Tools Design

The project aims to study how bacterial DNA organization can influence gene expression. In order to answer this question, the team decided to create a new tool based on CRISPR/Cas9 technology to bring together two DNA regions separated in space. This new tool is expected to assess the effect of DNA structure on gene expression. We designed a bring DNA closer tool (BDC tool) and a visualization tool.

In this part, we expose our tool design and our experimental strategy here.</p>

Molecular tools

In order to build our two tools, we need specific molecular tools with specific function, as a DNA recognition, a DNA binding function, dimerization and a fluorescence system.

DNA recognition and fixation

The two tools would have to recognize a specific target sequences on DNA and then bind to it. We chosed a based CRISPR/Cas9 recognition system but fir the Cas9 protein, we chosed a endonuclease deficient Cas9s (dCas9). The CRISPR/Cas9 system is a trending molecular tool for which the applications are rising. Our system will allow a new application to it. The team chose dCas9 for three reasons :

• its simplicity : once the tool efficient, it could be set up in few weeks.
• its adaptability : the tool would be adaptable to target any sequences in the genome only through the sgRNA sequence modification.
• its specificity : the use of orthologous dCas9s allow a specific sgRNA/dCas9 association and so a specific target sequence recognition by a specific orthologous dCas9.

Click here to discover all the potential applications and perspective related to our project.

Dimerization system

Our project aims to recognize specific sequences on the genome and then induced a spatial proximity. The team chose an induced dimerization system as FRB/FKBP12 system. In fact, this system allows a dimerization induced by rapamycin. To avoid any problem with rapamycin and E.coli, we decided to used an analog of rapamycin : rapalog (gratefully offered by Takara Clonethec). We then used a FRB mutated (FRB*) to be recognize by rapalog.

Fluorescence system

For the visualization tool, we need a fluorescence system to testify a spatial proximity modification. The team chose the tripartite split-GFP. This GFP is divide in three different parts. Two twenty amino acids parts corresponding to the 10th and 11th beta-sheets and a third parts corresponding to the other beta-sheets of the GFP. This 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 to have an accurate fluorescence.

Bring DNA Closer tool construction

We have designed a tool based on CRISPR/Cas9 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 dCas9s which will not interfere with each other. So we choosed two orthologous dCas9s which come from two different organisms T. denticola (TD) and S. pyogenes (SP). As they come from different organisms they recognize different sgRNAs and do not interfere as we wanted. We order from Addgene the plasmid coding for each one of these dCas9s and their sgRNAs.

To dimerize this two dCas9s we have chosen an inducible system using FRB and FKBP12 proteins. Originally found in mammals 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 gBlocks coding for the FRB* and FKBP12 optimized sequences and a linker in prevision to the fusion with their respective dCas9s.

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 giving us our BDC 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 orthologous 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