixture at a rate of several million droplets per experiment. After amplification, each gene will be expressed by coupled in vitro transcription/translation, then the enzymatic activity of the proteins produced in each droplet will be evaluated by adding a fluorinated substrate and the fluorescent fluoride reporter developed by the team. Thus, droplets containing an active defluorinating enzyme will fluoresce and can be sorted specifically on the basis of this signal. At the end of this project, new natural (strategy 1) or artificial (strategy 2) defluorination enzymes will have been identified and will not only be the subject of publications, but also of a valorization process if their performances allow it.

The full job profile is available at the following address:

Do not hesitate to contact Michaël RYCKELYNCK to the IBMC for further information.

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