Template:Team:Bonn:NetworkData

content.text= "Heat can be used to induce the expression of desired genes. This can be achieved via the linkage of heat-inducable promoters and the genomic sequence of the desired protein. These heat-inducable promoters are most commonly derived from the genes of heat-shock-proteins, whose cellular levels of concentrations are at least in part regulated via heat [33.1].</br></br>The main advantage for the use of heat lies in its simplicity, compared to other methods of induction. There is no need to modify the amino acid sequence of the targeted protein. Furthermore, the method of induction is rather simple, as it only requires the heat shock of the organism. However, therein also lies the biggest disadvantage of this method, as heat-shocking an organism triggers many, oftentimes undesired physiological reactions besides the expression of the desired gene [33.2][33.3]. Another disadvantage is that the usage of heat only offers induction of protein levels, and can not be used to modulate protein activity besides unspecific denaturation. Also, the spatiotemporal resolution of heat is rather low [33.2][33.3].</br><div class='content-image'><img src='https://static.igem.org/mediawiki/2013/d/df/Bonn-Heat-1.PNG'> </br>Diagram showing the activity of a heat-induced promoter in relation to the temperature of the applied heat-shock [33.3]</div></br>Furthermore, heat-induced promoters have a base level of activity, so through its usage only the effects of high level of protein on the organism can be examined [33.3] <h3>References</h3><a href='http://www.ncbi.nlm.nih.gov/pubmed/23912482'>[33.1] Zhang L. et al., &quot;Characterization of four heat-shock protein genes from Nile tilapia (Oreochromis niloticus) and demonstration of the inducible transcriptional activity of Hsp70 promoter.&quot; Fish Physiol Biochem. 2013 Aug 4</a></br> <a href='http://www.ncbi.nlm.nih.gov/pubmed/6322174'>[33.2] Bardwell JC, Craig EA. &quot;Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous.&quot; Proc Natl Acad Sci U S A. 1984 Feb;81(3):848-52</a></br><a href='http://www.ncbi.nlm.nih.gov/pubmed/10739675'>[33.3] Attila Ádám et al., &quot;Heat-Inducible Expression of a Reporter Gene Detected by Transient Assay in Zebrafish&quot;, Experimental Cell Research 256: 282-290 (2000)</a>";

content.summary= "A review on chemical induction and its advantages and disadvantages";  

content.text= "In nature there are many examples of voltage altering protein conformation such as channel proteins which can open or close^{<a href='#1'>[1]</a>} or enzymes which change their catalytic activity when the membrane potential changes.^{<a href='#2'>[2]</a>} Channel proteins require minimal changes in electrical potential and change conformation in submillisecond timescales.^{<a href='#3'>[3]</a>} However its usage in synthetic biology is yet to be demonstrated.<h2>References:</h2> <p><a name=1>1.</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412939/'> Venkataswarup Tiriveedhi, Melissa Miller, Peter Butko, and Min Li: <i>Autonomous Transmembrane Segment S4 of the Voltage Sensor Domain Partitions into the Lipid Membrane. </i>&quot;Biochimica et Biophysica Acta&quot; (2012)</a></p> <p><a name=2>2.</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2869078/'> Xiao Tao, Alice Lee, Walrati Limapichat, Dennis A. Dougherty, and Roderick MacKinnon: <i>A Gating Charge Transfer Center in Voltage Sensors. </i>&quot;Science&quot; (2010) Vol. 328 no. 5974 pp. 67-73 <p><a name=3>3.</a> <a href='http://www.sciencemag.org/content/314/5799/615.long'> Berkefeld H, Sailer CA, Bildl W, Rohde V, Thumfart JO, Eble S, Klugbauer N, Reisinger E, Bischofberger J, Oliver D, Knaus HG, Schulte U, Fakler B: BKCa-Cav channel complexes mediate rapid and localized Ca2+-activated K+ signaling. &quot;Science&quot; (2006)</a></p>";

content.type="Background";  

content.titleLong = "LOV-ipaA & VinD1";  

content.summary= "The LOV-ipaA -vinculin system is a combined system for light inducible heterodimerisation. This powerful tool, which allows photocontroled complex formation was establish by Lungu et al. in 2012.";

content.text= "The LOV-ipaA -vinculin system is a combined system for light inducible heterodimerisation. It consists out of a LOV domain, which undergoes conformational changes upon irradiation with blue light, and the ipaA-vinculin hybridization system. This two building blocks have be combined and described by Lungu et al. in 2012.</br></br> Lungu et al. (2008) where able to measure a 49-fold difference in target binding upon irradiation as compared to the dark state. However, they further modified the system by mutations of the LOV-ipaA construct and successfully weakend the baseline affinity for vinculin (initial design: 3.5 nM to 69 nM; mutant: 2.4 nM to >40µM affinity for vinculin) to reduce the dark state activity. </br></br> Lov-ipaA-VinD1 is a powerful tool which allows photocontroled complex formation. To establish this system Lungu et al. (2012)^{<a href=#ref36.1>36.1</a>} fused the Ja helix of the LOV Domain to ipaA.</br>To be more precise they used the LOV2 domain from Avena sativa photopropin 1 (AsLOV2), which – as previously shown – can be used to photomodulate the affinity of peptides for their binding partners (see Figure 1). </br><div class=contant-image><img src=https://static.igem.org/mediawiki/2013/0/02/Bonn_MS_Figure1_LOV.jpg></br>Figure 1: General design of AsLOV2 fusion proteins (Lungu et al. 2012)^{<a href=#ref36.1>36.1</a>}</div> </br>In other studies had been shown that the LOV domain can be fused to entire protein domains, allowing photomodulation of the protein binding. However, they stated that it might be of high importance to bring the LOV domain closer to ipaA, in order to allow photomodulation in this case, because ipaA is only a peptide and thus more flexible than folded domains.</br></br>Therefore, Lungu et al. (2012)^{<a href=#ref36.1>36.1</a>} identified similar amino acid sequences in the ipaA peptide and the Ja helix of the LOV Domain and used this combined with molecular modeling to create photomodulateable AsLOV2-ipaA (see Figure 2). </br><div class=contant-image><img src=https://static.igem.org/mediawiki/2013/5/54/Bonn_MS_Figure2_LOV-ipaA.jpg></br>Figure 2: Light-inducible LOV-ipaA construct (Lungu et al. 2012)^{<a href=#ref36.1>36.1</a>}</div></br>They were able to proof the functionality of the AsLOV2-ipaA system by heterodimerization in yeast (yeast two-hybrid system or Y2H) The yeast two-hybrid system can be used to monitor protein–protein interactions between two proteins. The system is based on a transcription factor, which is split into two separate fragments, called the binding domain (BD) and activating domain (AD). Each domain is fused to one protein and thus only if the proteins interact with each other BD and AD are close enough to initiate the transcription of a reporter gene.</br></br>The basic principle of the LOV-ipaA & VinD1 system works as follows. In the dark state the fusion product LOV-ipaA is not able to form a complex with vinculin, because LOV blocks ipaA sterically. However, activation of the LOV domain with blue light induces conformational changes in the fused molecule, which results in a movement of the Ja helix with the ipaA away from LOV. Thereby, ipaA becomes accessible for VinD1 and a Complex is formed.</br></br>The activation is reversible and the entire system can be genetically encoded. This two facts are the main advantages of this system in contrast to other typically used systems, which like for the chemical system for example, are based on in vivo covalently modified peptides, that can be activated by light induced cleavage. Moreover, the protein used are relatively small and thus should interfere as little as possible with the prokaryotic metabolism, the activity change form dark to light state is high, the system is completely genetically encoded and reversible. But, the most important property of this system is that it allows the light-controlled heterodimerisation of the two split variants of sspB, which is necessary for our system.</br></br><h2>References</h2></br><a name=ref36.1>36.1</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334866/>Lungu et al. (2012) Designing photoswitchable peptides using the AsLOV2 domain</a>";

