Difference between revisions of "Team:Freiburg/Project/Overview"

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The DiaCHIP is an innovative tool to screen for a broad range of antibodies present in serum. Antibodies can be an indicator for  an immune response towards an infection or a succesful vaccination. Antibodies also play a role in the diagnosis of autoimmune diseases. Especially the ability to differentiate between life threatening diseases and a mild infection within minutes bears the potential to save lifes.   
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The DiaCHIP is an innovative tool to screen for a broad range of antibodies present in serum. Antibodies can be an indicator for  an immune response towards an infection or a succesfull vaccination. Antibodies also play a role in the diagnosis of autoimmune diseases. Especially the ability to differentiate between life threatening diseases and a mild infection within minutes bears the potential to save lifes.   
 
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Spotting diseases by detecting correspondent antibodies in a patient's serum is an established method in  <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Diagnostics" title="diagnostics_today">modern diagnostics</a>. The DiaCHIP makes this possible using a single blood sample.
 
Spotting diseases by detecting correspondent antibodies in a patient's serum is an established method in  <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Diagnostics" title="diagnostics_today">modern diagnostics</a>. The DiaCHIP makes this possible using a single blood sample.
 
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The key feature in the concept of the DiaCHIP is the combination of on-demand protein synthesis and an innovative, label-free detection method in one device. This enables to overcome challenges in storage and handling that occur with currently available tests. Additionally, diagnoses can be received faster and in a more cost-efficient way than until nowadays. Nonetheless, the whole device is so simple that it can easily be rebuilt and utilized by future iGEM teams (LINK – new device).
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The key feature of the DiaCHIP concept is the combination of on-demand protein synthesis and a novel method of label-free detection packed in one device. The idea is to overcome challenges commonly found in protein array production and preservation. In addition results can be obtained in a time- and cost-efficient manner; with a device simple enough to be rebuilt by future iGEM teams(LINK – new device).
  
 
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Before being able to screen for antibody-antigen interactions, the antigens have to be synthesized and immobilized in a microarray arrangement. This is obtained by a copying mechanism converting a DNA template into a protein microarray by cell-free protein expression. This expression system based on a bacterial lysate prevents the need for genetically engineered organisms to produce every single antigen.  
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Pior to screening for antibody-antigen interactions, the antigens have to be synthesized and immobilized in a microarray set-up. Facilitated by a copy mechanism, which converts a DNA template into a protein microarray by cell-free protein expression. This expression system based on a bacterial lysate makes the need for genetically engineered organisms to produce each single antigen redudant.
 
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To obtain the DNA template, the respective sequences containing transcriptional and translational initiation sites, the antigen coding sequence and terminating regions have to be constructed and labeled with an amino group. An activated PDMS slide provides the basis for immobilization of the DNA by covalent binding of the amino group. Spotting the antigen coding sequences in a distinct pattern enables to retrace a detected binding event to a certain disease.
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In order to collect the DNA templates, the respective sequences containing transcriptional and translational initiation sites, the antigen coding sequence and terminating regions have to be constructed and labeled with an amino group. An activated PDMS slide provides the basis for immobilization of the DNA by covalent binding of the amino group. Spotting the antigen coding sequences in a distinct pattern enables to retrace a detected binding event to a certain disease.
 
The template slide is placed in close proximity to the future protein array enabling the expressed proteins to reach this other surface by diffusion. Complex chemistry ensures that target proteins are specifically immobilized on this surface, while components of the expression mix can be washed away before sample analysis.
 
The template slide is placed in close proximity to the future protein array enabling the expressed proteins to reach this other surface by diffusion. Complex chemistry ensures that target proteins are specifically immobilized on this surface, while components of the expression mix can be washed away before sample analysis.
  

Revision as of 17:09, 9 September 2015

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  • Building our own device

    The device orginally used, in collaboration with AG Roth, was a pricy machine based on rather simple physics. Therefore, we decided to build our own apparatus in a cost-efficient manner. We were able to produce reliable results with it and provided a construction plan. This plan will make it possible for future iGEM generations to built and use their own label-free protein array analysis tool.

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  • Communicating science

    Diagnosing diseases fast and reliable has not just been an issue among medical staff, it has also been subject to public interest. This has lead us to ask for people's opinions regarding the DiaCHIP. Although the device is labeled as a product of synthetic biology, which has been problematic for the broad public according to a survey initiated by the Leopoldina (National academy of science), we recieved lot of positive feedback.

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  • Modeling cellfree expression

    In order to optimize the DiaCHIP for future applications, we optimized the process of cell-free expression and diffusion over time. Making use of xxx parameters and xxx ordinary differential equations, we computed the size of the resulting antigen spots and identified the factors limiting cell-free expression in the DiaCHIP.

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  • Measuring our own blood

    One of the most promising results came from the detection of anti-tetanus antibodies in human blood serum. The DiaCHIP analysis made it possible for us to distinguish serum samples from an individual before and after vaccination. Samples taken three weeks after vaccination produced positive signals, compared to negative results prior to antigen exposure.

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Project overview: The DiaCHIP

DiaCHIP_Sabi

The DiaCHIP is an innovative tool to screen for a broad range of antibodies present in serum. Antibodies can be an indicator for an immune response towards an infection or a succesfull vaccination. Antibodies also play a role in the diagnosis of autoimmune diseases. Especially the ability to differentiate between life threatening diseases and a mild infection within minutes bears the potential to save lifes.
Spotting diseases by detecting correspondent antibodies in a patient's serum is an established method in modern diagnostics. The DiaCHIP makes this possible using a single blood sample.
The key feature of the DiaCHIP concept is the combination of on-demand protein synthesis and a novel method of label-free detection packed in one device. The idea is to overcome challenges commonly found in protein array production and preservation. In addition results can be obtained in a time- and cost-efficient manner; with a device simple enough to be rebuilt by future iGEM teams. (LINK – new device).

Step 1: Preparing the DiaCHIP by protein synthesis
Pior to screening for antibody-antigen interactions, the antigens have to be synthesized and immobilized in a microarray set-up. Facilitated by a copy mechanism, which converts a DNA template into a protein microarray by cell-free protein expression. This expression system based on a bacterial lysate makes the need for genetically engineered organisms to produce each single antigen redudant.
In order to collect the DNA templates, the respective sequences containing transcriptional and translational initiation sites, the antigen coding sequence and terminating regions have to be constructed and labeled with an amino group. An activated PDMS slide provides the basis for immobilization of the DNA by covalent binding of the amino group. Spotting the antigen coding sequences in a distinct pattern enables to retrace a detected binding event to a certain disease. The template slide is placed in close proximity to the future protein array enabling the expressed proteins to reach this other surface by diffusion. Complex chemistry ensures that target proteins are specifically immobilized on this surface, while components of the expression mix can be washed away before sample analysis.

Step 2: Measuring serum samples by iRIf
After preparation of the DiaCHIP, a patient’s serum sample can be flushed over the protein array. The binding of antibodies to the protein surface causes a minimal change in the thickness of the slide right at the corresponding antigen spot. This change can be measured without the need for a further label with an emerging method called iRIf (imaging reflectometric interference). Based on the interference of light beams reflected on different thin layers, binding events can be recorded in real-time.

After weeks of opimizing the different components of the DiaCHIP, we reveal our great results. The highlight of our project was reached with the successful detection of antibodies in our own blood!