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ERC

ProFF Report Summary

Project ID: 647275
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - ProFF (Programming in vitro evolution using molecular fitness functions)

Reporting period: 2015-09-01 to 2017-02-28

Summary of the context and overall objectives of the project

The ERC Consolidator “ProFF” targets the development of a new class of directed evolution strategies in order to facilitate the process of finding a new catalyst for a given reaction.
Designing a catalyst is extremely difficult. The best examples we know come from nature: enzymes are incredible catalysts, providing tremendous rate accelerations along with exquisite molecular control. Enzyme-like man-made catalysts made on purpose to accelerate a given reaction could solve many problems in molecular biology, biocatalysis and green chemistry, as well as to find some therapeutic applications.
Directed evolution is a set of techniques that mimick natural evolution in the lab and are used to obtain new molecules with improved or unnatural properties. While efficient to generate tailored enzymes, this approach is still very laborious. We want to use molecular programming concepts to make it faster, more autonomous and more efficient. Our idea is to pre-program a chemical system so as to bias the process of molecular evolution toward the desired target activity. Once designed, the molecular program, which includes sensors, DNA-based circuits and genetic amplificators, can be dispatched into billions of tiny compartments, where each candidate enzymatic mutant is then autonomously evaluated. To achieve this result, we must put together a number of experimental building blocks: generation of libraries of modified genes, expression of this genes into compartments, and build a molecular program able to detect the properties of the mutant and trigger amplification only for the ones with the desired properties. We are also looking at selection and evolution process in a more theoretical way.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

We have already demonstrated that it is possible to detect the activity of some enzymes, and to link this to the amplification efficiency of their gene, using a DNA-based molecular program. We have characterized the activity-fitness response function of such circuits using new reporting strategies to observe the production of primers by a selection circuit. We designed microfluidic chips to generate emulsions for compartmentalization, and performed some biochemical reactions in these emulsions. We also prepared bacterial extracts for optimized (high yield) in vitro expression of proteins and gel beads as a new way to link genotype and phenotype. We finaly studied the effect of co-compartmentalization on the evolution of a population performing self-selection processes and obtained the analytical relation.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The project is still in early stage and its main goals are not reached yet. However we have already contributed to the development of new architectures for molecular programs. Bistability in particular is a very important function for our purpose and we have reported a new, very simple strategy to achieve this function. We have also reported new way of localizing DNA-programmed reactions on the surface of microparticles, which will also constitute a building block for the ProFF project.
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