ESPCI web site
Lieu de travail Paris - Ile-de-France - France
Intitulé du sujet
Information transmission across generations : thermodynamics and evolutionary implications
Champs scientifiques : Physique Biochimie Biologie
Mots clés droplet microfluidics, thermodynamics, statistical physics, evolutionary biology, biophysics, non-euilibrium, protein engineering, molecular biology
Description
Enzymes catalyse chemical reactions in the crowded cellular environment. To perform their function they must recognize and process their substrate over a strong background of competing molecules. In most known reaction schemes, however, specificity comes at a cost in terms of reaction rate [1], leading to a trade-off that can be of fundamental evolutionary relevance. In this project we will address such trade-off using DNA polymerases, enzymes responsible for information transmission across generations, as a model. Polymerases act under constrains of very different origin. On the one hand the laws of thermodynamics impose a fundamental trade-off between the specificity of a chemical reaction and he reaction rate. On the other hand the rate of mutation is under strong evolutionary selection to produce just enough variability as required for adaptation. These constraints should not be thought of as independent : there is indication that, in RNA viruses, a trade off between replication speed and accuracy is actually setting the mutation rate [2]. The candidate will combine droplet microfluidics and next generation sequencing to measure both the error-rate and the relative activity of large (107) libraries of polymerase mutants using the Phi29 polymerase as a model. This will allow him to generate unprecedented data, allowing the measurement of the error-rate of every single-point mutant of the polymerase or of a significant fraction of double point mutants. This will provide a unique picture of the mechanism behind polymerase fidelity, a mechanism that is conserved across the different domains of life. We expect the following results :
1. An unprecedented description of the structural determinants of the error rate, clarifying the mechanism for selectivity and the regulation of mutation rate in polymerases.
2. A comparison of the error-rate and activity of different mutants and different homologs, casting light on their trade-off and correlating it with sequence information.
3. A study of such trade-off under different non-equilibrium conditions and in in relation to sequence dependence that will address the thermodynamic and information-theoretic aspects of the problem.
The experimental activity will be accompanied by the development of a theoretical framework bridging the existing theoretical literature on polymerase activity to the experiments. This will allow the candidate to use existing models as tools in data analysis but also to generalize his results beyond the polymerase system. Biological systems perform a variety of information processes at the molecular or cellular level, where fluctuations and energy transfers are of the order of the thermal energy [3]. Lessons learned on this model system might reveal principles to be applied elsewhere.
[1] Dan Tawfik (2014). Current Opinion in Chemical Biology. 21:73-80
[2] Fitzsimmons WJ et al. (2018) PLoS Biol 16(6) : e2006459
[3]Parrondo, JMR, et al. (2015). Nature physics 11(2) : 131
3i apsect of the project
Intersectoriality, Interdisciplinarity, International mobility
Far from being only a topic of great fundamental interest the activity/specificity trade off is an issue in industrial protein engineering projects. Protein engineering is a key biotechnological tool, whose impact has been recognized by the 2018 Nobel Prize in Chemistry. The tools used in the fellowship are essential ingredients in modern day protein engineering. During this fellowship the candidate will use :
droplet microfluidics : to study large libraries of polymerase mutants,
next generation sequencing techniques : to read the results,
bioinformatics tools : to analyze the data,
physics based models : to rationalize the results.
This complex toolset will enable him/her to pursue a career in the industrial biotechnology sector, should he choose to do so.
In order to explore the implications of selectivity/rate trade off in applications and to be exposed to research in the corporate environment the candidate will be hosted for a secondment period at the 4Catalyzer start-up incubator at the Rothberg Institute in Guilford, CT, with whom we have been collaborating over the last years. The incubator concentrates several startups in the industrial bio/medical technology sectors and will offer concrete career opportunities for the candidate.
In a further secondment period the candidate will visit the Small Biosystems Lab (SBL), directed by F. Ritort at the Unversity of Barcelona (Spain). The SBL is a single-molecule laboratory (Optical Tweezers) with a strong emphasis on the non-equilibrium aspects of molecular biophysics. Here, in collaboration with Ph.D. students at the SBL, the candidate will test the single-molecule behavior of a small set of mutants identified via the high-throughput techniques at the LBC.
Nature du financement
Financement public/privé
Précisions sur le financement
MSCA Cofund UPtoPARIS
Présentation établissement et labo d’accueil
ESPCI Paris
UMR8231 Chemistry, Biology & Innovation
Description of the research Unit/subunit
The institute Chemistry Biology Innovation (CBI) is a new UMR gathering 6 different teams working in experimental chemistry and biology with a common vocation : scientific creation and technological innovation at the frontiers of chemistry and biology.
The Laboratory of Biochemistry was created at ESPCI in September 2012 and is directed by Prof. Andrew Griffiths, one of the key figures in the development of phage-display for the selection of human antibodies for therapy. The LBC has developed very high-throughput single cell manipulation and analysis methods based on droplet microfluidics, with applications to diagnostics, directed evolution and bio-engineering. A major part of its current activity is focused on the coupling of these technologies with Next Generation Sequencing, using molecular barcoding strategies to decipher single cell genotypes and phenotypes. These efforts have led to the creation of several biotechnology start-up companies using droplet-based microfluidics (Raindance Technologies, HiFiBio, BioMillenia).
Name of the supervisor
Andrew Griffiths (Andrew.Griffiths@espci.fr)
Name of the co-supervisor
Marco Ribezzi (Marco.Ribezzi@espci.fr)
Site web :
Profil du candidat
The ideal candidate for this project would be a physicist by training with interest in statistical physics and an interest for experimental work. Previous experience in biochemistry and wet lab work would be a plus but it is not required. The experimental setup is currently working at the LBC and the relevant experimental protocols have been validated. Based on previous experience with research interns an undergraduate student can master the relevant experimental techniques in less than 6 months.
The project requires combining the experimental activity with data analysis and modeling. This is the part of the project that will require the most development. As for most intense experimental projects the suitable candidate will ideally have good time managing, flexibility and communication skills. These are not only key aspects of experimental activity but are transferable skills to be used in sectors beyond academia. As such they will also be addressed in the complementary training of the candidate.
Date limite de candidature
31/03/2019
Applications must be submitted on the UPtoPARIS website : https://www.upto.paris/
More information is available in the "How to apply" section of the website
!! BEGINNING OF THESIS BETWEEN MARCH AND SEPTEMBER 2019 !!