Difference between revisions of "Team:Paris Bettencourt/Engineering"
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<p><i>The concept of the MoClo toolkit</i></p> | <p><i>The concept of the MoClo toolkit</i></p> | ||
| − | <p>A MoClo is a modular | + | <p>A MoClo kit is a modular cloning concept based on Golden Gate assembly. EpiFlex is a Moclo toolikit developed specifically for parts that function in <i>S.epidermidis</i></p> |
| − | <p>The aim of this toolkit is to allow cloning of | + | <p>The aim of this toolkit is to allow the cloning of transcriptional units (TU) in a direct and rapid fashion. It also allows efficient construction of many different TUs or even genetic circuits by assembling various combinations of parts or TUs which have standard fusion sites.</p> |
| − | <p>The concept of | + | <p>The concept of developing a kit is to furnish a set of parts, such as a promoter, RBS, CDS, or terminator, all stored in plasmids.A backbone receiving the differents parts of TUs to allows scientists to assemble their own genes choosing the parts they want like pieces of a puzzle.</p> |
| − | <p>The plasmid | + | <p>The plasmid containing individual parts is called a level 0 plasmid (p0). The plasmids containing different parts assembled creating a TU are called level 1 plasmids (p1), and finally the level 2 plasmids (p2) are plasmids containing several TUs to create a genetic circuit or metabolic pathway (Fig.1).</p> |
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Revision as of 20:59, 27 October 2020
Description
The little history of EpiFlex genesis...
After investigating the skin microbiome composition with the Quaranskin study, the iGEM Paris Bettencourt team aspired to engineer the microbiome. The final outlook being, the ability to control populations dynamics of the skin microbiome in order to maintain its equilibrium and avoid dysbiosis induced pathologies.This is where we envision the role of synthetic biology in the probiotic arena.
To pursue this, we focused on a commensal bacteria of the skin microbiome to use as a chassis for synthetic biology: Staphylococcus epidermidis. On this quest we discovered that this bacterium has few tools available for efficient expression of recombinant DNA and genetic engineering. This generated the idea to develop a MoClo toolkit forS.epidermidis, the EpiFlex toolkit. Here we hope to establish a robust and useful set of genetic parts for standardized rational design of genetic circuits within this cool microbe for the staphylococcus and synthetic biology community alike.
The concept of the MoClo toolkit
A MoClo kit is a modular cloning concept based on Golden Gate assembly. EpiFlex is a Moclo toolikit developed specifically for parts that function in S.epidermidis
The aim of this toolkit is to allow the cloning of transcriptional units (TU) in a direct and rapid fashion. It also allows efficient construction of many different TUs or even genetic circuits by assembling various combinations of parts or TUs which have standard fusion sites.
The concept of developing a kit is to furnish a set of parts, such as a promoter, RBS, CDS, or terminator, all stored in plasmids.A backbone receiving the differents parts of TUs to allows scientists to assemble their own genes choosing the parts they want like pieces of a puzzle.
The plasmid containing individual parts is called a level 0 plasmid (p0). The plasmids containing different parts assembled creating a TU are called level 1 plasmids (p1), and finally the level 2 plasmids (p2) are plasmids containing several TUs to create a genetic circuit or metabolic pathway (Fig.1).
The p0, p1 and p2 plamsmids are containing respectively Ampicilin, Kanamycin and chloramphenicol resistance genes
Each part is designed adding both sides fusion sites (depending on the position inside the TU it whill be) and BsaI restriction sites allowing to be assembled with other part in the p1 backbone. These sequences (Part + Fusion site + BsaI recognition site) also present on both sides p0_fusion sites and BbsI restriction sites allowing to insert the part in the p0 backbone. The p0 backbone has a GFP cassette in between the two The p1 backbones has on both side of the pairs of p0_fusion sites and BbsI restriction sites allowing us to verify the insertion of the part, and so the removal of the GFP cassette by seeing the color of the colony containg the plamsid after transofrmation. In the same way, the p1 backbone are designed with a GFP cassette in between pairs of A/H fusion sites and BbsI restriction site needed to link to the first and the last part of the TU. It also presents level 2 fusion site and BbsI restriction site allowing to assemble different TUs in the p2 backbone following the same principle as the TU assembly.
On figure 3.a are the list of all the part and backbone available in our kit. For now X p1 has been assembled, the TUs correspondants are shown in figure 3.b.
The p1 has been caracterized and is part of our EpiGlow project. More information on the EpiGlow as an EpiFlex Proof of Concept can be found here