Project description

The iGEM Paris-Saclay project is part of the Foundational Advance track, and aims to study the effects of DNA topology on gene expression in E. coli. The purpose is to answer this question: Does bringing a strong promoter closer to a weak promoter influences the expression level of genes located downstream?

We have designed a new tool based on the CRISPR/Cas9 system to bring two specific DNA regions closer. This system is composed of two different dCas9 proteins fused with each part of the FRB / FKBP12 dimerization system. Each dCas9 will target a specific DNA sequence, for example one on a chromosome area and another one on another chromosome area. The dimerization system will promote the joining of the two dCas9s when rapalog is added in the medium. In order to assess whether or not this system works, we have also designed a new tool to visualize the interaction between both dCas9s. This tool is composed of a split GFP attached to two dCas9s. These two small GFP tags will interact with the complementary GFP detector only if the two dCas9s are close enough to interact.

To learn more about our goal:

Guideline of the project

When we set up our project, we knew it would be tough to obtain our final tools. That is why we organized our work to begin with intermediate devices.

In the laboratory, we focused on the tool used to visualize the interaction between both dCas9s. For that purpose, we designed a biobrick in order to characterize the assembly of the split GFP. This biobrick is composed of one part of the FRB / FKBP12 system fused to the other part of the tripartite split-GFP system (GFP 10 / GFP 11) plus GFP 1.9 in the same plasmid.

Furthermore, a model was built in order to determine the optimal distance between the two dCas9s proteins for the GFP to fluoresce. This model was based on two devices: the pSB1C3 plasmid composed of the tripatrite split GFP plus two dCas9s and another plasmid composed of the two target sequences of the dCas9 and the two sgRNAs coding sequences. For this second plasmid, we wanted to test several distances (50 bp, 75 bp, 100 bp and 150 bp) between the two target sequences of the dCas9s, in order to determine the best distance for the tripartite split GFP to fluoresce.

Perspective

In this project we design a tool to study the relation between DNA structure and gene expression. We propose many applications of our tool in genome study and in industry. Using dCas9 in this system gives us the advantage of specifically targeting the desired sequence and changing DNA structure. Indeed, it would be possible to design specific sgRNA to target specific sequences.

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

Achievements

Bronze

1. Register for iGEM, have a great summer, and attend the Giant Jamboree.
2. Meet all deliverables on the Requirements page.
3. Create a page on your team wiki with clear attribution of each aspect of your project.
4. Document at least one new standard BioBrick Part or Device central to your project and submit this part to the iGEM Registry (submissions must adhere to the iGEM Registry guidelines).

Silver

1. Experimentally validate that at least [http://parts.igem.org/Part:BBa_K2039000 one new BioBrick Part] or Device of your own design and construction works as expected. Document the characterization of this part in the Main Page section of that Part’s/Device’s Registry entry. Submit this new part to the iGEM Parts Registry. This working part must be different from the part documented in bronze medal criterion. [IN PROGRESS]
2. Convince the judges you have helped any registered iGEM team from high school, a different track, another university, or another institution in a significant way.
3. iGEM projects involve important questions beyond the lab bench, for example relating to (but not limited to) ethics, sustainability, social justice, safety, security, and intellectual property rights. Demonstrate how your team has identified, investigated, and addressed one or more of these issues in the context of your project.

Gold

1. Human Practice: expand on your silver medal activity by demonstrating how you have integrated the investigated issues into the design and/or execution of your project.
2. Improve the function OR characterization of an existing BioBrick Part or Device and enter this information in the Registry. Please see the Registry help page on how to document a contribution to an existing part.
3. Demonstrate a functional proof of concept of your project. Your proof of concept must consist of a BioBrick device; a single BioBrick part cannot constitute a proof of concept. (biological materials may not be taken outside the lab).
4. Show your project working under real-world conditions. To achieve this criterion, you should demonstrate your whole system, or a functional proof of concept working under simulated conditions in the lab (biological materials may not be taken outside the lab).