Team:Lyon-INSA-ENS/Project/Ethics

A race between two strategies to obtain the Prcn-csgBAEFG

In E. coli the curli-producing system is organized in two divergeant operons with the genes csgA, csgB and csgC on one side and csgD, csgE, csgF and csgG on the other one. That is why, to achieve the first approach of the project, it is necessary to begin by the creation of three "sub-parts": the first one would be the part with the sole promoter PrcnA, the second would be the part with the genes csgA and csgB and final one would be the part with the genes csgE, csgF and csgG.

The synthesis of this first DNA part (the cobalt-inducible promoter Prcn-csgBAEFG, which is designed to induce formation of a biofilm and to promote cell adhesion) was conceived as a competition between two methods: a completely synthetic approach, and a "manual" method involving PCR steps followed by ligations.

• The first approach consist in ordering the whole part at a private company (Genecust) after a computer design.


• The second approach was to made it directly at the bench, this approach included three steps: first the amplification by PCR of each of the sub parts, second a mutagenesis step to remove all the natural internal EcoRI sites located in the sub parts, and finally the ligation of these parts.

Both approches were initiated at the same time, and if the second one allowed us to obtain PCR amplifications of the correct size and a complete Prcn-csgBAEFG construction, unfortunately sequencing analysis revealed unexpected mutations that were not removed before reception of the whole part made by Genecust. Briefly the "click and order" strategy wins the race !