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| − | The 2015 iGEM team of TU Eindhoven aims to develop a modular sensor system based on | + | The 2015 iGEM team of TU Eindhoven aims to develop a modular sensor system based on Clickable Outer Membrane Proteins (COMPs) in E. coli. The COMPs contain an unnatural amino acid (pAzF) with an azide functional group. Last year's iGEM 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. | 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. | ||
Revision as of 00:43, 19 September 2015
Dig Deeper
Read more about the structural elements of COMBs.
Read more about the structural elements of COMBs.
Next Chapter
Explore how we tested our COMBs experimentally.
Explore how we tested our COMBs experimentally.
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. The COMPs contain an unnatural amino acid (pAzF) with an azide functional group. Last year's iGEM 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.