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
Tripartit Split-GFP and FRB-FKBP12 dimerization systems
Preliminary we have designed two biobricks to test the FRB*/FKBP12 interaction and the tripartite GFP system. FRB* was fused with one subunit of GFP (GFP 11) and FKBP12 was fused with another one (GFP10). Then, we also put the GFP 1.9 gene in pSB1C3 to form the tripartite.
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 asess the system. The system would be tested by measuring GFP fluorescence with rapalog and without (rapamycin analog). 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 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 BDC tool with the tripartite split-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 dCas9s for 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 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.
