Idea

We are committed to constantly improve our detection methods. Therefore, we already thought ahead and came up with an alternative approach for the detection of pathogens. The current method uses the quorum sensing system pathogens and is thus limited to bacteria that secrete autoinducers. Our alternative detection system uses biomolecules tagged with a reporter that bind to the surface of the cell and reveal its presence.

The specific binding of galectin-3 enables the construction of such a detection system. Parts of the lipopolysaccharide structure (LPS) of Pseudomonas aeruginosa can be bound by galectin-3. A fusion protein of galectin-3 and a reporter protein, such as a fluorescent protein, can be built with which Pseudomonas aeruginosa can be detected.

A galectin-3-RFP fusion protein is built and expressed in E. coli. A his-tag and a snap-tag for purification are included. The fusion protein can then be incorporated into a cell-free biosensor system. Such biosensors have many advantages over systems that use living cells; storage, for example, is much easier. From a safety and social acceptance perspective, it is also advantageous if the sensor system does not contain live genetically modified organisms.

To detect P. aeruginosa cells, an agar chip could be used to sample a solid surface. The chip is then be immersed in a detection solution containing the galectin-3-RFP fusion protein. After a washing step, the galectin-3 would remain bound to the pathogen while illumination with 588 nm, the excitation frequency of RFP, in a modified version of our measurement device reveals the location of the cells. The picture taken by the measurement device can then be analyzed by our software Measurarty.

Background

Galectins are proteins of the lectin family, which posess carbonhydrate recognition domains binding specifically to β-galactoside sugar residues. In humans, 10 different galectines have been identified, among which is galectin-3.

Galectin-3 has a size of about 31 kDA and is encoded by a single gene, LGALS3. It has many physiological functions, such as cell adhesion, cell growth and differentiation, and contributes to the development of cancer, inflammation, fibrosis and others.

Human galectin-3 is a protein of the lectin-family that was shown to bind the LPS of multiple human pathogens. Some of them, including pseudomonas aeruginosa protect themselves against the human immune system by mimicking the lipopolysaccharides (LPS) present on human erythrocytes.

By making fusion proteins of galectin-3 with fluorescent reporter proteins, pathogens can be labelled and made visible by fluorescence-microscopy.

Achievements

The galectin-3-YFP fusion protein part was successfully built and transformed into E. coli rosetta. The cells were cultivated in a fermentation during which the fusion protein was expressed. Subsequently, the fusion protein was purified using the binding of the his-tag to a nickel NTA column and a äkta protein purification system.

The fusion protein showed a yellow fluorescence.

References