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The project aim to study how bacterial (e.coli) 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 linking tool and a visualization tool.

Here, we will expose you our experimental strategy, as well as, the biobricks we have designed to do so.

Linking tool construction

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.

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

Then, the team has considered to establish a composite biobrick composed of the three biobricks in the same pSB1C3 plasmid.

Characterization strategy

Tripartit Split-GFP and FRB/FKBP12 dimerization systems

Assessment of the minimal distance to have fluorescence

Assessment of the DNA regions brought closer

Gene expression tests