content.titleShort = "project summary";  

content.summary= "We engineer a photoswitachble protein degradation system relying on the ClpXP protease system an a LOV domain from avena sativa";  

content.text= "";  

content.type="Project";  

content.i = 38;  

content.parents=[37];  

content.childs=[40,39];  

content.titleShort = "Outlook";  

content.titleLong = "Outlook";  

content.summary= "Possible applications of our project in the future";  

content.text= "<p>At the moment our system is only working in sspB-deficient bacteria as the natural occurrence of sspB in E. coli interferes with our split-sspB system. This problem could be solved by using the core region of sspB and the ssrA tag from the Caulobacter crescentus (Cc) bacteria. That way proteins with the Cc-ssrA tag can be degraded by the ClpXP protease after a light stimulus while the natural protein degradation homeostasis is not influenced. In order to do so, a split version of Cc-sspB would have to be generated and implemented.</p><p>With the Cc versions the kill-switch system could be used as a general lab safety tool for genetically engineered organisms (GEO). Despite of current safety measures, it can never be guaranteed that GEOs escape the laboratories. The kill switch would in that case ensure that the escaped organisms die and do not pose a threat to people and nature.</p><p>The next step would be to implement our degradation system in eukaryotes such as yeast. The ClpXP protease and the light-regulated sspB can be expressed in yeast. Proteins that were tagged with ssrA would then be degraded at a light signal. That way the kill-switch is also possible in eukaryotes.</p> ";

content.type="Project";  

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>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.parents=[38];  

content.childs=[41,74,43];  

content.titleLong = "General information about C. crescentus and the ClpXP protein degradation system";

content.summary= "Here you can find a brief introduction to Caulobacter crescentus and its ClpXP protease system.";

content.text= "Caulobacter crescentus is a Gram-negative &alpha;-protobacterium often found in fresh water lakes or in the sea. Its cell division cycle is a favoured object of study due to its remarkable asymmetry. ^{<a href=’[40.1]’>[40.1]</a>}</br>ClpXP is a protease that degrades proteins tagged with the ssrA peptide. sspB&alpha; is an adaptor protein that can accelerate protein degradation by tethering ssrA-tagged proteins towards the ClpX subunit of the ClpXP protease.</br>For further information, please browse the related articles. </br></br><h2>References</h2></br><a href=’[40.1]’>[40.1]</a><a href=’ http://www.amazon.com/Brock-Biology-Microorganisms-13th-Edition/dp/032164963X’> Brock Microbiology of Microorganisms, Madigan et al., Pearson, German edition Vol. 13, 2012</a>";

content.parents=[40];  

content.childs=[];  

content.summary= "This article is about the ClpXP protease system in C. crescentus and regulation of proteolysis via ClpXP. It also provides information about similarities and differences between C. crescentus and E. coli orthologs of related proteins.";

content.text= "<div class='content-image'><img src='https://static.igem.org/mediawiki/2013/d/de/Bonn-41-CC-ClpXP.png'>ClpXP mediated proteolysis ^{<a href='[41.3]'>[41.3]</a>}</div>ClpXP is an AAA+ protease which is of particular importance for proper cell-cycle progression. It consists of a hexameric ClpX subunit, which recognizes and unfolds tagged proteins, while ATP is hydrolyzed, and a ClpP subunit, that contains the actual peptidase domain. ^{<a href=’[41.1]’>[41.1]</a>,<a href=’[41.2]’>[41.2]</a>} Its structure is highly conserved, such that E. coli and C. crescentus orthologs are very similar. </br>Specific activity of the ClpXP protease is mediated by sspB and ssrA. ssrA is a short peptide consisting of fourteen amino acids in C. crescentus. Proteins which need to be degraded, e.g. for regulatory purpose or due to errors in their structure, are tagged with the ssrA peptide that can be recognized by the ClpX subunit. sspB is a dimeric adaptor protein in C. crescentus which tethers ssrA-tagged proteins to the ClpXP protease and, in this way, accelerates protein degradation. </br>Whilst sspB protein structure is well- conserved among many microorganisms, the structure of ^CCsspB&alpha; (i.e., the C. crescentus ortholog of sspB) shows significant differences compared to other orthologs. For example, ^CCsspB&alpha; and ^ECsspB (i.e., the E. coli ortholog) only show up with sequence identities of 16%, while CCsspB is still able to specifically bind to ^ECClpXP.^{<a href=’[41.2]’> [41.2]</a>} ssrA tags can also be very different among microorganismic species. </br>This makes it possible to establish a protein degradation system in E. coli involving ^CCsspB&alpha; and ^ECClpXP, as long as the ssrA tag can be recognized by both ^CCsspB&alpha; and ^ECClpXP. </br></br><h2>References</h2></br><a href=’[41.1]’>[41.1]</a><a href=’ http://www.ncbi.nlm.nih.gov/pubmed/17937918’> Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</a></br><a href=’[41.2]’>[41.2]</a><a href=’http://www.ncbi.nlm.nih.gov/pubmed/20014030’> Versatile modes of peptide recognition by the ClpX N domain mediate alternative adaptor-binding specificities in different bacterial species, Chowdhury et al., Protein Science, 2010, PMID: 20014030</a></br><a href='[41.3]'>[41.3]</a><a href='http://www.biochem.umass.edu/faculty/peter-chien'>Peter Chien, Department of Biochemistry and Molecular Biology (official website), http://www.biochem.umass.edu/faculty/peter-chien, picture URL: cchttp://www.biochem.umass.edu/sites/biochem/files/resize/ClpX-300x268.jpg</a>";

content.parents=[74];  

content.childs=[];  

content.summary= "This article deals with the structure of sspB&alpha; and conformational details of its binding to ssrA and ClpXP during tethering.";

content.text= "<div class='content-image'><img src='https://static.igem.org/mediawiki/2013/thumb/4/42/Bonn_OutlookCCSspB1_Version2.png/726px-Bonn_OutlookCCSspB1_Version2.png'>sspB structure and its conservation among C. crescentus, E. coli and H. influenzae ^{<a href='[42.1]'>[42.1]</a>}</div>The sspB&alpha; dimeric structure is stabilized by two &alpha;-helices in interaction, as part B of the figure above shows, each of them located at the N-terminus of either sspB&alpha; molecule. The subsequent parts of the protein form a domain consisting of two &beta;-sheet structures, together building up the ssrA binding site. An unstructured area at the C-terminus being referred to as the XB module forms the ClpX binding part of the protein. It is connected to the rest of the molecule via a linker domain. ^{<a href='[42.2]'>[42.2]</a>} </br>Chien et al. ^{<a href='[42.1]'>[42.1]</a>} compared crystal structures of C. crescentus sspB&alpha; and its E. coli and H. influenzae sspB orthologs, discovering that in sspB&alpha; the &alpha;-helices are significantly longer, more twisted and cover a larger cross section area than the other two sspB orthologs. Also considering that &beta;-sheets are rotated by around 20&deg; in comparison to E. coli and H. influenzae orthologs, this leads to an antiparallel orientation of the two ssrA tagged protein bound to the ssrA binding sites of an sspB&alpha; dimer in C. crescentus, while they are parallel in &gamma;-protobacterial sspB.</br></br><div class='content-image'><img src='https://static.igem.org/mediawiki/2013/6/6c/Bonn_OutlookCCSspB2.png' align=left>By measuring GFP fluorescence intensity, decrease of GFP-^CCssrA concentration (1) without sspB&alpha; added, (2) with mutated sspB&alpha;(Q74A) added , (3) with wildtype sspB&alpha; added can be visualized. ^{<a href='[42.1]'>[42.1]</a>}</div>Chien et al. point out that although there are the remarkable differences in protein structure between sspB&alpha; and its &gamma;-protobacterial ortholog, they show up with similar effectiveness in binding proteins tagged with the related ssrA peptide. But it turned out in their research that effectiveness of sspB&alpha; binding to the protein which needs to be tethered to the ClpXP protease strongly depends on which ssrA ortholog the protein is tagged with. sspB&alpha; binds firmly to ^CCssrA, with an affinity being 175 times as large as for binding to ^ECssrA (i.e. the E. coli ortholog). By comparing the crystal structures of both sspB&alpha; and the compound of sspB&alpha; and ^CCssrA, Chien et al. further proved that binding of sspB&alpha; to ^CCssrA does not lead to significant changes of its 3D conformation.</br></br><h2>References</h2></br><a href='[42.1]'>[42.1]</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/17937918'>Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</a></br><a href='[42.2]'>[42.2]</a><a href='http://www.ncbi.nlm.nih.gov/pubmed/14967151'> Bivalent tethering of sspB to ClpXP is required for efficient substrate delivery: a protein design study, Bolon DN et al., Mol Cell, 2004, PMID: 14967151</a>";

