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| − | {{Team:Paris_Saclay/project_header}} | + | {{Team:Paris_Saclay/project_header|titre=Experimental Strategy}} |
| | + | <html><style>header{background-image: url("https://static.igem.org/mediawiki/2016/8/8f/T--Paris_Saclay--banniere_project.jpg");} |
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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 bring DNA closer tool and a visualization tool.
| + | We designed a bring DNA closer tool (BDC tool) and a visualization tool, as mentioned [[Team:Paris_Saclay/Design#design|perviously]]. In order to characterize our tools, we set up the experimental strategy explained bellow. |
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| − | Here, we will expose you our experimental strategy, as well as, the biobricks we have designed to do so.
| + | =Characterization strategy= |
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| − | =Bring DNA Closer tool construction= | + | ==Tripartite Split-GFP and FRB*-FKBP12 dimerization systems== |
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| − | '''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. ''' | + | First, we designed two biobricks to test the FRB*/FKBP12 interaction and the tripartite GFP system. FRB* was fused with one subunit of the gene encoding the GFP (GFP 11) and FKBP12 was fused with another subunit (GFP10) '''[Fig1]'''. Then, we also put the gene encoding the last subunit (GFP 1-9) in the plasmid pSB1C3 to form the tripartite GFP '''[Fig2]'''. |
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| − | 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.
| + | [[Image:Paris_Saclay--design4.png|frameless|upright=2.5|center|]] |
| | + | <center>'''Figure 1''': Tripartite split-GFP and FRB*/FKBP12 functional assessment. ''Biobricks corresponding to intermediate characterization. FRB* is fused with GFP11 and FKBP12 is fused with GFP10.''</center> |
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| − | [[Image:Image1design.jpg|frameless|center|upright=3|alt=dCas9 mecanism]]
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| − | 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)
| + | In order to test the system, we built a plasmid containing three biobricks to express the full system '''[Fig2]'''. Then we transformed it in ''E. coli'' to assess the system. The system would be tested by measuring GFP fluorescence level under two different conditions '''[Fig1]''': a growth medium containing rapalog (rapamycin analog) and a growth medium without it. We also planed to test it in bacteria containing just two parts (FRB-GFP11 and FKBP12-GFP10) instead of three (so without GFP1-9). |
| − | 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)
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| − | 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.
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| − | <br>Using these two systems (dCas9 recognition and FRB/FKBP12 dimerization) we design our new tool based on the two following biobricks :
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| − | [[Image:Image2design.jpg|frameless|center|upright=3|alt=dCas9 mecanism]] | + | [[Image:Paris_Saclay--design5.png|frameless|upright=2.5|center|]] |
| | + | <center>'''Figure 2''': Biobrick design containing all the parts to characterize tripartite split-GFP and FRB*/FKBP12. ''All our biobricks in one plasmid and no interference.''</center> |
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| − | These two biobricks will be assembled in pSB1C3 plasmid give us our get DNA closer tool which will function as bellow :
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| − | [[Image:Image3design.jpg|frameless|center|upright=2|alt=dCas9 mecanism]]
| + | This construction would give us the first results, validate the functionality of the tripartite GFP and the dimerization of FRB* and FKBP12. |
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| − | =Visualization tool construction= | + | ==Assessment of the minimal distance to have fluorescence== |
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| − | 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.
| + | One of the goals of our project is to assess the system composed of the BDC tool and the tripartite split-GFP. To evaluate the effect of the bring DNA closer tool, we have to know the minimal distance needed to observe fluorescence emission. |
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| − | To build this new tool, two other ortholog nuclease function deficient Cas9s (dCas9s) : N.meningitidis and S.thermophilus, will be fused to fluorescent proteins.
| + | This question was also the core of our [[Team:Paris_Saclay/Model#modelisation|model]], which answers the question: ''What is the optimal distance between the two dCas9s to observe fluorescence?'' |
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| − | We also decide to use dCas9 system for this tool in order to have detection of a accurate and unique sequence in the genome.
| + | This question is essential because the distance between the dCas9s may cause major problems. First, steric hindrance and dCas9 footprint may avoid GFP assembling if we target sequences that are too close. Secondly, the proteins size could prevent the GFP parts from assembling if they are too far away. As a result, fluorescence emission would be detected only if the proteins, as well as the DNA regions, are at a precise range of distance. |
| − | It will be fulfilling with a new and unreleased in the iGEM competition tripartite slip-GFP.
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| − | 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.
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| − | 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.
| + | To experimentally assess such distance, we decided to design different plasmids containing the visualization target sequences separated from each other by different number of base pairs '''[Fig3]'''. To do so, we designed specific primers to carry out reverse PCR and obtain, from a plasmid in which the target sequences are distant by 1kB, different plasmids where the number of base pairs between the target sequences is reduced. |
| | + | This plasmid was thought to be expressed with the plasmid pSB1C3 containing the BioBricks 3, 4 and 5 (cf [[Team:Paris_Saclay/Design#design|design]] page). |
| | + | The target sequences would have been separated by: |
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| − | The team has designed three biobricks to achieve this part of the project.
