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This year, we aim to develop a modular sensor system based on the clickable outer membrane proteins (COMPs), developed by last year’s iGEM team. These COMPs contain a non-natural amino acid with an azide functional group. The team has shown that DNA functionalized with a DBCO-group could be clicked on these proteins using the SPAAC click chemistry reaction. For more information on last year’s project, we refer to their iGEM wiki page (see <a target="_blank" href="https://2014.igem.org/Team:TU_Eindhoven">iGEM TU Eindhoven 2014</a>). Instead of using COMPx, we will be working with the OmpX variant.
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Sensitive, accurate and quick detection tools are the key to problems within all fields of society. Already a lot of research is done to accomplish this, but these tools are not yet always available. Many sensor systems are designed for the detection of a specific molecule, but a universal approach is still lacking.  
 
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To modify OmpX into signaling proteins, we need both an extracellular sensory domain and an intracellular domain that can relay the signal. The extracellular part will be a pair of dual aptamers, each clicked on a different membrane protein. This results in two outer membrane proteins to be brought to close proximity if the aptamer’s ligand is present. For the intracellular part, we consider a number of signaling methods that respond to a change in proximity. As proof of concept we use a split luciferase and BRET (Bioluminescence resonance energy transfer). The split luciferase can only emit light when its two parts are connected. BRET is another promising possibilty when we use NanoLuc and mNeonGreen, since these two proteins have no affinity towards each other. Too much intracellular interaction could influence the proximity of the proteins even with the absence of the ligand. After the testing phase an intracellular TEV protease system could be used to release a transcription factor in order to activate protein transcription.
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The 2015 iGEM team of TU Eindhoven aims to develop a modular sensor system based on clickable outer membrane proteins (COMPs) in E. Coli. In order to accomplish this, one part of the system relies on the iGEM project of last year’s team. The COMPs contain a unnatural amino acid (pAzF) with an azide functional group. The team has shown that DNA functionalized with a DBCO-group could be clicked on these proteins using the SPAAC click chemistry reaction. For more information on last year’s project, we refer to their iGEM wiki page (see <a target="_blank" href="https://2014.igem.org/Team:TU_Eindhoven">iGEM TU Eindhoven 2014</a>). Instead of using the protein COMPx as a scaffold for click chemistry, we will be working with the protein OmpX.
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To modify OmpX into signaling proteins, we need both an extracellular sensory domain as well as an intracellular domain that can relay the signal. A pair of dual aptamers will form the extracellular sensor domain, clicked on different OmpX proteins. This results in two OmpX variants to be brought in close proximity if the aptamer’s ligand is present. For the intracellular part, we consider a number of signaling methods that respond to a change in proximity. As proof of concept we make use of a luminescent signal constituted by a split luciferase or a BRET (Bioluminescence Resonance Energy Transfer) system. The split luciferase can only emit light when its two parts are connected and a substrate is present. Whenever the OmpX proteins are actively brought together due to the extracellular clicked aptamers, the split luciferases are more likely to connect and relay a signal. BRET is the other promising possibilty when we use the BRET pair NanoLuc and mNeonGreen. These two proteins have no affinity towards each other which is favourable, because too much intracellular interaction could influence the proximity of the proteins even without the presence of a ligand. After the testing phase with a luminescent signal, an intracellular TEV protease system could be used. This TEV protease could possibly release a transcription factor in order to activate protein transcription. In this case more characteristics of E. Coli can be exploited, making the sensor system even more universally applicable.
 
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Figure 1: Schematic overview of the device: the left part depicts the situation where no ligand is bound, the middle shows thrombin bound by the aptamers and the right parts shows that the signal is transducted across the membrane by binding of the two intracellular components.  
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Figure 1: Schematic overview of the sensor device: the left part of the image depicts the situation where no ligand is around, the middle part shows a marker bound by the aptamers and the right part shows the signal transduction across the outer membrane of E. Coli to enable an interaction of the two intracellular components.  
 
