Template:Team:Bonn:NetworkData

content.i = 38;

content.i = 38;

content.parents=[37];

content.parents=[37];

content.titleShort = "Outlook";

content.titleShort = "Outlook";

content.titleLong = "Outlook";

content.titleLong = "Outlook";

content.parents = [38];

content.parents = [38];

content.childs = [];

content.childs = [];

content.summary = "The opportunity of a light switchable system in which a tagged protein is degraded by the protease ClpXP in eukaryotes offers various applications. A puplication from Grilly et al. gives information about the practicability of an application in eukaryotes";

content.summary = "The opportunity of a light switchable system in which a tagged protein is degraded by the protease ClpXP in eukaryotes offers various applications. A puplication from Grilly et al. gives information about the practicability of an application in eukaryotes";

content.text = "The application of our system in eukaryotes can yield many advantages.</br>As the ClpXP degradation system does not exist in eukaryotes naturally it does not get influenced by the eukaryotic cell. Besides no homologue to the protease ClpXP has been found in yeast [39.1]. An advantage is that in yeast many genes can get expressed quite easier than in prokaryotes. Moreover a functionality of our system in yeast may offer applications in other eukaryotic model organisms.</br>Hence a degradation system in which degradation of a tagged protein by ClpXP is switchable by light in eukaryotes enables to produce several more proteins light-dependently which can lead to various new applications.</br></br>Grilly et al. made it possible to use the ClpXP degradation system in eukaryotes [39.1].</br>Grilly et al. used the <i>E.coli</i> degradation machinery ClpXP to construct a <i>S.cerevisiae</i> strain that enables a tunable degradation of a tagged protein. A ClpXP system was created which degrades tagged proteins specifically and is tunable by changing concentrations of IPTG in the medium.</br></br>The genes ClpX and ClpP which code for the ClpXP protease were integrated into the yeast genome. However the genes ClpX and ClpP had to be modified with ten silent mutations until they worked in the yeast. The genes were integrated each together with an IPTG inducible promoter.</br>Now there was a system in the yeast genome which produces ClpXP only when IPTG was present.</br>The protein which was chose to be degraded by the ClpXP system was the yeast-enhanced green protein yEGFP. As the rate of fluorescence is measurable the yEGFP worked as an reporter. The yEGFP gene was integrated into the genome and tagged with a ssrA tag.</br>The promoter used was GAL1. GAL1 is induced by galactose and repressed by glucose. Thus the yeast produced yEGFP and was fluorescent in the present of galactose and did not produce yEGFP in the present of glucose.</br>The coexpression of the tagged yEGFP and the degradation machinery (IPTG and Galactose in the medium) leaded to an almost complete loss of fluorescence whereas if an untagged yEGFP was expressed the fluorescence did not decreased. This establishes that the degradation machinery works specifically to target proteins.</br>The influence of different concentrations of IPTG on the activity of ClpXP was studied.</br>By increasing the concentration of IPTG the rate of degradation of the tagged protein yEGFP increased, the fluorescence became less in a shorter time (figure 1). Thus the activity of the degradation system was tunable by concentrations of IPTG.</br></br><div class='content-image'><img src='https://static.igem.org/mediawiki/2013/7/74/Bonn.case39-bild2.PNG'[https://static.igem.org/mediawiki/2013/7/74/Bonn.case39-bild2.PNG]></br></br&gt;Figure 1: The decrease of fluorescence of <i>S.cerevisiae</i> cells in time with different concentrations of IPTG in the medium [39.1]</div></br></br>All in all considering the results of the work of Grilly et al. one can say that the degradation machinery ClpXP works in yeasts with little modifications. An important observation is that ClpXP is not toxic for yeast, the growth rate and morphology of the cells were unchanged [39.1]. Additionally it is possible to create systems in eukaryotes that make a tuning of the ClpXP activity possible.</br>In this system the activity of ClpXP is controlled by the concentration of IPTG whereas in our system ClpXP is controlled via sspB. SspB is not used in eukaryotes yet. However, sspB should be expressed without many problems because of it small size of 164 amino acids. Furthermore Grilly et al. state in their outlook that the coexpression of sspB might increase degradation [39.1].</br>The other components used in our system, LOV-ipA and VinD1, have already been used in eukaryotes.</br>Hence a case can be made that our system should also work with little modifications in eukaryotes. Grilly et al. state that the system should function with any protein which can support the short ssrA and that they expect this degradation system to work with many different applications [39.1].</br></br><h3>References<h3></br><a href=‘http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1943424/‘>[39.1] Chris Grilly, Jesse Stricker, Wyming Lee Pang, Matthew R. Bennett, Jeff Hasty (2007), <i>A synthetic gene network for tuning protein degradation in Saccharomyces cerevisiae</i>, University of California San Diego</a>";

