Team:Aachen/Project/Gal3

From 2014.igem.org

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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=420px}}
{{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=420px}}
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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''.
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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''.
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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 [https://2014.igem.org/Team:Aachen/Safety biosafety] and social acceptance perspective, it is also advantageous if the sensor system does not contain live genetically modified organisms.
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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 [https://2014.igem.org/Team:Aachen/Safety biosafety] and social acceptance perspective, it is also advantageous if the sensor system does not contain live genetically modified organisms.
{{Team:Aachen/FigureFloatRight|align=center|Aachen_14-10-09_Cell_Free_Biosensor_iNB.png|width=500px}}
{{Team:Aachen/FigureFloatRight|align=center|Aachen_14-10-09_Cell_Free_Biosensor_iNB.png|width=500px}}
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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''.
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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''.
{{Team:Aachen/BlockSeparator}}
{{Team:Aachen/BlockSeparator}}
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<span class="anchor" id="naturalfunctions"></span>
<span class="anchor" id="naturalfunctions"></span>
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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.  
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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&nbsp;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.
Galectin-3 has a size of about 31&nbsp;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.
Human galectin-3 is a protein of the lectin-family that was shown to bind the LPS of multiple human pathogens.
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Some of them, including ''pseudomonas aeruginosa'' protect themselves against the human immune system by mimicking the lipopolysaccharides (LPS) present on human erythrocytes.  
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Some of them, including ''Pseudomonas&nbsp;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.
By making fusion proteins of galectin-3 with fluorescent reporter proteins, pathogens can be labelled and made visible by fluorescence-microscopy.
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<span class="anchor" id="gal3achievements"></span>
<span class="anchor" id="gal3achievements"></span>
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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.
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Due to the generous support of Sophia Böcker and Prof.&nbsp;Elling of the Helmholtz Institute for Biomedical Engineering, we got access to an pET17-derived expression plasmid for a His- and SNAP-tagged YFP-galectin-3 fusion protein. We transformed the fusion protein into ''E.&nbsp;coli''&nbsp;Rosetta cells and conducted a batch fermentation to obtain large amounts of protein (FIGURE).
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With the help of David Schönauer and Alan Mertens from the Institute for Biotechnolgy we then purified the fusion protein using by FPLC.
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 +
 
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We attempted to do an experiment to test the binding of the Gal-3 fusion protein to the LPS of [TARGET_ORGANISMS]  as shown previously [LITERATURE-REFERENCE], but we were unable to obtain useful results because our fluorescence microscope was not sensitive enough.
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After we received the collection of pSBX-expression vectors from Team Heidelberg, we used Gibson assembly to make K1319020 from K1319003 and pSBX1A3, which is the translational unit for a mRFP-Gal3 fusion protein with a C-terminal 6xHis tag:
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<center>{{Team:Aachen/Figure|File:Aachen_K1319020.png|width=500px|title=K1319009|This BioBrick is a construction intermediate of K1319003 (gal3), E1010 (mRFP), K1319007 (6xHis tag) to K1319020 (translational unit of the fusion protein).}}</center>
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We also cloned our K1319003 into the pET17 expression vector and expressed all combinations of fusion proteins in E.&nbsp;coli&nbsp;BL21(DE3). A SDS-PAGE showed that all fusion proteins were fully translated:
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{{Team:Aachen/FigureFloat|File:Aachen_Gal3_Expression.png|width=350px|title=SDS-PAGE of K1319020 expression|subtitle=The fusion protein was fully translated to the correct molecular mass of 74&nbsp;kDa.}}
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The desired fusion protein showed yellow fluorescence.
 
<center>
<center>

Revision as of 15:41, 16 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
'



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 14-10-15 Medal Cellocks iNB.png

Achievements

Due to the generous support of Sophia Böcker and Prof. Elling of the Helmholtz Institute for Biomedical Engineering, we got access to an pET17-derived expression plasmid for a His- and SNAP-tagged YFP-galectin-3 fusion protein. We transformed the fusion protein into E. coli Rosetta cells and conducted a batch fermentation to obtain large amounts of protein (FIGURE).


With the help of David Schönauer and Alan Mertens from the Institute for Biotechnolgy we then purified the fusion protein using by FPLC.


We attempted to do an experiment to test the binding of the Gal-3 fusion protein to the LPS of [TARGET_ORGANISMS] as shown previously [LITERATURE-REFERENCE], but we were unable to obtain useful results because our fluorescence microscope was not sensitive enough.

After we received the collection of pSBX-expression vectors from Team Heidelberg, we used Gibson assembly to make K1319020 from K1319003 and pSBX1A3, which is the translational unit for a mRFP-Gal3 fusion protein with a C-terminal 6xHis tag:

500px
K1319009

We also cloned our K1319003 into the pET17 expression vector and expressed all combinations of fusion proteins in E. coli BL21(DE3). A SDS-PAGE showed that all fusion proteins were fully translated:

350px
SDS-PAGE of K1319020 expression
The fusion protein was fully translated to the correct molecular mass of 74 kDa.


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.