Difference between revisions of "Team:FAU Erlangen/Tour21"
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One of the prerequisites for our goal was the colocalization of the DNA binding protein and the histone modifying protein. This could be achieved by a fusion protein combining those two, as in the work of Mendenhall et al. (2013). However, this method would force its users to synthesize new fusion proteins for every new locus and every new modification they want to analyze. A more flexible approach lies in the non-covalent bond between biotin and streptavidin. Although this would have allowed us to design interchangeable fusion proteins for easier large-scale applications, we were unwilling to commit to the trade-off between flexibility and stability, as the bond in question, albeit strong, doesn't react well to low pH or mechanical forces (Chivers et al., 2010). To get the best of both worlds, we chose to utilize the recently established SpyTag/SpyCatcher system, which provides an isopeptide bond to connect two proteins and hold them together. | One of the prerequisites for our goal was the colocalization of the DNA binding protein and the histone modifying protein. This could be achieved by a fusion protein combining those two, as in the work of Mendenhall et al. (2013). However, this method would force its users to synthesize new fusion proteins for every new locus and every new modification they want to analyze. A more flexible approach lies in the non-covalent bond between biotin and streptavidin. Although this would have allowed us to design interchangeable fusion proteins for easier large-scale applications, we were unwilling to commit to the trade-off between flexibility and stability, as the bond in question, albeit strong, doesn't react well to low pH or mechanical forces (Chivers et al., 2010). To get the best of both worlds, we chose to utilize the recently established SpyTag/SpyCatcher system, which provides an isopeptide bond to connect two proteins and hold them together. | ||
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The SpyTag/SpyCatcher system as it is used in our project was developed in 2013 by the group of Associate Professor Mark Howarth from the University of Oxford. It (the system, not the university) is derived from the adhesin domain CnaB2 of a fibronectin-binding protein of Streptococcus pyogenes, a Gram-positive species of bacteria pathogenic to humans (Zakeri et al.). A spontaneous reaction within the mostly hydrophobic protein core irreversibly binds the nitrogen atom of a lysin's side chain to an oxygen atom of an aspartate's side chain. This intramolecular bond can be turned into an intermolecular bond by splitting the protein in two, which is exactly what Howarth's group did (Fig 1). | The SpyTag/SpyCatcher system as it is used in our project was developed in 2013 by the group of Associate Professor Mark Howarth from the University of Oxford. It (the system, not the university) is derived from the adhesin domain CnaB2 of a fibronectin-binding protein of Streptococcus pyogenes, a Gram-positive species of bacteria pathogenic to humans (Zakeri et al.). A spontaneous reaction within the mostly hydrophobic protein core irreversibly binds the nitrogen atom of a lysin's side chain to an oxygen atom of an aspartate's side chain. This intramolecular bond can be turned into an intermolecular bond by splitting the protein in two, which is exactly what Howarth's group did (Fig 1). | ||
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− | + | <a class="popupImg alignRight" style="width:350px" target="_blank" href="https://static.igem.org/mediawiki/2015/f/f1/FAU_SpyFig.jpeg" title="Residues forming the isopeptide bond before and after the separation. (Veggiani et al., 2014)"> | |
− | + | <img src="https://static.igem.org/mediawiki/2015/f/f1/FAU_SpyFig.jpeg" style="width:350px" /> | |
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During the course of several steps, the Catcher was optimized and shortened from 138 to 84 amino acids (Zakeri et al. 2012, and Li et al., 2014). The Catcher we decided to use shares its 113 amino acids with the protein from the Registry part BBa_K1159200, which was submitted in 2013 by the team from the TU Munich. | During the course of several steps, the Catcher was optimized and shortened from 138 to 84 amino acids (Zakeri et al. 2012, and Li et al., 2014). The Catcher we decided to use shares its 113 amino acids with the protein from the Registry part BBa_K1159200, which was submitted in 2013 by the team from the TU Munich. | ||
Revision as of 20:05, 17 September 2015
One of the prerequisites for our goal was the colocalization of the DNA binding protein and the histone modifying protein. This could be achieved by a fusion protein combining those two, as in the work of Mendenhall et al. (2013). However, this method would force its users to synthesize new fusion proteins for every new locus and every new modification they want to analyze. A more flexible approach lies in the non-covalent bond between biotin and streptavidin. Although this would have allowed us to design interchangeable fusion proteins for easier large-scale applications, we were unwilling to commit to the trade-off between flexibility and stability, as the bond in question, albeit strong, doesn't react well to low pH or mechanical forces (Chivers et al., 2010). To get the best of both worlds, we chose to utilize the recently established SpyTag/SpyCatcher system, which provides an isopeptide bond to connect two proteins and hold them together.
The SpyTag/SpyCatcher system as it is used in our project was developed in 2013 by the group of Associate Professor Mark Howarth from the University of Oxford. It (the system, not the university) is derived from the adhesin domain CnaB2 of a fibronectin-binding protein of Streptococcus pyogenes, a Gram-positive species of bacteria pathogenic to humans (Zakeri et al.). A spontaneous reaction within the mostly hydrophobic protein core irreversibly binds the nitrogen atom of a lysin's side chain to an oxygen atom of an aspartate's side chain. This intramolecular bond can be turned into an intermolecular bond by splitting the protein in two, which is exactly what Howarth's group did (Fig 1).