We designed a bring DNA closer tool (BDC tool) and a visualization tool, as mentioned there. In order to characterize our tools, we set up an experimental strategy exposed bellow.
Characterization strategy
Tripartite Split-GFP and FRB-FKBP12 dimerization systems
Preliminary, 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). Then, we also put the gene endoing the last subunit (GFP 1-9) in the plasmid pSB1C3 to form the tripartite GFP.
In order to test the system, we built a plasmid containing three biobricks to express the full system. 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: 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).
This construction would give give us our first results and validate the functionality of the tripartite GFP and check the 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 BDC tool with 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.
This question was also the core of our model, which answers the question: What is the optimal distance between the two dCas9s to observe fluorescence?
This question is essential because the distance between the dCas9 may cause major problems. First, the steric hindrance and the dCas9 footprint may avoid the GFP assembling if we target sequences that are too close. Secondly, the protein sizes we chose avoid GFP 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 distant between a precise range of distances.
To assess experimentally such distance, we decided to design different plasmids containing the visualization target sequences separated from each other by different number of base pairs. 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 numer of base pairs between the target sequences is reduced.
This plasmid would have been expressed with the plasmid pSB1C3 containing the BioBricks 3, 4 and 5.
The target sequences would have been separated by:
- 1kB
- 500pB
- 150pB
- 75pB
- 50pB
Assessment of the DNA regions brought closer
In order to test our BDC tool, all the biobricks should been expressed in E. coli, as well as all the sgRNAs corresponding to each dCas9s. After, the team would measure GFP fluorescence variations with and without rapalog.
Gene expression tests
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 as luciferase and Beta-Galactosidase for instance.
