Difference between revisions of "Team:Paris Bettencourt/Engineering"

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: LoremIpsum.

Figure 2: LoremIpsum.

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 protein-based 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: LoremIpsum.