content.parents=[40];  

content.childs=[];  

content.titleShort = "SspB Split";  

content.titleLong = "SspB Split in C. crescentus";  

content.summary= "The protein degradation system in Caulobacter crescentus resembles the system in E. coli, but the respective sequences of ssrA and SspB differ. Thus the different specifities can be used to introduce the ccSspB split system in wildtyp E. coli without disturbing the native processes in it.";  

content.text= "The protein degradation system in Caulobacter crescentus resembles the system in E. coli, but the respective sequences of ssrA and SspB differ ^{<a href=#[43.1]>[43.1]</a>}. Thus ccssrA only binds ccSspB but not E. coli SspB. ^{<a href=#[43.2]>[43.2]</a>} ^{<a href=#[43.3]>[43.3]</a>} ^{<a href=#[43.4]>[43.4]</a>} However, proteins tagged with ccssrA can be degraded by E. coli ClpXP. Therefore the utilization of ccSspB and ccssrA in E. coli has the advantage that SspB+ strains can be used. ^{<a href=#[43.1]>[43.1]</a>} </br> In order to use this with the SspB split system, the fusion proteins ccSspBΔ10-FRB and FKBP12-SspB[XB] (E. coli) were incubated with GFP-ccDAS+4 and E. coli ClpXP in vitro. Without rapamycin there was no degradation detected. Equally, addition of E. coli SspB showed no degradation. Addition of rapamycin led to a reduction of GFP-ccDAS+4 of around 12% in 180 seconds. Compared to the E. coli split system (around 30 % in 180 seconds) this system is less fast but can, at least in vitro, be used with sspB-wildtype E. coli. ^{<a href=#[43.1]>[43.1]</a>} </br> As the results of the E.coli and the C. crescentus system in vitro show many similarities and the E. coli system works in vivo. It may be possible to use the C. crescentus in vivo as well. </br> <img src='https://static.igem.org/mediawiki/2013/8/82/Bonn-ccSspB.jpg'> ^{<a href=#[43.1]>[43.1]</a>} <h2>References:</h2> </br> <a name=[43.1]>[43.1]</a> <a href='http://dspace.mit.edu/bitstream/handle/1721.1/58089/654116495.pdf?sequence=1'> Understanding and Harnessing Energy-Dependent Proteolysis for Controlled Protein Degradation in Bacteria, J. Davis, Massachusetts Institute of Technology, april 2010 </a> </br> <a name=[43.2]>[43.2]</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC58509/'> Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis, Flynn et al, Proc Natl Acad Sci USA 2001 Sep 11, PMID: 11535833 </a> </br> <a name=[43.3]>[43.3]</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/17937918'> Structure and substrate specifity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al, Structure 2007 Oct, PMID: 17937918 <a/> </br> <a name=[43.4]>[43.4]</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2581644/'> Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP, Griffith and Grossman, Mol Microbiol. 2008 Nov, PMID: 18811726 <a/> </br>";  

content.type="Project";  

content.summary= "Proteins that need to be degraded by the ClpXP protease have to be tagged with the ssrA peptide previosly. Here is some information on the structure of ssrA.";

content.text= "<div class='content-image'><img src='https://static.igem.org/mediawiki/2013/c/c4/Bonn_OutlookCCSsrA.png' align=right width=800>Peptide array for testing ssrA on amino residues relevant for sspB&alpha; binding; in every row one of the amino acids was replaced by any other amino acid one by one; the columns denote the amino acids put in after replacement; the darker the spot, the more stable is the binding of ssrA to sspB&alpha; ^{<a href='[44.1]'>[44.1]</a>} </div></br>Chien et al. ^{<a href='[44.1]'>[44.1]</a>} tested the 14-amino acid peptide ssrA (AANDNFAEEFAVAA, ^{<a href='[44.2]'>[44.2]</a>}) on the residues crucial for binding to sspB&alpha; by singly replacing the first twelve amino acids from N-terminus by any other amino acid and testing the mutated peptides in a peptide array. They found out, that residues 6-12 could be replaced by any other amino acid without reducing binding effectiveness, while residues 1-5 appeared to be responsible for specific binding to sspB&alpha;. They figured out that N3, D4 and N5 were outstanding, as they were the most intolerant amino acids to mutation; therefore they named this sequence the NDN motif, which is the sspB&alpha; binding site.</br>Griffith and Grossman ^{<a href='[44.3]'>[44.3]</a>} engineered a protein degradation system in Bacillus subtilis, using the B. subtilis ClpXP protease for degradation of proteins tagged with modified ssrA tags from E. coli or C. crescentus. These ssrA tags will only deliver a protein to the ClpXP protease if E. coli sspB or C. crescentus sspB&alpha;, respectively, is present, as ^ECsspB can only detect ^ECssrA and ^CCsspB&alpha; can only detect ^CCssrA, which makes it possible to specifically regulate protein degradation by using two different promotors for the two sspB gene orthologs. </br>We used a similarly engineered system in E. coli for our project: By expressing ^CCssrA and ^CCsspB&alpha; in E. coli, it became possible for us to specifically establish a light-inducible protein degradation system by connecting ^CCsspB&alpha; to the light-sensitive LOV domain.</br></br><h2>References</h2></br><a href='[44.1]'>[44.1]</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/17937918'>Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</a></br><a href='[44.2]'>[44.2]</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/20014030'>Versatile modes of peptide recognition by the ClpX N domain mediate alternative adaptor-binding specificities in different bacterial species, Chowdhury et al., Protein Science, 2010, PMID: 20014030</a></br><a href='[44.3]'>[44.3]</a><a href='http://www.ncbi.nlm.nih.gov/pubmed/18811726'>Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP, Griffith and Grossman, Moleculare Microbiology, 2008, PMID: 18811726 </a>";  

content.text= "The design of our System enables several advantages";  

content.text= "";  

content.summary= "Since the system is commonly used and reliable its easily applicable to a wide variety of proteins";

content.text= "";  

content.summary= "Fusing only a 15Aa small tag to the desired protein leads to low influence on its native function";

content.text= "";  

content.text= "";  

content.text= "";  

