Toggle navigation Home Team Team Media Collaborations Sponsors Acknowledgements Project Background Design CRISPR/Cas9 Strategy Experiments Notebook Results Perspective Interlab Study Parts Parts Basic Parts Composite Parts Human Pratices Overview Societal Issues of CRISPR/Cas9 Responsible Research and Innovation GMO regulation Integrated Practices Engagement Model Attributions Safety {{{titre}}} Project Overview Recent observations lead to the idea that genes, not in the same operon but spatially close, are highly co-transcribed, even in the absence of regulatory factors at their promoter regions. alt text The iGEM Paris-Saclay project aims to study the effects of DNA topology on gene expression in E.coli by answering to this question: Does bringing a strong promoter closer to a weak promoter influence the expression level of genes located downstream? alt text We have designed a new tool based on CRISPR/Cas9 system to bring two specific DNA regions closer. This system is composed of two different dCas9 fused with each part of FRB / FKBP12 dimerization system. Each dCas9 will target a specific DNA sequence, one on the chromosome and one on a plasmid, whereas dimerization system will promote the joining of the two dCas9 when rapalog is added. alt text In order to assess whether or not this system works, we have also designed a new tool to visualize the interaction between both dCas9. This tool is composed of a split GFP attached to two dCas9. These two small GFP tags will interact with the complementary GFP detector only if the two dCas9 are closed enough to interact. If we obtain a highest expression level of the weak promoter with our two tools, it could lead to several useful applications. For example, we would be able to use this tool to enhance gene expression of any endogenous genes due to CRISPR/Cas9 specificity. Indeed, it would be possible to design specific sgRNA but user should be aware about off-target activity of the CRISPR/Cas9 system.
Recent observations lead to the idea that genes, not in the same operon but spatially close, are highly co-transcribed, even in the absence of regulatory factors at their promoter regions.
The iGEM Paris-Saclay project aims to study the effects of DNA topology on gene expression in E.coli by answering to this question: Does bringing a strong promoter closer to a weak promoter influence the expression level of genes located downstream?
We have designed a new tool based on CRISPR/Cas9 system to bring two specific DNA regions closer. This system is composed of two different dCas9 fused with each part of FRB / FKBP12 dimerization system. Each dCas9 will target a specific DNA sequence, one on the chromosome and one on a plasmid, whereas dimerization system will promote the joining of the two dCas9 when rapalog is added.
In order to assess whether or not this system works, we have also designed a new tool to visualize the interaction between both dCas9. This tool is composed of a split GFP attached to two dCas9. These two small GFP tags will interact with the complementary GFP detector only if the two dCas9 are closed enough to interact.
If we obtain a highest expression level of the weak promoter with our two tools, it could lead to several useful applications. For example, we would be able to use this tool to enhance gene expression of any endogenous genes due to CRISPR/Cas9 specificity. Indeed, it would be possible to design specific sgRNA but user should be aware about off-target activity of the CRISPR/Cas9 system.