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The device we aim to develop can have a wide range of applications, due to its inherent modularity. A particular application which we will consider during our iGEM project is the use of the system within the gastrointestinal tract. It is known that disturbances within the immune system in the intestines are associated with many different pathologies, such as Crohn’s disease and intestinal cancer. Moreover, aptamers for the signaling molecules of the immune system, cytokines, have been examined within the scientific community. In the future, we hope the system could thus be used to detect immunological disturbances within the intestines and serve as a means to diagnosing certain pathologies in early stages of the disease. The application in the gastrointestinal tract isn't limited to human disease only; Q fever, a local problem, could benefit from earlier detection with our system as well. Nowadays, Q fever is a huge problem in the area around Eindhoven and other parts of the Netherlands. Spreading of this disease from farm to farm or even from goats to humans can be prevented when detection is possible in an earlier stage. Furthermore, the system can be used to create an interactive pesticide sensor system or microfluidic device that allows testing of multiple biomarkers at once. When used on the field, the system could release pesticides only when it senses particular fungi. This can reduce the use of pesticides drastically, which will be beneficial for both nature and the economy.
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The device we aim to develop can have a wide range of applications, due to its inherent modularity and incorporation in E. Coli. A particular application which we considered during our iGEM project is the use of the system within the gastrointestinal tract. It is known that disturbances within the immune system in the intestines are associated with many different pathologies, such as Crohn’s disease and intestinal cancer. Moreover, aptamers for the signaling molecules of the immune system, named cytokines, have been extensively examined within the scientific community. In the future, we hope the system could thus be used to detect immunological disturbances within the intestines and possibly even serve as a means to diagnosing certain pathologies in early stages of the disease. The application of COMBs is not limited to human diseases only; Q fever, a local problem here in Eindhoven, could benefit from earlier detection with our system as well. Since Q fever is a huge problem in the Netherlands, the need for sensitive and accurate diagnostic systems is bigger than ever. Spreading of this disease from farm to farm or even from goats to humans can be prevented when detection is possible in an earlier stage. Furthermore, the system can be used to create an interactive pesticide sensor system. When used on the field, the system could release pesticides only when it senses particular fungi. This can reduce the use of pesticides drastically, which will be beneficial for both nature and the economy.
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In the future, COMBs could be used for the several applications adressed. For example the fight against several diseases and reduction of pesticide use have significance importance, making the research for a universal GMO biosensor more relevant than ever.
 
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Revision as of 23:45, 18 September 2015





Description



Overview



Sensitive, accurate and quick detection tools are the key to problems within all fields of society. Already a lot of research is done to accomplish this, but these tools are not yet always available. Many sensor systems are designed for the detection of a specific molecule, but a universal approach is still lacking.

The 2015 iGEM team of TU Eindhoven aims to develop a modular sensor system based on clickable outer membrane proteins (COMPs) in E. Coli. In order to accomplish this, one part of the system relies on the iGEM project of last year’s team. The COMPs contain a unnatural amino acid (pAzF) with an azide functional group. The team has shown that DNA functionalized with a DBCO-group could be clicked on these proteins using the SPAAC click chemistry reaction. For more information on last year’s project, we refer to their iGEM wiki page (see iGEM TU Eindhoven 2014). Instead of using the protein COMPx as a scaffold for click chemistry, we will be working with the protein OmpX.
To modify OmpX into signaling proteins, we need both an extracellular sensory domain as well as an intracellular domain that can relay the signal. A pair of dual aptamers will form the extracellular sensor domain, clicked on different OmpX proteins. This results in two OmpX variants to be brought in close proximity if the aptamer’s ligand is present. For the intracellular part, we consider a number of signaling methods that respond to a change in proximity. As proof of concept we make use of a luminescent signal constituted by a split luciferase or a BRET (Bioluminescence Resonance Energy Transfer) system. The split luciferase can only emit light when its two parts are connected and a substrate is present. Whenever the OmpX proteins are actively brought together due to the extracellular clicked aptamers, the split luciferases are more likely to connect and relay a signal. BRET is the other promising possibilty when we use the BRET pair NanoLuc and mNeonGreen. These two proteins have no affinity towards each other which is favourable, because too much intracellular interaction could influence the proximity of the proteins even without the presence of a ligand. After the testing phase with a luminescent signal, an intracellular TEV protease system could be used. This TEV protease could possibly release a transcription factor in order to activate protein transcription. In this case more characteristics of E. Coli can be exploited, making the sensor system even more universally applicable.

Figure 1: Schematic overview of the sensor device: the left part of the image depicts the situation where no ligand is around, the middle part shows a marker bound by the aptamers and the right part shows the signal transduction across the outer membrane of E. Coli to enable an interaction of the two intracellular components.


Future Applications



The device we aim to develop can have a wide range of applications, due to its inherent modularity and incorporation in E. Coli. A particular application which we considered during our iGEM project is the use of the system within the gastrointestinal tract. It is known that disturbances within the immune system in the intestines are associated with many different pathologies, such as Crohn’s disease and intestinal cancer. Moreover, aptamers for the signaling molecules of the immune system, named cytokines, have been extensively examined within the scientific community. In the future, we hope the system could thus be used to detect immunological disturbances within the intestines and possibly even serve as a means to diagnosing certain pathologies in early stages of the disease. The application of COMBs is not limited to human diseases only; Q fever, a local problem here in Eindhoven, could benefit from earlier detection with our system as well. Since Q fever is a huge problem in the Netherlands, the need for sensitive and accurate diagnostic systems is bigger than ever. Spreading of this disease from farm to farm or even from goats to humans can be prevented when detection is possible in an earlier stage. Furthermore, the system can be used to create an interactive pesticide sensor system. When used on the field, the system could release pesticides only when it senses particular fungi. This can reduce the use of pesticides drastically, which will be beneficial for both nature and the economy. In the future, COMBs could be used for the several applications adressed. For example the fight against several diseases and reduction of pesticide use have significance importance, making the research for a universal GMO biosensor more relevant than ever.