Team:Aachen/Project/Gal3

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{{Team:Aachen/FigureFloat|Aachen_14-10-09_Pseudomonas_LPS_iNB.png|title=Cell wall composition of ''Pseudomonas aeruginosa''|subtitle=Gram-negative bacteria have two cell membranes. The LPS are embedded in the outer membrane and are composed of a lipid and an O polysaccharide.|width=388px}}
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{{Team:Aachen/FigureFloat|Aachen_14-10-09_Pseudomonas_LPS_iNB.png|title=Cell wall composition of ''Pseudomonas aeruginosa''|subtitle=Gram-negative bacteria have two cell membranes. The LPS are embedded in the outer membrane and are composed of a lipid and an O polysaccharide.|width=385px}}
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. Specifically, the O polysaccharide (see figure on the left) of the LPS is recognized by galectin-3. A fusion protein of galectin-3 and a reporter protein, such as a fluorescent protein, can be built and applied in the detection of ''Pseudomonas aeruginosa''.
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. Specifically, the O polysaccharide (see figure on the left) of the LPS is recognized by galectin-3. A fusion protein of galectin-3 and a reporter protein, such as a fluorescent protein, can be built and applied in the detection of ''Pseudomonas aeruginosa''.

Revision as of 06:35, 14 October 2014

Galectin-3

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 involves biomolecules tagged with a fluorescent reporter that bind to the surface of the cell and reveal its presence.



Aachen 14-10-13 Galectin-3-YFP iNB.png

An Alternative Sensing Molecule

Aachen 14-10-09 Pseudomonas LPS iNB.png
Cell wall composition of Pseudomonas aeruginosa
Gram-negative bacteria have two cell membranes. The LPS are embedded in the outer membrane and are composed of a lipid and an O polysaccharide.

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. Specifically, the O polysaccharide (see figure on the left) of the LPS is recognized by galectin-3. A fusion protein of galectin-3 and a reporter protein, such as a fluorescent protein, can be built and applied in the detection of Pseudomonas aeruginosa.

In our approach, a galectin-3-YFP 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 biosafety and social acceptance perspective, it is also advantageous if the sensor system does not contain live genetically modified organisms.

Aachen 14-10-09 Cell Free Biosensor iNB.png
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To detect P. aeruginosa cells, an agar chip could be used to sample a solid surface. However, other materials but agar can be considered to collect the pathogens. The cell stick to the sampling chip which is then immersed in a detection buffer containing the galectin-3-YFP fusion protein. Excess protein is removed during washing in a suitable buffer. The galectin-3 remains bound to the pathogen and illumination with 514 nm, the excitation frequency of YFP, 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.

Aachen 14-10-13 Galectin-3 iNB.png

Natural Functions of Galectin-3

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.

Aachen 14-10-13 Gal3-YFP Pellet iNB.png

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 protein purification system.

The desired fusion protein showed yellow fluorescence.

Aachen 14-10-04 Expression Pellets iMO.png
Pellets of different fusion proteins
These pellets resulted from the expression of a variety of fusion proteins in uninduced and IPTG-induced cultures.