Difference between revisions of "Template:Team:TU Eindhoven/Description HTML"

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−	The Recognition Element – aptamers	+	Description
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−	Since the discovery of the nucleic acid structure, nucleic acids were long thought to have a single function – storage of the heredity information as genetic instructions. Our perception on the function of DNA and RNA changed radically, however, as it was discovered that small RNA molecules could fold into a three-dimensional structure, exposing a surface onto which other small molecules could perfectly fit.	+	This year, we aim to develop a modular system for signaling pathways 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 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>). We will be working with the OmpX variant.
−	Soon after the discovery that nucleic acids could have interaction with other molecules, Craig Tuerk and Larry Gold described a procedure, called SELEX, to isolate high-affinity nucleic acid ligands for proteins through a Darwinian-like evolution process carried out in vitro [1]. This procedure enabled researchers to discover oligonucleotides with high affinities and a perfect fit for arbitrary proteins, cells, small molecules and even viruses. After this perfect fit, these synthetic oligonucleotides were called ‘aptamers’ – stemming from the Latin aptus which can be translated into ‘fit’.	+
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−	The Rise of Aptamers	+
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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, which can only emit light when the two parts are connected. We are also looking into the use of BRET between NanoLuc and mNeonGreen, since these 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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		+	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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−	25 Years after the discovery of Tuerk’s and Gold’s invention of Systemic Evolution of Ligands by Exponential enrichment (SELEX), aptamers have become available for hundreds of ligands, including proteins, viruses, other small molecules and even whole cells. As such, aptamers have become a viable alternative for biology’s traditional recognition elements, antibodies.
−	Aptamers also provide numerous advantages over antibodies. As mentioned, aptamers are oligonucleotides whereas antibodies are proteins. As such, aptamers have a remarkable stability in a wide range of pH and temperatures, have higher shelf lifes, are non-toxic and lack immunogenicity [2][3]. Other advantages stem from the fact that aptamers are generated in vitro, whereas antibodies are generated through in vivo enrichment followed by purification through monoclonal cell lines [4]. As a result, generation of aptamers is less laborious, production costs are lower and reproducibility is higher. Finally, in contrast to antibodies, aptamers can be generated against virtually any molecule, including toxins and poor immunogenic targets [5]. This places aptamers amongst the most powerful tools in biotechnology [6].
−	A major limitation of aptamers in comparison with antibodies is their stability in vivo, where nucleic acids are rapidly degraded. Aptamers which have been used as therapeutic agents, for example, suffered from a half-life of 2 minutes [7]. This problem has, however, partially been overcome by using chemical modifications to the aptamers. An example of these modifications are Spiegelmers [6]. These oligonucleotides’ backbones contain L-Ribose instead of R-Ribose– “spiegel” means mirror. These mirrored aptamers are more stable in vivo, because they suffer less from degradation.
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Revision as of 12:10, 22 July 2015

Description

This year, we aim to develop a modular system for signaling pathways 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 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). We will be working with the OmpX variant.
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, which can only emit light when the two parts are connected. We are also looking into the use of BRET between NanoLuc and mNeonGreen, since these 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.

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.