content.text= "";  

content.summary= "There are several applications for our systems e.g. a light inducible Kill Switch or investigation of protein function";

content.text= "<h1>Applications</h1><p>Our system offers several different applications sicne the ssrA tag can be added theoretically to every protein. Hence it allows the user to any protein and eliminate it just by switching on blue light!</p><p>A possible application is a ssrA-labeled toxin. In this case the cells would only be viable if blue light is present and the toxin is degraded.  Alternatively one could create a system in which an antitoxin is degraded in the presence of blue light while the toxin is encoded on another plasmid. The cells would be killed in the present of blue light. These so called &quot;kill-switches&quot; could be used on the one hand for lab security as cells could be created which could only survive in a laboratory where only red light is present, however, they would die in nature since daylight is energetic enough to trigger antitoxin degradation and thus kill escaped bacteria. On the other hand a kill-switch can be used in safe environmental applications using bacteria which can only survive under daylight exposure and die at night when no light is present anymore.</b>Besides our system can be used to investigate the function of a certain gene. By switching degradation of a gene product on and off, the cell&apos;s activity and phenotype can be studied with and without the protein of interest.</p><p>The advantages in contrast to other protein degradation systems are that our system is fast and has little unwanted side effects.</b>Our systems is faster since it only takes milliseconds until the blue light penetrates the cells. Comparable protein degradation systems are induced by small activator molecules which need time to reach their target.</b>The ssrA tag consists of only 15 amino acids and hence changes the protein sequence only very little and an effect on the conformation is unlikely. This creates a high variety of proteins which can be degraded. Additionally there are no external activator molecules in our system which could have an effect on the results.</p>";  

content.titleLong = "Kill-switch systems using stress-induced toxin-antitoxin modules in Escherichia coli";

content.summary= "Toxin-antitoxin systems are composed by an antitoxin and a toxin coding gene. Connecting our light inducible protein degradation system to the antitoxin via an ssrA-tag allows light induced cell death, as predominance of the toxin in a bacterium activates a cell death pathway.";

content.text= "Our system of light inducible protein degradation can be utilized to degrade any specific protein and is thus usable in a light induced kill-switch system. For this application a connection between the degradation system and a toxin-antitoxin module like MazEF or ccdA/ccdB is needed. Either the toxin or the antitoxin could be light inducibly degraded by adding an ssrA-tag, which is detected by our degradation system, to its genetical code: <ul><li><b>Using the degradation of the toxin:</b> For that purpose the insertion of a plasmid containing the ssrA-tagged toxin encoding gene is needed. Since the predominance of the toxin activates a cell death pathway in bacteria, a bacterium containing a module that allows light inducible degradation of the toxin would only be viable, when the toxin is degraded. In darkness the toxin overexpression is no longer compensated and aggregation of it leads to cell death. Apart from the use in lab security such a kill-switch system would also be useful for environmental applications of bacteria, since it opens up the possibility of deploying bacteria for only one day and ensures their passing by nightfall. For example bacteria could be used to perform the ecological stabilization of a lake but after one night any genetically modified bacteria would be dead.</li><li><b>Using the degradation of the antitoxin:</b> Two plasmids are needed: The first one to express the toxin and the second one to express the ssra-tagged antitoxin, in such manner that the amounts of the toxin and the antitoxin are in equilibrium. Once light induces the degradation system, the antitoxin is degraded and the predominant toxin will kill the bacterium. </li></ul> Regarding our idea to improve lab security by implementing a kill-switch system, both described ways seem possible. Usage of the former would require cultivating and working with the bacteria under constant blue light, as darkness would kill them. Realization of the latter would require no usage of any blue light in the lab since bacteria which get into touch with daylight our any blue light would be killed. Due to the high light sensivity of our degradation system it can most likely be induced by daylight, which renders the former killswitch system useless. Bacteria which escape from the lab could survive simply through contact with daylight. Consequently we focused on the second system (the degradation of the antitoxin).</br> With MazEF we described a light inducible kill-switch system via the insertion of plasmids into bacteria. However a final kill-switch system would have to be introduced into the genomic DNA since plasmids in bacteria can be ejected, for instance via cell division, whereas a genomic DNA mutation is less likely to occur. Nevertheless the risk of a loss of function cannot be eliminated , which is why a secure system should countain much more than one kill-switch system to compensate the malfunction of a single kill-switch system. Therefore, we consider the MazEF kill-switch system to be part of a much larger security system for genetically engineered bacteria. </br>";

 

content.i = 55;  

content.parents=[53];  

content.childs=[];  

content.summary= "To understand the role of a specific gene or DNA region is one of the big challenges in lifescience research. Our system, which allows the fast and convenient elimination of defined proteins, is a new improved technique, with many advantages.";

content.text= "To understand the role of a specific gene or DNA region is one of the big challenges in lifescience research. Our system, which allows the fast and convenient elimination of defined proteins, is a new improved technique, with many advantages. It allows the control of protein activity with respect to time and space, is fast, robust, can be used for different proteins and changes the protein sequence only little, because the ssrA tag consist only out of 15 amino acids. Recently technologies like gene knockout or knockdown had been developed, which allow to investigate the role of a particular gene or DNA region by comparing the knockout organism to a wildtype with a similar genetic background. </br></br> A knockout means that a particular gene is deleted from the genome of an organism. This organism might be bacteria or yeast, but also eukaryotic cells, plants or even animals. To create a knockout organism recombinant DNA is inserted into a gene (Bartke, 2006^{<a href=#ref55.3>55.3</a>}). When a genes sequence is interrupted, it may still be translated, but the resulting protein will be nonfunctional. Moreover, it is possible to knockout the gene only in defined tissues or at defined time points. This technique is called a conditional knockout. </br></br> On the other hand the knockdown, does not eliminate the specific gene on DNA, but on RNA level. Here interfering RNAs (siRNA) are inserted into the cell, leading to the degradation of the genes mRNA (Pratt and MacRae, 2009^{<a href=#ref55.1>55.1</a>}) and hence no protein can be produced. </br></br> In contrast to the knockout and knockdown, our system allows the expression of the gene and the translation into functional protein. However, irradiation with blue light leads to the fast elimination of the particular protein. Therefore, one of the major advantages of our system is its speed, not only in comparison to knockout and knockdown, but also in comparison to other protein level systems. Comparing our system, to other protein level systems, like the system developed by Davis et al.^{<a href=#ref55.2>55.2</a>} in 2011, which are induced by small molecules our system would still be faster due to the use of light. In small molecule systems it takes some time until the small molecules reach their target in the case of light this happens within milliseconds. Furthermore, with our system it is not necessary to add any kind of activator molecules, which might effect the results, to the cells. This enables researchers to investigate cell activity with and with out the protein in direct comparison, while the only interference is one light puls.</br></br><h2>References</h2></br></br><p><a name=ref55.1>55.1</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709356/>Pratt and MacRae (2009) The RNA-induced silencing complex: a versatile gene-silencing machine.</a></p> </br> <p><a name=ref55.2 >55.2</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220803/>Davis et al. (2011) Small-molecule control of protein degradation using split adaptors.</a></p></br><p><a name=ref55.3 >55.3</a> <a href=http://www.sciencedirect.com/science/article/pii/S0531556506002798>Bartke (2006) New findings in transgenic, gene knockout and mutant mice.</a></p>";  

content.type="Project";  

