Difference between revisions of "Team:Hannover/Description"
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<div class="col-sm-8 text-left main-content"> | <div class="col-sm-8 text-left main-content"> | ||
<h1>Description</h1> | <h1>Description</h1> | ||
| − | + | <h2>TALebots</h2> | |
| − | < | + | <div class="jumbotron"> |
| − | <p> | + | <div class="container text-center"> |
| − | </p><p> | + | <h1>Our Project</h1> |
| − | + | <p>Übersichtsseite Arthur</p> | |
| − | </p><p> | + | </div> |
| − | + | </div> | |
| − | </p><p> | + | |
| − | Our aim is to develop a circular TAL-effector with the help of a linker in order to stabilize the protein. Thereby, TAL-effectors could be utilized on a daily basis and enable new techniques of genetic engineering in the lab. | + | <div><h2>Overview</h2> |
| − | </p> | + | <p>Twenty years ago, modifying the human genome seemed impossible. But new technologies, such as CRISPR-Cas9, Zink finger and TALEs (transcription activator-like effectors) have made modifying DNA possible and even pretty quick. Several articles in the press also show that those new techniques could help develop new treatments for diseases, fight the Zika virus (Specter, 2016) and even cure cancer (Office, 2015). </p> |
| + | <p>In fact, Layla, one little girl in Britain, was already saved from leukemia using genome editing with TALENs which are TALEs combined with a nuclease (Qasim, 2015). Waseem Qasim and his team from the University College of London had the idea to engineer donor blood cells and implant them into Layla. Using TALENs as molecular scissors, they disabled two genes in the donor cells to prevent a tissue rejection and making the cells invisible for antibodies which are given to the patient as a therapy (Qasim, 2015). </p> | ||
| + | <p>In 2016, those modified blood cells from Cellectis were also used to cure a second baby from cancer (Hirschler, 2016). </p> | ||
| + | <p>In order to enable more ways to use TALEs or TALENs to cure cancer, other diseases or discover other treatment options, those proteins need to be as stable as they can be. This is we want to step in. TALEs are not very stable and proteases can destroy the link between amino acids, which form the protein. This instability leads to the problem that the purification of TAL-effectors as well as the in-vitro application in the lab is difficult to perform. For this reason, TAL-effector proteins are excluded from a huge field of application, because a lot of genetic work usually takes place in vitro. </p> | ||
| + | <p>Our aim is to develop a circular TAL-effector with the help of a linker in order to stabilize the protein. Thereby, TAL-effectors could be utilized on a daily basis and enable new techniques of genetic engineering in the lab. Circularizing a protein was first introduced to iGEM by the team from Heidelberg in 2014 with their “Ring of Fire”. This led us to the idea that a circularization of TALEs in not impossible. </p> | ||
| + | |||
| + | <h2>Background</h2> | ||
| + | <h3>TAL effectors</h3> | ||
| + | <p>Transcription activator-like effectors (TALEs) are type III-secreted effector proteins of Xanthomonas ssp. They are used to induces target plant genes and support bacterial colonization of plant tissue. Xanthomonas injects the protein into plants by using a type III secretion system. Inside the plant cell, the protein reaches the nucleus and binds to promoters of targeted genes. In this way, they activate transcription. </p> | ||
| + | <p>Those proteins are able to bind to a specific DNA sequence determined by a central domain of variable number of repeats which also define specificity. (Boch J. , 2011) The repeats are very similar, but contain a variable pair of amino acids at position 12/13 which determine the DNA-binding specificity. The bond of the protein with the DNA is caused by a bond of each repeat with one DNA base pair and the interaction of amino acid 13 with the base of the DNA-leading strand. In addition, non-canonical repeats in the N-terminal region interact with the DNA in general. The repeats of the TALE can be rearranged to generate a bond to any desired DNA sequence. Furthermore, TAL effectors contain a nuclear localization signal and an acidic transcriptional activation domain, too. </p> | ||
| + | <img src="TODO“/> TODO Bild vom TALE (Jens um Erlaubnis fragen) <!-- TODO --> | ||
| + | <p>In order to use TALEs for biotechnology, catalytic domains of endonucleases as FokI can be fused to the TALE protein. By inducing sequence specific double-strand breaks, TALEs are an effective tool for genome editing. In addition, TALEs naturally regulate gene expression by replacing the TATA-binding protein or recruiting transcription factors. (Miller, 2011). </p> | ||
| + | <p>The TALE system is indeed very similar to the CRISPR/Cas9 technology. But nevertheless, TALEs offer more accurate genome editing “in test tubes” and other organisms. In addition, TALE proteins offer a significant advantage compared to usual procedures e.g. with restriction enzymes: Trough an easy change of the amino acid sequence, the protein can be customized to any DNA sequence. The function of TALEs could be proven in vivo in cell cultures and also in animals. This is why TALEs save time and effort in the lab, allowing multiple ways to alter a genome. </p> | ||
| + | |||
| + | <h3>Team Heidelberg: Ring of Fire</h3> | ||
| + | <p>In 2014, the iGEM team Heidelberg conducted their project “Ring of Fire” (Heidelberg, 2014) and successfully circularized GFP and dihydrofolate reductase by ligating the N- and C-terminal ends through inteins and Sortase circularization. Inteins were integrated into proteins. After protein translation, they perform an autocatalytic splicing reaction and link the ends of the protein together. They also used the intein Npu DnaE which is naturally split into two fragments and reassembles.</p> | ||
| + | <p>They successful showed that circularization enhances resistance against proteases and denaturation.</p> | ||
