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The project aim to study how bacterial DNA organization can influence gene expression. In order to answer our question, the team decide to create a new tool based on CRISPR-Cas9 to bring together two distant DNA regions. This new tool is expected to assess the effect of DNA structure on gene expression. As a results, we have designed a bring DNA closer tool (BDC tool) and a visualization tool.

Here, we will expose you our tool design and we will expose you our experimental stratgy here.

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:

Visualization tool construction

Every system need an efficient control, as a result, a new part of the project has been setting up and the team has designed the visualization tool. To build this new tool, two other ortholog nuclease function deficient Cas9s (dCas9s) : N.meningitidis and S.thermophilus, will be fused to fluorescent proteins. We also decide to use dCas9 system for this tool in order to have detection of a accurate and unique sequence in the genome. It will be fulfilling with a new and unreleased in the iGEM competition tripartite slip-GFP.

The tripartit split-GFP is composed of two twenty amino-acids long GFP tags (GFP10 and GFP11) and a third complementary subsection (GFP1-9). The tags will be fused to the two dCas9 previously quoted. A functional GFP will be achieved when the tools would be close enought to allow the three slip-GFP parts reunion and the fluorescence emission. This fluorescence system avoids poor folding and/or self-assembly background fluorescence. With this system, only two sgRNAs associate with their dCas9s fused to their specific GFP tags will be necessary instead of nearly 30 with mundane GFP due to background fluorescence.

The team has designed three biobricks to achieve this part of the project:

• Biobrick n°3 : N.meningitidis fused to GFP-10 expressed by a constitutive promoter, a RBS and a double terminator
• Biobrick n°4 : S.thermophilus fused to GFP-11 expressed by a constitutive promoter, a RBS and a double terminator
• Biobrick n°5 : The third part of the GFP 1-9 expressed by a constitutive promoter, a RBS and a double terminator

The biobricks were inserted into pSB1C3 using the iGEM process : restriction sites EcoRI and PstI.

Then, the team has considered to establish a composite biobrick composed of the three biobricks in the same pSB1C3 plasmid. This plasmid would have been build using the iGEM restriction site technique.

The dCas9 technique allows the target of specific sequences. In fact, this technique its adaptable to any DNA sequences on the genome through the single guide RNA (sgRNA). Those sgRNAs are associated with their cognate ortholog dCas9s mostly thanks to their palindromic associate motif (PAM).

The team has chosen to express the sgRNAs on another plasmid pZA11 which is compatible with pSB1C3.

References

Bayle, J. H., Grimley, J. S., Stankunas, K., Gestwicki, J. E., Wandless, T. J., & Crabtree, G. R. (2006). Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity. Chemistry and Biology, 13(1), 99–107. http://doi.org/10.1016/j.chembiol.2005.10.017

Cui, B., Wang, Y., Song, Y., Wang, T., Li, C., Wei, Y., … Shen, X. (2014). Bioluminescence resonance energy transfer system for measuring dynamic protein-protein interactions in bacteria. mBio, 5(3), 1–10. http://doi.org/10.1128/mBio.01050-14

Liberles, S. D., Diver, S. T., Austin, D. J., & Schreiber, S. L. (1997). Inducible gene expression and protein translocation using nontoxic ligands identified by a mammalian three-hybrid screen. Proceedings of the National Academy of Sciences of the United States of America, 94(15), 7825–7830. http://doi.org/10.1073/pnas.94.15.7825