content.text= "Riboswitches can be identified as a subtopic of transcriptional and translational regulation. They are based on self-regulating mRNA, achieved by combination with an aptamer region and a ligand-binding region. Ligands can be sugars, nucleotides, metal ions or other small molecules. This enables riboswitches to bind special metabolisms in order to induce conformational changes. These conformational changes can block or free the ribosomal binding site and therefore inhibit or activate translation of the mRNA into a polypeptide. Moreover it is able to control transcription by sequestering or releasing termination sequences. In addition to that the aptamer structures can mask or unmask ribozyme binding-sites, which enables a regulated RNA-degradation^{<a href=#66.1>[66.1]</a>}.</br></br> <div class='content-image'><img src='https://static.igem.org/mediawiki/2013/8/8f/Bonn.Riboswitches.jpg'></br>&quot;Diversity of Riboswitches and Mechanisms of Gene Control in BacteriaMechanisms of modulation of gene expression are highly divergent in prokaryotes and involve control of transcription, translation, splicing, and mRNA stability&quot;^{<a href=#66.1>[66.1]</a>}</div><br/><div class='content-image'><img src='https://static.igem.org/mediawiki/2013/f/ff/Bonn.Riboswitches3.jpg'></br>&quot;Structural Principles of Ligand Recognition by Riboswitches(A–C) Schematic representations of a 'straight' junctional fold&quot;<sup><a href=#66.1>[66.1]</a></div> </br></br> <p><a name=66.1>[66.1]</a> <a href='http://www.sciencedirect.com/science/article/pii/S0092867412015462'> A Decade of Riboswitches Alexander Serganov, Evgeny Nudler, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA;"

content.titleLong = "A kill-switch system using the stress-induced toxin-antitoxin module MazEF in Escherichia coli";

content.summary= "The toxin-antitoxin system MazEF is composed by an upstream gene <i>mazE</i>, encoding a labile antitoxin, and a downstream gene <i>mazF</i>, that encodes a stable toxin. Connecting our light inducible protein degradation system to the antitoxin MazE allows light inducible cell death, as predominance of MazF in a bacterium activates cell death pathway.";

content.text= "Our system of light inducible protein degradation can be utilized to degrade any specific protein and is therefore usable to realize a light inducible kill-switch system. A connection of protein degradation to a cell death pathway is represented by the stress-induced toxin-antitoxin module <i>mazEF</i> in Escherichia coli. <i>mazEF</i> is located on the chromosome in E.coli which is associated with programmed cell death. The toxin-antitoxin system is composed by an upstream gene <i>mazE</i>, encoding a labile antitoxin, and a downstream gene mazF, that encodes a stable toxin. </br>The product of <i>mazF</i> cleaves mRNAs and tmRNAs at a specific site, which leads to an inhibition of translation. MazF shows a specific cleaving mechanism, which is not well understood yet, but shows that there is also protein synthesis which is unaffected by MazF. These proteins are presumably part of a cell death pathway. </br>The effect of <i>mazF</i> is suppressed by the Product of <i>mazE</i> which is degraded by the Protease ClpAP in bacteria. As a result of stressful conditions expression of the chromosomal <i>mazEF</i> module is reduced which leads to an imbalance between the products of <i>mazF</i> and <i>mazE</i>: When expression is lowered the stable toxin of <i>mazF</i> still persists while the labile antitoxin of <i>mazE</i> is degraded and can no longer suppress the effect of <i>mazF</i> leading to acute toxicity and cell death. </br><i>MazEF</i>-mediated cell death in E. coli can be caused by:<ul><li>extreme amino acid starvation^{<a href = '#1'>[17.1]</a>}</li><li> inhibition of transcription and/or translation by antibiotics such as rifampin, chloramphenicol, and spectinomycin under specific growth conditions^{<a href = '#1'>[17.1]</a>}</li><li>inhibition of translation by the Doc protein of prophage P1^{<a href = '#1'>[17.1]</a>}</li><li>DNA damage caused by thymine starvation as well as by mitomycin C, nalidixic acid, and UV irradiation^{<a href = '#1'>[17.1]</a>}</li><li>oxidative stress (H2O2)^{<a href = '#1'>[17.1]</a>}</li></ul>Amitai et al. tested in 2004 the Hypothesis of Pedersen et al.^{<a href = '#2'>[17.2]</a>}, that chromosomal toxin-antitoxin systems may rather cause a state of reversible bacteriostasis than programmed cell death^{<a href = '#1'>[17.1]</a>}.Therefore E.coli strain MC4100 &#916<i>mazE</i>F relA1 lacIq was cotransformated with:<ul><li>pBad-<i>mazF</i></li><li>pQE-&#916his-<i>mazE</i></li></ul><i>mazF</i>-expression can be induced by the addition of Arabinose via the pBad promoter of the first plasmid. The transformation of the second plasmid results firstly in the repression of <i>mazE</i> expression, whereas when IPTG is added <i>mazE</i> production is induced.</br><div class='content-image' align='center' height=501 width=410><a href='https://static.igem.org/mediawiki/2013/a/ac/Team_Bonn_MazF_1.png'><img src='https://static.igem.org/mediawiki/2013/a/ac/Team_Bonn_MazF_1.png' height=491 width=400></a></br><i>Ability of E. coli cells that had been ectopically overexpressing MazF in liquid medium to form colonies when ectopically overexpressing MazE on plates. The cultures were grown in LB medium (A) or M9 minimal medium with 0.5% glycerol (B) at 37°C to midlogarithmic phase (OD600, 0.5)^{<a href = '#1'>[17.1]</a>}.</i></div>Using these tools, Amitai et al. tested the effect of MazE overproduction on MazF-overproducing bacteria during growth in liquid medium.</br>The E.coli strain was incubated in LB medium. After <i>mazF</i> expression was induced by adding arabinose two samples were taken at several time points. To repress <i>mazF</i> expression to both of them glucose was added. In addition IPTG was added to one culture to induce <i>mazE</i> expression. The two cultures were compared via the level of protein synthesis and OD600.</br>Finally Amitai et al. confirmed the assumption that the overproduction of MazE after until 6h under overproduction of MazF could resuscitate E.coli cells in LB medium (Fig. 1A)^{<a href = '#1'>[17.1]</a>}, but the longer MazF was induced the less cells could be resuscitated by MazE.</br>Whereas MazE overproduction can reverse the inhibitory effect of MazF on translation, it cannot reverse the effect of MazF on colony formation, which is shown in figure 2. Only 1h after the induction of MazE expression, the rate of translation was restored to nearly 100% (Fig.2 Aa, Ab, Ac) but the bacteriocidic effect could not be reversed (Fig.2 Ba, Bb, Bc).</br>Additionally, Amtai et al. found out, that in M9 medium MazE was less able to reverse the effects of MazF overexpression than in LB medium (Fig.1B vs. 1A). it was concluded that there is a point of no return, when MazE is inable to resuscitate a MazF damaged cell, which occurs earlier in M9 medium than in LB medium.</br>Based on their results a model of the MazEF mechanism was built: A <i>mazF</i>-mediated cascade leads to a cell death pathway, but can nevertheless be stopped at several intermediary steps by e.g. <i>mazE</i>. When a point of no return is reached, the cascade cannot be stopped anymore.<div class='content-image' align='center' height=827 width=784><a href='https://static.igem.org/mediawiki/2013/9/9e/Team_Bonn_MazF_2.png'><img src='https://static.igem.org/mediawiki/2013/9/9e/Team_Bonn_MazF_2.png' height=817 width=764></a></br><i>Effect of MazE overproduction during growth in liquid medium on the ability of MazF-overproducing E. coli cells to synthesize proteins and form colonies. To induce <i>mazE</i> expression, IPTG was added to the bacterial culture at 1 h (Aa and Ba), 4 h (Ab and Bb), and 6 h (Ac and Bc) after <i>mazF</i> induction at time zero. The effects of the ectopic overexpression of MazE were measured at 1 and 3 h after the induction of <i>mazE</i> expression.^{<a href = '#1'>[17.1]</a>}.</i></div></br>Back to our project and to the idea of a light inducible kill-switch system:</br>As we described in the previous paragraph for both of our kill-switch systems using the MazEF module either MazF or MazE could be degraded:<ul><li>Using the degradation of MazF:</b> For that purpose the insertion of a plasmid containing the ssrA-tagged toxin encoding gene is needed. Since the predominance of the toxin activates a cell death pathway in bacteria, a bacterium containing a module that allows light inducible degradation of the MazF would only be viable, when it is degraded. In darkness the MazF overexpression is no longer compensated and aggregation of it leads to cell death. </li><li><b>Using the degradation of the antitoxin:</b> Two plasmids are needed: The first one to express the MazF and the second one to express the ssra-tagged MazE, in such manner that the amounts of the <i>MazF</i> and the <i>MazE</i> are in equilibrium. Once light induces the degradation system, the MazE is degraded and the predominant toxin will kill the bacterium.</li></ul>As we explained in the general kill-switch system text we finally focused on the second system (via the degradation of MazE).</br>With the design of a MazEF kill-switch system the possibility of resuscitating bacteria in the way Amitai et al. showed has to be considered. A predominant MazF could kill a bacterium in LB within about two hours, but it needs to be predominant over a long period (>7h) to induce its death without it being resuscitated by renewed <i>mazE</i> expression (Fig.1A)with more than 50% probability .</br>Certainly these facts seem to be unfavourable for the realization of a kill switch system via MazEF, but fortunately our system of heterodimerization (Lungu et al.) allows long continuous degradation^{<a href = '#3'>[17.3]</a>}, due to the high stability of the light induced hererodimer. Therefore likely a short exposure time will result in prolonged protein degradation sufficing for bacterial death. Additionally, Amitai et al. showed that the less nutrition is available for a bacterium, the earlier the point of no return is reached. If a bacterium escapes the lab, it will likely have less nutrition available than in LB medium. It might reach the point of no return earlier.</br>We described a light inducible MazEF kill-switch system via the insertion of plasmids into bacteria. However a final kill-switch system would have to be implemented in the genomic DNA since plasmids in bacteria can be ejected, for instance via cell division, whereas a genomic DNA mutation is less likely to occur. Nevertheless risk of a loss of function cannot be eliminated , which is why a secure system should countain much more than one kill-switch system to compensate the malfunction of a single kill-switch system. Therefore, we consider the MazEF kill-switch system to be part of a much larger security system for genetically engineered bacteria.<h2>References</h2><a name = '1'>^[17.1]</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC532418/'>MazF-Mediated Cell Death in Escherichia coli: a Point of No Return, Shahar Amitai et al., Journal of Bacteriology Vol. 186, No. 24, 2004, p.8295–8300.</a></br><a name = '2'>^[17.2]</a> <a href = 'http://www.ncbi.nlm.nih.gov/pubmed/?term=Rapid+induction+and+reversal+of+bacteriostatic+conditions+by+controlled+expression+of+toxins+and+antitoxins'>Rapid induction and reversal of bacteriostatic conditions by controlled expression of toxins and antitoxins, Pedersen et al., Molecular Microbiology 45, 2002, 501–510.</a></br><a name = '3'>^[17.3]</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334866/'>Designing Photoswitchable Peptides Using the AsLOV2 Domain, Oana I. Lungu et al., Chem Biol. 2012, 19(4):507-17.</a>";

