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 make 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).

Strain Improvement

Make our strain auxotrophic

In order to avoid strain dispersion and problems GMO can generate, we aim to make our strain dependable of some culture conditions. For that purpose, we decided to make our strain auxotrophic that is deleting a gene of amino acid biosynthesis. Thanks to that knock out, the strain would need a culture medium with that amino acid and would die as soon as there is not the amino acid anymore.
In practice, we would have used the "Quick & Easy E. coli Gene Deletion Kit". We would have designed a sequence with an antibiotic resistance gene such as Tetracycline, some features provided by the Kit and an homologous sequence to the gene we want to delete. By recombination, the whole sequence would have been inserted in the gene. And then by excision, we would have removed the resistance to avoid ethical issues.

This is the sequence of the part we wanted to construct :

C G A G C A G G T C A G C T T T G C G C A A G C C G T A A C C C A G G G G T T G G G C A A A A A T C A A T T A A C C C T C A C T A A A G G G C G A T G A A A T C T A A C A A T G C G C T C A T C G T C A T C C T C G G C A C C G T C A C C C T G G A T G C T G T A G G C A T A G G C T T G G T T A T G C C G G T A C T G C C G G G C C T C T T G C G G G A T A T C G T C C A T T C C G A C A G C A T C G C C A G T C A C T A T G G C G T G C T G C T A G C G C T A T A T G C G T T G A T G C A A T T T C T A T G C G C A C C C G T T C T C G G A G C A C T G T C C G A C C G C T T T G G C C G C C G C C C A G T C C T G C T C G C T T C G C T A C T T G G A G C C A C T A T C G A C T A C G C G A T C A T G G C G A C C A C A C C C G T C C T G T G G A T C C T C T A C G C C G G A C G C A T C G T G G C C G G C A T C A C C G G C G C C A C A G G T G C G G T T G C T G G C G C C T A T A T C G C C G A C A T C A C C G A T G G G G A A G A T C G G G C T C G C C A C T T C G G G C T C A T G A G C G C T T G T T T C G G C G T G G G T A T G G T G G C A G G C C C C G T G G C C G G G G G A C T G T T G G G C G C C A T C T C C T T G C A T G C A C C A T T C C T T G C G G C G G C G G T G C T C A A C G G C C T C A A C C T A C T A C T G G G C T G C T T C C T A A T G C A G G A G T C G C A T A A G G G A G A G C G T C G A C C G A T G C C C T T G A G A G C C T T C A A C C C A G T C A G C T C C T T C C G G T G G G C G C G G G G C A T G A C T A T C G T C G C C G C A C T T A T G A C T G T C T T C T T T A T C A T G C A A C T C G T A G G A C A G G T G C C G G C A G C G C T C T G G G T C A T T T T C G G C G A G G A C C G C T T T C G C T G G A G C G C G A C G A T G A T C G G C C T G T C G C T T G C G G T A T T C G G A A T C T T G C A C G C C C T C G C T C A A G C C T T C G T C A C T G G C C C C G C C A C C A A A C G T T T C G G C G A G A A G C A G G C C A T T A T C G C C G G C A T G G C G G C C G A C G C G C T G G G C T A C G T C T T G C T G G C G T T C G C G A C G C G A G G C T G G A T G G C C T T C C C C A T T A T G A T T C T T C T C G C T T C C G G C G G C A T C G G G A T G C C C G C G T T G C A G G C C A T G C T G T C C A G G C A G G T A G A T G A C G A C C A T C A G G G A C A G C T T C A A G G A T C G C T C G C G G C T C T T A C C A G C C T A A C T T C G A T C A T T G G A C C G C T G A T C G T C A C G G C G A T T T A T G C C G C C T C G G C G A G C A C A T G G A A C G G G T T G G C A T G G A T T G T A G G C G C C G C C C T A T A C C T T G T C T G C C T C C C C G C G T T G C G T C G C G G T G C A T G G A G C C G G G C C A C C T C G A C C G A G C C C T A T A G T G A G T C G T A T T A G A G C T G G C A G A A C G T G C T G A T T T A C C C T T G C T T T C A C A T A A T C T G C C C G C C G

Transforming a bacteria (MG1655) using the kit should delete a threonine biosynthesis gene (thrC), making it auxotrophic for threonine : the constructed strain shouldn't be able to grow on medium without threonine.
We designed by adding parts of the thrC gene and the cassettes provided by the kit : beginning of thrC-cassette-tetR-cassette-end of thrC
That would have taken more than a couple of weeks so it remains just a project.

Insert the transporter gene directly in the efflux pomp gene

We could have two problems with our strain :
- the transporter features are located on a plasmid which can be not so stable
- there is a Kanamycine resistance gene in the chromosome which knocks out the efflux pomp gene

There is a unique solution to these two issues : insert the transporter features in the efflux pomp gene.
We would use the same Kit as previously, but we would not use a resistance gene that we would remove afterward, but directly our transporter gene instead. Then we would select the strains that respond to cobalt (Co) concentration.