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| − | * '''Biobrick n°3 :''' ''N.meningitidis'' fused to GFP-10 expressed by a constitutive promoter, a RBS and a double terminator
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| − | * '''Biobrick n°4 :''' ''S.thermophilus'' fused to GFP-11 expressed by a constitutive promoter, a RBS and a double terminator
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| − | * '''Biobrick n°5 :''' The third part of the GFP 1-9 expressed by a constitutive promoter, a RBS and a double terminator
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| − | The biobricks were inserted into pSB1C3 using the iGEM process : restriction sites EcoRI and PstI.
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| − | [[File:T--Paris_Saclay--visualization_biobricks.jpeg|frameless|center|upright=2.5|]]
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| − | 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.
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| − | [[File:T--Paris_Saclay--composite_visualization_biobrick.jpeg|upright=2.5|frameless|center|]]
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| − | [[File:T--Paris_Saclay--visualization2.jpeg|frameless|center|upright=2.5|]]
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| − | 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).
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| − | The team has chosen to express the sgRNAs on another plasmid pZA11 which is compatible with pSB1C3.
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| − | [[File:T--Paris Saclay--visualization sgRNA.jpeg|upright=2.5|frameless|center|]]
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| − | =Characterization strategy=
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| − | =====Tripartit Split-GFP and FRB/FKBP12 dimerization systems=====
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| − | <br>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).
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| − | <br>
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| − | [[Image:Image4design.jpg|frameless|upright=3|center|]]
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| − | 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.
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| − | [[Image:Image5design.jpg|frameless|upright=3|center|]]
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| − | This construction will give us our first results and validate the functionality of tripartite GFP and dimerization of FRB* and FKBP12.
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| − | =====Assessment of the minimal distance to have fluorescence=====
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| − | One of the goal of our project is to assess the system bring DNA closer tool with the tri-partite GFP.
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| − | To assess the effect of the bring DNA closer tool, we have to know the minimal distance needed to such fluorescence emission.
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| − | This question was also the core of our [[Team:Paris_Saclay/Model#modelisation|modeling]] part which answer the question “what is the optimal distance between the two dCas9 for fluorescence?”
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| − | 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.
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| − | 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.
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| − | This plasmid would have been express with the composite biobrick composed of the biobricks 3, 4 and 5.
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| − | The target sequence would have been separate from :
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| | *1kB | | *1kB |
| − | *500pB | + | *500bp |
| − | *150pB | + | *150bp |
| − | *75pB | + | *75bp |
| − | *50pB | + | *50bp |
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| − | [[File:T--Paris_Saclay--distance_assessment.jpeg|upright=3|frameless|center|]]
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| − | =====Assessment of the DNA regions brought closer===== | + | [[File:T--Paris_Saclay--distance_assessment.jpeg|upright=2.7|frameless|center|]] |
| | + | <center>'''Figure 3''': Plasmid design to assess the minimal distance needed to have GFP fluorescence and fully characterize the tripartite split-GFP. ''It would be expressed with the visualization tool BioBrick. The different RT-PCRs would allow us to have different distances between the two target sequences.''</center> |
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| − | =====Gene expression tests===== | + | ==Assessment of the DNA regions brought closer== |
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| | + | In order to test our BDC tool, all the biobricks previously quoted in the [[Team:Paris_Saclay/Design#design|design]] page should be expressed in ''E. coli'', as well as all the sgRNAs (corresponding to the target sequences and their cognate dCas9s). Later, the team would measure GFP fluorescence levels in growth medium with or without rapalog. The emission of any fluorescence by the tripartite split-GFP would validate our BDC tool '''[Fig4]''' as it would mean the two target sequences are close. |
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| | + | [[File:T--Paris_Saclay--BDCtool_characterization.jpeg|frameless|center|upright=2.5|]] |
| | + | [[File:T--Paris_Saclay--BDCtool_characterization_continuation.jpeg|frameless|center|upright=2.5|]] |
| | + | <center>'''Figure 4''': BDC tool characterization by the visualization tool mechanism. ''Using both tools together.''</center> |
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| − | =References= | + | =Gene expression tests= |
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| − | 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
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| − | 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
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| − | 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
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| | + | In order to test a possible influence of the spatial proximity in gene expression, we would test the expression of two different reporter genes. With the aim of having more accurate variation measurements, we should use enzymes such as luciferase or beta-galactosidase. |
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| − | {{Team:Paris_Saclay/footer}} | + | {{Team:Paris_Saclay/project_footer}} |