content.titleShort = "ccdA/ccdB";  

content.summary= "this is a summary6";  

content.text= "The ccd module is a toxin-antitoxin (TA) system similar to the mazE/mazF system. The module is located on the F Plasmid in Escherichia coli bacteria and essential for their survival. Normally the toxin ccdB is inactivated by the presence of the antitoxin ccdA in the form of a ccdAB complex. If ccdA is no longer available, ccdB inhibits DNA gyrase which leads to cell death. Gyrase is a type IIA topoisomerase and is able to produce negative DNA supercoiling by making a double-strand break in the DNA and religating it. The gyrase enzyme consists of two subunits: the C-terminal GyrA domain that wraps around the DNA strand and the N-terminal GyrB domain that catalyses the ATP-dependant supercoiling of the DNA. CcdB stabilizes the gyrase cleavage complex by binding to the GyrA domain and thus blocks the catalytic function of the gyrase. That means that the gyrase remains bound to the DNA and the cleaved DNA is not religated. DNA- and RNA polymerases can’t copy the DNA anymore and cell proliferation as well as protein biosynthesis is stopped. The double-stranded breaks in the DNA initiate cell death.<p>Because gyrases are specific to bacteria such as E. coli it is also a target for some anti-bacterial medications e.g. ciprofloxacin (CFX). As can be seen in the data below, CcdB proves to be as effective as CFX at inducing DNA cleavage ^{<a href=#1>[1]</a>}^{<a href=#2>[2]</a>}</p><div class='content-image'align='center'><img src='https://static.igem.org/mediawiki/2013/2/2f/Sspb_CFX_compared.jpg' width='300'>Comparison of the effect of CcdB and CFX on gyrase activity. N: negatively supercoiled DNA, L: linear DNA, SC: supercoiled DNA. A higher concentration of CcdB/CFX leads to more cleaved (linear) DNA^{<a href= http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635281/figure/fig2/>[source]</a>}</div><div class='content-image' align='center'><img src='https://static.igem.org/mediawiki/2013/e/e4/Bonn_Ccdb_and_ccda.jpg' width='300'> A higher concentration of ccdB leads to blocking of gyrase and positively supercoiled DNA is cleaved to linear DNA instead of being processed to negatively supercoiled DNA. ^{<a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460896/figure/pone-0046499-g003/>[source]</a>}</div><p>We used this system to build a light-induced kill-switch. Therefore we added the ssrA tag to the antitoxin ccdA. When the bacteria a emitted to light, ccdA is degraded and ccdB is set free and can bind to the gyrase. and cell death is initiated. Like in most TA systems, the toxin ccdB is relatively stable, while the antitoxin ccdA is vulnerable to degradation.</p><h2>References</h2><p><a name='1'>1.</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635281/'>A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site, Andrew B. Smith and Anthony Maxwell, Nucleic Acids Res. 2006 October; 34(17): 4667–4676, PMC 1635281</a></p><p><a name='2'>2.</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460896/'> A Common Origin for the Bacterial Toxin-Antitoxin Systems parD and ccd, Suggested by Analyses of Toxin/Target and Toxin/Antitoxin Interactions, Andew B. Smith et al, PLoS One. 2012; 7(9): e46499, PMCID: PMC3460896</a></p>";

content.summary= "The operon-model is a very popular one. The operon allows an organism to regulate the expression of specific genes and therefore the production of corresponding proteins, depending on the concentration of a specific substrate (&quot;substrate-induction&quot;) or the lack of an important product (&quot;product-repression&quot;).";

