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 divergent operons with the structural genes csgA, csgB and csgC on one side and csgD (regulation), csgE, csgF and csgG (secretion) on the other one. The sought-after property, adherence via curli, can be boosted by two different way: creating an independent curli synthesis pathway (first option), or activating the existing cryptic curli synthetis pathway which can be found in most laboratory E. coli strains (second option).

To achieve the first option consisting in the creation of a single independent curli operon, we tried two methods in parallel: a completely synthetic approach, and a classic method involving mutagenesis to get rid of three disturbing internal iGEM restriction sites, and PCR steps followed by ligations.

• The first approach consists in carefully designing the part from in silico data, and ordering the whole part at a private company (Genecust).


• 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 or PstI sites located in the sub parts, and finally the ligation of these parts.

Both approaches 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.

The "Design, click and order" strategy wins the race !

In the second option, we used two available biobricks to activate the curli pathway via the top of the regulatory cascade: cloning of the superactivator ompR234 gene under control of the constitutive promoter BBa_J23119 should allow to activate the main curli activator CsgD (See diagram).