Sofía Fraile

I am the lab technician. I have a degree in Biological Sciences from the Autonoma University of Madrid and I have been in the laboratory for over 20 years tuning up multiple techniques of molecular biology. Among other tasks, I keep the stock of laboratory strains plus the new pSEVA collection. “I am the person who knows where all things in the lab”.

In the last time, we are looking for different strategies to create artificial communities of bacteria modifying the adhesive properties of P. putida. This could have an enormous biotechnological potencial.

I created in the lab the nanoPad platform, an important factory in order to produce and select highly specific adhesins. These adhesins are monomeric recombinant antibodies or nanobodies. The nanobodies against different proteins are using as tools to detect and manipulate different biological activities as well as the adhesive properties of P.putida.

These monomeric recombinant antibodies can be packaged into phage M13 to achieve new rounds of panning, secreted into the extracellular medium, expressed intracellularlly or bacteria surface expression, allowing us the binding with other bacteria or solid surfaces.
In order to achieve this goal, we need to generate outer membrane anchoring system with intimins and with autotransporters. Both models can express surface proteins and we have them in the laboratory.

Intimins are 930-residue proteins required for intimate attachment of enterohemorraghic and enteropathognic coli to mammalian cells.
The N-terminal domains are anchored on OM and there are 4 domains exposed on the surface.
We removed 3 of them and changed for a tag (e-tag) for detection in Western-blot and the adhesine (nanobody) in Sfi-Not.

Autotransporters have an structure of Beta domain anchored in the OM with 45KD that expresses out a passenger domain. This can be different sizes.

We use as model the Beta-domain of IgA protease from Neisseria Gonorrea. In our autotransporter tool, we change the passenger domain to the adhesin (nanobody) in Sfi-Not and an e-tag for detection in W.

sofíafraile

Schematic representation of the use of nanobodies as adhesins in P. putida. The OM anchoring system used is intimin. A. Bacteria binding to a substrate or solid surface. B. Adhesión bacteria with other bacteria.
See more in: Fraile S et al. Proteomics. 2013 Oct;13(18-19):2766-75.