content.text= "In absence of the substrate, a special repressor binds to the operator DNA sequence in order to inhibit the transcription by the DNA polymerase. The substrate can bind to an allosteric center of the repressor, which leads to a change of conformation. Now the repressor lost his affinity to the operator DNA and cannot inhibit the transcription anymore. This is called &quot;substrate induction&quot;.^{<a href=#71.1>[71.1]</a>} </br> </br><div class='content-imgage'> <img src=https://static.igem.org/mediawiki/2013/0/0f/BonnLacOperon.jpg></br><i>lac </i>operon^{<a href=#71.2>[71.2]</a>} </br> <a href='geneticsengineering.blogspot.com'> </a> </div> </br> </br> &quot;Product repression&quot; works the other way around. As long as there is an excess of substrate and a lack of product, the repressor has an low affinity to the operator DNA and isn’t able to inhibit the transcription. If there is enough product, it can bind to the allosteric center of the repressor, which improves itŽs affinity to the operator DNA. Now it is able to inhibit transcription.^{<a href=#71.3>[71.3]</a>}</br> </br><div class='content-image'> <img src='https://static.igem.org/mediawiki/2013/e/ed/BonnTrpOperon.jpg'></br>Tryptophan operon^{<a href=#71.4>[71.4]</a>}</div></br> </br></br><p><a name=71.1>[71.1] </a><a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3722110/'>A single mutation in the core domain of the lac repressor reduces leakiness, Pietro Gatti-Lafranconi, Willem P Dijkman, Sean RA Devenish, and Florian Hollfelder corresponding author.</a></p><p><a name=71.2>[71.2]</a> <a href='http://geneticsengineering.blogspot.de/'>geneticsengineering.blogspot </a></p><p><a name=71.3>[71.3] </a><a href='http://www.ncbi.nlm.nih.gov/pubmed/22713856'> Influence of the feedback loops in the trp operon of B. subtilis on the system dynamic response and noise amplitude. Zamora-Chimal C, Santillán M, Rodríguez-González J.</a></p><p><a name=71.4>[71.4]</a> <a href='http://bio1903.nicerweb.com/Locked/media/ch18/18_21bTrpOperon_2.jpg'> bio1903.nicerweb.com/Locked/media/ch18/18_21bTrpOperon_2.jpg </a>";

content.text= "The family of the &quot;zinc finger&quot; proteins has a different approach of Transcription regulation. They contain a zinc ion as a cofactor. Zinc finger proteins have DNA binding and dimerization domain. They can be differentiated because of different loops. On the one hand, they can bind to almost every part of DNA and on the other hand they can bind to several receptors. So the activating or repressing effect isnŽt defined by the zinc finger itself, but by the effector Protein it is binding to.<sup> <a href=#72.1>[72.1]</a> </br></br> <div class='content-image'> <img src='https://static.igem.org/mediawiki/2013/4/4b/BonnZincFinger.jpg' width='400' height='400'></br>&quot;The zinc ion (green) is coordinated by two histidine and two cysteine amino acid residues&quot;^{<a href=#72.2>[72.2]}</div></br></br> <p><a name=72.1>[72.1]</a> <a href=http://www.pnas.org/content/early/2013/09/11/1303625110.long> Transcription factor ZBED6 affects gene expression,proliferation, and cell death in pancreatic beta cellsXuan Wang, Lin Jiang,Ola Wallerman, Ulla Engström, Adam Ameur, Rajesh Kumar, Gupt, YuQi, Leif Andersson and Nils Welsh Science for Life Laboratory, Department of Medical Cell Biology, and Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123 Uppsala, Sweden; Ludwig Institute for Cancer Research Ltd., Science for Life Laboratory, Uppsala University, SE-751 24 Uppsala, Sweden; andScience for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, SE-75185 Uppsala, Sweden </a> </p> </br> <p><a name=#72.2>[72.2]</a><a href='http://en.wikipedia.org/wiki/Zinc_finger'>Cartoon representation of the zinc-finger motif of proteins</a></p>";

content.summary= "TALEs enable an easy and modular assembly of proteins binding specific desired DNA sequences";

content.text= "These proteins bind promoter sequences. Their DNA binding domain consists of several tandem repeats that are able to bind specific domains of the DNA. These tandem repeats can easily be engineered, so the user can define the domain to bind to. Very similar to the zinc finger they actually do not regulate transcription, but bind effector proteinswhich are able to activate transcription. The great advance, in comparison with the zinc finger domain, is itŽs easy way of engineering. Scientists can very specificly regulate transcription by the use of TALEs.</br></br><p> <a href='http://onlinelibrary.wiley.com/doi/10.1111/jipb.12091/abstract'> Site-Specific Gene Targeting Using Transcription Activator-Like Effector (TALE)-Based Nuclease in Brassica oleracea: Zijian Sun†,Nianzu Li†, Guodong Huang, Junqiang Xu, Yu Pan, Zhimin Wang, Qinglin Tang, Ming Song*, Xiaojia Wang> </a> </p>";

content.parents=[40];  

content.childs=[42,44];  

content.summary= "This article gives a brief overview of the roles of ssrA and sspB&alpha; for specific function of the ClpXP protease system in C. crescentus.";  

content.text= "ssrA and sspB are peptides that mediate proteolysis via the ClpXP protease system in bacteria. In this article and the related articles, focus is laid on their orthologs in C. crescentus, being referred to as ^CCssrA and <sup>CC>/sup>sspB&alpha;, respectively, omitting &quot;^CC&quot; when obvious out of context. The ClpXP protease has an important function in regulation of the cell division cycle by effective proteolysis of short-lived regulatory proteins.</br>A protein which needs to be degraded will be tagged with the amino acid peptide ^CCssrA, which is added at its C-terminus during translation. ^{<a href='[74.1]'>[74.1]</a>,<a href='[74.2]'>[74.2]</a>} The ClpX subunit of the ClpXP protease recognizes the ssrA tag by specific binding and unfolds the tagged protein, in which ATP is hydrolyzed. In C. crescentus, the ssrA tag has a length of 14 amino acids, while the E. coli ortholog is only eleven amino acids long. </br>sspB&alpha; is a dimeric protein that serves as a tether which brings the ssrA-tagged protein and the ClpXP protease together and therefore accelerates protein degradation. It simultaneously binds to both the ssrA tag and the ClpX subunit and in this way brings the tagged protein in close contact with the protease. </br></br><h2>References</h2></br><a href='[74.1]'>[74.1]</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/11535833'>Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis, Flynn et al., Proceedings of the National Academy of Sciences of the United States of America, 2001, PMID: 11535833</a></br><a href='[74.2]'>[74.2]</a><a href='http://www.ncbi.nlm.nih.gov/pubmed/17937918'> Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</a>";

content.i = 100;  

content.titleShort = "Human Practice";  

content.titleLong = "Human Practice in General";  

content.summary= "The major goal in Human Practice is to make synthetic biology easily understandable and interesting for everybody. For that purpose our project's design follows the well-known franchise of Star Wars; our project name &quot;LOV WARS&quot; was born. The constant references to the Star Wars movies facilitate the access to synthetic biology to a person that is not familiar with the subject. With the most famous icon of Star Wars being the laser sword it offers a great opportunity to present our light induction system in an entertaining way. We put great effort into adapting every aspect of our public presence to Star Wars.";  

content.text= "<p>The major goal in Human Practice is to make synthetic biology easily understandable and interesting for everybody. For that purpose our project's design follows the well-known franchise of Star Wars; our project name 'LOV WARS' was born. The constant references to the Star Wars movies facilitate the access to synthetic biology to a person that is not familiar with the subject. With the most famous icon of Star Wars being the laser sword it offers a great opportunity to present our light induction system in an entertaining way. We put great effort into adapting every aspect of our public presence to Star Wars.</p><p>Therefore we introduced a <a onclick=node(110)>flash game</a> and a <a onclick=node(109)>comic series</a> called LOV Wars in which we explain basic concepts of synthetic biology and our project. The comic and the LOV Wars shooter arouse interest in people that would normally never engage with the field. That way we can spread the possibilities and advantages that modern genetically engineered organisms offer and clear possible misunderstandings or prejudices against the subject.</p>For the same purpose we organized multiple events for general public at which we presented the tools used in synthetic biology and our project; over the last six months we <a onclick=node(108)>visited several High Schools</a> in Bonn and nearby and gave presentations. In addition we had an information booth in our city center informing passersby about the risks and benefits of synthetic biology with the aid of several posters and flyers. A big highlight was a <a onclick=node(106)>Science slam</a> we arranged. In front of a big audience six scientists from different fields presented an interesting aspect of their academic field. The Science Slam was a great success in terms of getting people in touch with science and without exception received very good critiques.<br> At every opportunity we handed out questionnaires in which we evaluated the public's opinion about synthetic biology and whether our human practice events were informative and interesting.";  

