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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 experimental strategy.

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.

Characterization strategy

Tripartit Split-GFP and FRB/FKBP12 dimerization systems

Preliminary we have designed two biobricks to test the FRB*/FKBP12 interaction and the tripartite GFP. FRB* has been fused with one subunit of GFP (GFP 11) and FKBP12 has been fused with another one (GFP10).

In order to test the system we built a plasmid containing three biobricks to express the full system. Then we transform it in E.coli to asess the system.

This construction will give us our first results and validate the functionality of tripartite GFP and dimerization of FRB* and FKBP12.

Assessment of the minimal distance to have fluorescence

One of the goal of our project is to assess the system bring DNA closer tool with the tri-partite GFP. To assess the effect of the bring DNA closer tool, we have to know the minimal distance needed to such fluorescence emission.

This question was also the core of our modeling part which answer the question: “What is the optimal distance between the two dCas9 for fluorescence?”

This question is essential because the distance between the dCas9 may cause major problem. First, the steric hindrance and the dCas9 footprint may avoid the GFP assembling for target sequence too close. Second, the proteins size we have chosen avoid GFP assembling if there are too far away. As a result, fluorescence emission would be detect only if the proteins, as well as, the DNA regions are distant between a precise range of distance.

To assess experimentally such distant, the team has decided to design different plasmids containing the visualization target sequences separate from each other with different distances. To do so, the team has designed specific primers to carry out RT-PCR and obtain from a plasmid in which the target sequences are distant with 1kB, different plasmids. This plasmid would have been express with the composite biobrick composed of the biobricks 3, 4 and 5. The target sequence would have been separate from :

• 1kB
• 500pB
• 150pB
• 75pB
• 50pB

Assessment of the DNA regions brought closer

In order to test our BDC tool, all the biobricks should been express in E. coli, as well as all the sgRNAs corresponding to each dCas9s. After, the team would have measure the GFP fluorescence variations in absence or not of rapalog.

Gene expression tests

In order to test a possible influence of the spatial proximity in gene expression. The team would have test the expression of two different reporter genes. In the aim to have more accurate variation measurements, we should have used enzymes as luciferase and Beta-Galactosidase.

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