| + | <p>To perform our experiments, we were able to use the vector construct of the team Heidelberg. For the purpose of optimizing it and stabilizing the circulation, we changed the intein linker sequence.</p> | ||
| + | <img src="TODO“/> TODO Logo Heidelberg <!-- TODO --> | ||
| + | |||
| + | <h3>Cyclization of TALEs with the help of self-splicing Inteins</h3> | ||
| + | <p>A circular protein has been shown to be thermal stable and resistant against digestion by exo-proteases or reducing environments (Williams, 2002). This is achieved by a smaller size and the reduction of conformational entropy in the denatured state (Muir, 2003).</p> | ||
| + | <p>Like the team from Heidelberg, we focused on Inteins to perform the cyclisation. Inteins are protein domains which perform a cis-splicing reaction followed by their own excision and the break of two peptide bonds. A new bond is formed between the remaining parts (also called exteins). Recent studies discovered Inteins that are split into two halves and only regain activity when binding together, for example the Npu Intein (Wood & Camarero, 2014). In this way, two proteins can be ligated through Intein-splicing. If a protein is fused to a C-Intein next to a C-Extein and a N-Intein next to a N-Extein, trans-splicing causes the formation of a peptide bond between C-Extein and N-Extein as well as the excision of the combined intein (Muir, 2003).</p> | ||
| + | <img src="TODO“/> TODO Schaubild als Übersicht<!-- TODO --> | ||
| + | |||
| + | </div> | ||
</div> | </div> | ||
Revision as of 05:48, 14 October 2016
Projects
- Description
- Design
- Modelling
- Proof
- Demonstrate
- Results
- Description
- Design
- Modelling
Description
TALebots
Our Project
Übersichtsseite Arthur
Overview
Twenty years ago, modifying the human genome seemed impossible. But new technologies, such as CRISPR-Cas9, Zink finger and TALEs (transcription activator-like effectors) have made modifying DNA possible and even pretty quick. Several articles in the press also show that those new techniques could help develop new treatments for diseases, fight the Zika virus (Specter, 2016) and even cure cancer (Office, 2015).
In fact, Layla, one little girl in Britain, was already saved from leukemia using genome editing with TALENs which are TALEs combined with a nuclease (Qasim, 2015). Waseem Qasim and his team from the University College of London had the idea to engineer donor blood cells and implant them into Layla. Using TALENs as molecular scissors, they disabled two genes in the donor cells to prevent a tissue rejection and making the cells invisible for antibodies which are given to the patient as a therapy (Qasim, 2015).
In 2016, those modified blood cells from Cellectis were also used to cure a second baby from cancer (Hirschler, 2016).
In order to enable more ways to use TALEs or TALENs to cure cancer, other diseases or discover other treatment options, those proteins need to be as stable as they can be. This is we want to step in. TALEs are not very stable and proteases can destroy the link between amino acids, which form the protein. This instability leads to the problem that the purification of TAL-effectors as well as the in-vitro application in the lab is difficult to perform. For this reason, TAL-effector proteins are excluded from a huge field of application, because a lot of genetic work usually takes place in vitro.
Our aim is to develop a circular TAL-effector with the help of a linker in order to stabilize the protein. Thereby, TAL-effectors could be utilized on a daily basis and enable new techniques of genetic engineering in the lab. Circularizing a protein was first introduced to iGEM by the team from Heidelberg in 2014 with their “Ring of Fire”. This led us to the idea that a circularization of TALEs in not impossible.
Background
TAL effectors
Transcription activator-like effectors (TALEs) are type III-secreted effector proteins of Xanthomonas ssp. They are used to induces target plant genes and support bacterial colonization of plant tissue. Xanthomonas injects the protein into plants by using a type III secretion system. Inside the plant cell, the protein reaches the nucleus and binds to promoters of targeted genes. In this way, they activate transcription.
Those proteins are able to bind to a specific DNA sequence determined by a central domain of variable number of repeats which also define specificity. (Boch J. , 2011) The repeats are very similar, but contain a variable pair of amino acids at position 12/13 which determine the DNA-binding specificity. The bond of the protein with the DNA is caused by a bond of each repeat with one DNA base pair and the interaction of amino acid 13 with the base of the DNA-leading strand. In addition, non-canonical repeats in the N-terminal region interact with the DNA in general. The repeats of the TALE can be rearranged to generate a bond to any desired DNA sequence. Furthermore, TAL effectors contain a nuclear localization signal and an acidic transcriptional activation domain, too.
Cyclization of TALEs with the help of self-splicing Inteins
A circular protein has been shown to be thermal stable and resistant against digestion by exo-proteases or reducing environments (Williams, 2002). This is achieved by a smaller size and the reduction of conformational entropy in the denatured state (Muir, 2003).
Like the team from Heidelberg, we focused on Inteins to perform the cyclisation. Inteins are protein domains which perform a cis-splicing reaction followed by their own excision and the break of two peptide bonds. A new bond is formed between the remaining parts (also called exteins). Recent studies discovered Inteins that are split into two halves and only regain activity when binding together, for example the Npu Intein (Wood & Camarero, 2014). In this way, two proteins can be ligated through Intein-splicing. If a protein is fused to a C-Intein next to a C-Extein and a N-Intein next to a N-Extein, trans-splicing causes the formation of a peptide bond between C-Extein and N-Extein as well as the excision of the combined intein (Muir, 2003).
