{{{titre}}}

Project Design

Bring DNA Closer tool construction

We have designed a tool based on Crispr/Ca9 property to target precisely a sequence. We imagine a system using dCas9 that dimerize under an induction signal to bring two DNA strain closer.

A dCas9 is a protein which recognize precisely a DNA sequence with dead nuclease activity. We choosed it for the high adaptability of this system, as it target DNA through a sgRNA it is easy to customize the target sequence. But as we need to target two different sequences we also need to work with dCas9 which will not interfere with each other. So we choosed two orthologous dCas9 which come from two different organisms T. denticola (TD) and S. pyogenes (SP). As they come from different organisms they recognize different sgRNA and do not interfere as we want. We order from Addgene the plasmid coding for each one of these dCas9 and its sgRNA.

To dimerize this two dCas9 we have chosen an inducible system using FRB and FKBP12 proteins. Originally found in mammal this two proteins form an heterodimer when rapamycin is added, it is particularly used in protein interaction studies (Cui et al., 2014). However rapamycin is toxic for bacteria. But studies have shown that a mutated FRB (FRB*) stills allow dimerization with an analog of rapamycin non toxic called rapalog. The mutations implied are: T2098L, K2095P, W2101F.(Bayle et al., 2006; Liberles, Diver, Austin, & Schreiber, 1997).

A biobrick coding FRB with mutation T2098L was already in the parts registry (iGEM Part_ J18926) but it was not available. Moreover it contains only one mutation on the 3 described in the literature. So we decided to work with the fully mutant FRB. Rapalog and plasmid with mutant FRB and FKBP12 were offered to us by Takara Clontech. But like we mentioned previously this system is used in mammal cells, so we decide to optimize the sequences for an expression in E.coli with the Jcat plateforme. So we finally order GBlock of our optimized sequences and a linker in prevision to the fusion with their respective dCas9.

Using these two systems (dCas9 recognition and FRB/FKBP12 dimerization) we design our new tool based on the two following biobricks:

These two biobricks will be assembled in pSB1C3 plasmid give us our get DNA closer tool which will function as bellow: