Team:Paris Bettencourt/Engineering

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EpiFlex, a MoClo Toolkit for S. epidermidis

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 for S.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).

Figure 1. Concept of MoClo : All the parts individually stored in p0 are assembled in a TU stored in a P1 using Golden Gate assembly. In the same way, several TUs of different p1 can be assembled in a p2 (inset). Blue tint squares and green tint squares are respectively level 1 fusion sites allowing assembly the p1 and level 2 fusion sites for assembly in the p2.

Figure 2. Design of the backbones and parts : A. Design of the backbone of the plasmid of level 0 (p0), level 1 (p1), level 2 (p2). Grey tint squares are level 0 fusion sites (FSp0), blue tint squares are level 1 fusion sites (FSp1) and green tint squares are level 2 fusion sites. Pink tint and blue antibiotic resistance genes are respectively E. coli and S. epidermis ones B. Design of a part to be assembled in the p0 backbone using Golden Gate assembly. Here is an exemple of a part with the fusion sites FSp1_A and FSp1_B, so the part is designed to be in the 1st position in the p1 TU. These fusion sites of p1 (in blue) has to be modified in function of the position of the part in the TU.

Plasmids and Parts Design

The p0, p1 and p2 plasmids contain ampicillin, kanamycin and chloramphenicol resistance genes respectively (Fig.2) for selection in E. coli.

Each part is designed by flanking the ends with level 1 fusion sites (depending on its relative position inside the TU), and BsaI restriction sites. This allows the assembly of multiple parts in the p1 backbone. These sequences (Part + level 1 fusion site + BsaI recognition site) are also surrounded on both sides by p0_fusion sites and BbsI restriction sites allowing to insert the part into the p0 backbone.

The p0 backbone has a GFP dropout cassette in between the the pairs of p0 fusion sites and BbsI restriction sites allowing us to verify the insertion of the part. The removal of the GFP cassette is a visual indicator of successful transformants, as the lack of a green suggests successful interruption of the cassette.

In the same way, the p1 backbones 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 has a level 2 fusion site and BbsI restriction site allowing the assembly of different TUs in the p2 backbone following the same principle as the TU assembly.

We chose a fluorescent reporter as a cloning selection marker to avoid the extra cost of reagents needed in traditional screening methods such as blue-white screening which requires X-gal.

An important feature of our p1 and p2 backbones is the fact that they're E. coli -> S. epidermidis shuttle vectors. This means that they contain origins of replication for both E. coli and S. epidermidis as well antibiotic resistance selection for each microbe. This is important for a two step cloning workflow as E. coli is an easy host for cloning and plasmid amplification before seeing the performance of the cassette in S. epidermidis. The difficulty of cloning into S. epidermidis is further illustrated in project EpiGlow page here.

Figure 3. Scheme of EpiFlex part design map : Each DNA part can be designed with terminal fusion sites that correspond to a particular position within a TU, with a total of 7 possible positions as the kit currently stands.

Table 1 shows the nomenclature of the parts and the backbones in the MoClo toolkit for now. Each part is added to the toolkit after being designed to be EpiFlex compatible, should follow the same nomencalture.

For now one p1 has been successfully assembled using the EpiFlex toolkit, with a TU coding for the mCherry protein. This plasmid has been characterized and this is part of the EpiGlow project which is a proof of concept of EpiFlex.You'll find more information about this project on the EpiGlow page here.

Figure 4. Nomenclature and plasmids assembled : A. Nomenclature of the plasmids. pPBEF : plasmid Paris Bettencourt EpiFlex. FS : Fusion Site. B. As an example, a table of the plasmids designed and assembled to create a TU coding for mCherry used in the EpiGrow Project.