content.text = "The application of our system in eukaryotes can yield many advantages.</br>As the ClpXP degradation system does not exist in eukaryotes naturally it does not get influenced by the eukaryotic cell. Besides no homologue to the protease ClpXP has been found in yeast [39.1]. An advantage is that in yeast many genes can get expressed quite easier than in prokaryotes. Moreover a functionality of our system in yeast may offer applications in other eukaryotic model organisms.</br>Hence a degradation system in which degradation of a tagged protein by ClpXP is switchable by light in eukaryotes enables to produce several more proteins light-dependently which can lead to various new applications.</br></br>Grilly et al. made it possible to use the ClpXP degradation system in eukaryotes [39.1].</br>Grilly et al. used the <i>E.coli</i> degradation machinery ClpXP to construct a <i>S.cerevisiae</i> strain that enables a tunable degradation of a tagged protein. A ClpXP system was created which degrades tagged proteins specifically and is tunable by changing concentrations of IPTG in the medium.</br></br>The genes ClpX and ClpP which code for the ClpXP protease were integrated into the yeast genome. However the genes ClpX and ClpP had to be modified with ten silent mutations until they worked in the yeast. The genes were integrated each together with an IPTG inducible promoter.</br>Now there was a system in the yeast genome which produces ClpXP only when IPTG was present.</br>The protein which was chose to be degraded by the ClpXP system was the yeast-enhanced green protein yEGFP. As the rate of fluorescence is measurable the yEGFP worked as an reporter. The yEGFP gene was integrated into the genome and tagged with a ssrA tag.</br>The promoter used was GAL1. GAL1 is induced by galactose and repressed by glucose. Thus the yeast produced yEGFP and was fluorescent in the present of galactose and did not produce yEGFP in the present of glucose.</br>The coexpression of the tagged yEGFP and the degradation machinery (IPTG and Galactose in the medium) leaded to an almost complete loss of fluorescence whereas if an untagged yEGFP was expressed the fluorescence did not decreased. This establishes that the degradation machinery works specifically to target proteins.</br>The influence of different concentrations of IPTG on the activity of ClpXP was studied.</br>By increasing the concentration of IPTG the rate of degradation of the tagged protein yEGFP increased, the fluorescence became less in a shorter time (figure 1). Thus the activity of the degradation system was tunable by concentrations of IPTG.</br></br><div class='content-image'><img src='https://static.igem.org/mediawiki/2013/7/74/Bonn.case39-bild2.PNG'[https://static.igem.org/mediawiki/2013/7/74/Bonn.case39-bild2.PNG]></br></br&gt;Figure 1: The decrease of fluorescence of <i>S.cerevisiae</i> cells in time with different concentrations of IPTG in the medium [39.1]</div></br></br>All in all considering the results of the work of Grilly et al. one can say that the degradation machinery ClpXP works in yeasts with little modifications. An important observation is that ClpXP is not toxic for yeast, the growth rate and morphology of the cells were unchanged [39.1]. Additionally it is possible to create systems in eukaryotes that make a tuning of the ClpXP activity possible.</br>In this system the activity of ClpXP is controlled by the concentration of IPTG whereas in our system ClpXP is controlled via sspB. SspB is not used in eukaryotes yet. However, sspB should be expressed without many problems because of it small size of 164 amino acids. Furthermore Grilly et al. state in their outlook that the coexpression of sspB might increase degradation [39.1].</br>The other components used in our system, LOV-ipA and VinD1, have already been used in eukaryotes.</br>Hence a case can be made that our system should also work with little modifications in eukaryotes. Grilly et al. state that the system should function with any protein which can support the short ssrA and that they expect this degradation system to work with many different applications [39.1].</br></br><h3>References<h3></br><a href=‘http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1943424/‘>[39.1] Chris Grilly, Jesse Stricker, Wyming Lee Pang, Matthew R. Bennett, Jeff Hasty (2007), <i>A synthetic gene network for tuning protein degradation in Saccharomyces cerevisiae</i>, University of California San Diego</a>";