Project description

The iGEM Paris-Saclay project is part of the Foundational Advance track, and aims to study the effects of DNA topology on gene expression in E.coli. The purpose is to answer to this question: Does bringing a strong promoter closer to a weak promoter influence the expression level of genes located downstream?

We have designed a new tool based on CRISPR/Cas9 system to bring two specific DNA regions closer. This system is composed of two different dCas9s fused with each part of FRB / FKBP12 dimerization system. Each dCas9 will target a specific DNA sequence, one on the chromosome and one on a plasmid, whereas dimerization system will promote the joining of the two dCas9s when rapalog is added. In order to assess whether or not this system works, we have also designed a new tool to visualize the interaction between both dCas9s. This tool is composed of a split GFP attached to two dCas9s. These two small GFP tags will interact with the complementary GFP detector only if the two dCas9s are close enough to interact.

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Guideline of the project

In the laboratory, we first focused on the tool used to visualize the interaction between both dCas9s. For that purpose, we designed a biobrick in order to characterize the assembly of the split GFP. This biobrick is composed of one part of FRB / FKBP12 system fused to the other part of tripartite split-GFP system (GFP 10 / GFP 11) plus GFP 1.9 in the same plasmid. This biobrick was characterized by measuring GFP activity. Furthermore, a model was built in order to determine the optimal distance between the two dCas9s proteins for GFP to fluoresce. In order to test this model, we designed one biobrick composed of tripatrite split-GFP plus two dCas9s and another biobrick composed of the two target sequences of the dCas9 and the two sgRNAs coding sequence. For this second biobrick, we wanted to test several distances (50 bp, 75 bp, 100 bp and 150 bp) between the two target sequences of the dCas9, in order to determine the best distance for tripartite split-GFP to fluorescence, regarding to the established a model.

Perspective

If we obtain a higher expression level of the weak promoter with our two tools, it could lead to several useful applications. For example, we would be able to use this tool to enhance gene expression of any endogenous genes due to CRISPR/Cas9 specificity. Indeed, it would be possible to design specific sgRNAs but the user should be aware about off-target activity of the CRISPR/Cas9 system.

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Achievements

TODO SUR LE DRIVE IL Y A FICHIER PPT AVEC DES IMAGES CONTENANT LES CRITÈRES DES MEDAILLES VALIDÉS (VERT) OU NON (ROUGE), CA SERA MIS SOUS FORME D'IMAGES AVEC LE BON DESIGN : A VOIR S'IL Y A PAS MOYEN DE CODER UN TRUC SYMPA QUI FERAIT DES RENVOIS VERS LES DIFFERENTS CRITERES VALIDÉS, CA PEUT ÊTRE SYMPA