content.type="Human Practice";  

content.summary= "The iGEM Teams Bonn and Bielefeld were this year represented at the 4th BIO.NRW (PhD) Student Convention in the Esprit-Arena in Düsseldorf. Here our ambassadors Marc and Dustin were able to successfully introduce the current project of light induced protein dregadation in a short presentation. ";

content.text= "One hundred undergraduates, graduate students and PhD students from the life sciences came on Friday 12 July 2013 in the Esprit Arena in Dusseldorf, to train with professionals from academia and industry in two days for a successful start of their career. The fourth BIO.NRW ( PhD ) Student Convention, offered an exiting combination of lectures and workshops, regarding non-scientific knowledge and soft skills to complement the expertise of the young researchers. </br></br> We decided to use the opportunity to evaluate the level of knowledge about synthetic biology and iGEM a scientific community. Further, we used our chance to give a presentation and inform the young scientists about synthetic biology, iGEM and our project. In this environment it was a grate experience to discuses our ideas with a scientific community, not only during our presentation but also during the evening event with a barbecue on the sidelines in the Esprit Arena. </br></br> The Presentation of our iGEM project by Dustin Dankelman and Marc Schulte and the iGEM team from Bielefeld started the Convention together with three keynote lectures.The three keynote lectures by representatives of famous companies provided the participants with a practical and intuitive representation of the work of the fields and developments in the industry. Dr. Holger Bengs of the BCNP Consultants GmbH gave the interested participants at the beginning an overview of the biotech industry. In the second lecture, Dr. Barbara Maertens , Head of protein expression of the Cube Biotech GmbH, talked about her jump from a global biotech company to a start-up. The third keynote lecture with the topic research and development in the global pharmaceutical industry was held by Dr. Thomas Lauterbach, Head of Clinical Operations Europe at the biopharmaceutical company UCB. The second day was dedicated to a customized workshop program. </br></br> To evaluate how many scientist know about iGEM, how they think about synthetic biology in general and useful our presentation was, we have designed a questionnaire. At this point we would like to thank all the young scientist, who helped use with their feedback, to address this questions as shown below. <h2>Pictures</h2><div class=subpage-text><table><tr>           <th><a href=https://static.igem.org/mediawiki/2013/a/ac/Bonn_MS_BioNRW1.jpg><img src=https://static.igem.org/mediawiki/2013/a/ac/Bonn_MS_BioNRW1.jpg width=260px></a></th>           <th><a href=https://static.igem.org/mediawiki/2013/7/7b/Bonn_MS_BioNRW2.jpg><img src=https://static.igem.org/mediawiki/2013/7/7b/Bonn_MS_BioNRW2.jpgwidth=260px></a></th>           <th><a href=https://static.igem.org/mediawiki/2013/d/d0/Bonn_MS_BioNRW3.JPG><img src=https://static.igem.org/mediawiki/2013/d/d0/Bonn_MS_BioNRW3.JPG width=260px></a></th></tr><tr>           <th><a href=https://static.igem.org/mediawiki/2013/f/f1/Bonn_MS_BioNRW4.JPG><img src=https://static.igem.org/mediawiki/2013/f/f1/Bonn_MS_BioNRW4.JPG width=260px></a></th>           <th><a href=https://static.igem.org/mediawiki/2013/1/1d/Bonn_MS_BioNRW5.jpg><img src=https://static.igem.org/mediawiki/2013/1/1d/Bonn_MS_BioNRW5.jpg width=260px></a></th>           <th><a href=https://static.igem.org/mediawiki/2013/0/03/Bonn_MS_BioNRW6.JPG><img src=https://static.igem.org/mediawiki/2013/0/03/Bonn_MS_BioNRW6.JPG width=260px></a></th>           </tr>         </table></br>           <h2> Analysis of the questionnaires: </h2></br>             72 people have been asked:</br><img src=https://static.igem.org/mediawiki/2013/6/63/DoYouKnowWhatIgemIs.jpg width=800px></br></br></br><img src=https://static.igem.org/mediawiki/2013/b/b7/HowDoYouRate.jpg width=800px></br>Please klick here to see our <a href=https://static.igem.org/mediawiki/2013/1/16/Fragebogen_iGem.pdf>questionnaire</a>";

content.type="Human Practice";  

content.summary= "our day in Berlin. We met important people and other iGEM teams";  

content.text= "iGEM Bonn 2013 was invited to join the Biocom AG Kongress &quot;Biotechnologie 2020+&quot; (biotechnology 2020+) in Berlin on June 27th. We gladly accepted this invitation, as it presented us with a chance to get in touch with all the other german iGEM teams and gain from the feedback the professional audience could give us on our ideas.</br></br>Among the attendees were directors and leading scientists of Max-Planck and Fraunhofer institutes, and also government representatives. Their response not only to our ideas but also to the design of our poster was very positive, with some criticism regarding the lack of self-gathered data confirming the functionality of the system. Some of the more applianceoriented scientists pointed our thinkings about possible applications towards new directions such as pro-drug design and specific location targeting.</br></br>At least as rewarding was the chance to talk to members of other iGEM teams. Was it a talk with the more experienced to learn about new techniques, or a chat with the less experienced during which we could share our own knowledge, one could always either give or gain valuable knowledge. In the end, we believe most iGEM teams, us very much included, found the meeting to have been an inspiring and fun possibility to improve on their own projects. <table><tr><th><div class='content-image'><img src='https://2013.igem.org/File:Bonn_Berlin_Niklas.JPG'>Niklas (left) in a discussion with a member of team Darmstadt</div></th><th><div class='content-image'><img src='https://2013.igem.org/File:Bonn_Poster_Berlin.JPG'>Our Poster for the congress</th><th><div class='content-image'><img src='https://2013.igem.org/File:Bonn_Berlin_Poster_Media.JPG'>Our Poster in place</div></th></tr><tr><th><div class='content-image'><img src='https://2013.igem.org/File:Bonn_Berlin_Poster_explanation_Max.JPG'></th><div class='content-image'><img src='https://2013.igem.org/File:Bonn_Berlin_all_the_iGEMs.JPG'></tr></table>";

content.summary= "We implemented many ideas in the design of our wiki to enable fast and easy insight in our project";

content.type="Human Practice";  

content.summary = "We organized a science slam to offer an experience of science for everyone. And the event met with great approval!";

content.text = "<h1>Science Slam</h1><p>Our project aims to make versatile control of biological machines easily possible for everybody. During conception we wondered how our human practice work could represent that thought and decided to organize a science slam – an event which makes science available to everybody!</p><p>We believe that science must not only stay in laboratories and lecture halls but reach lots of people out there. And without a doubt, the walls of the reputed “ivory tower” couldn’t stand throughout the evening of our event!</p><p>Furthermore, the science slam provided a platform for our slammers to share the passion for their project with more than just their colleagues and thus help to popularize their field. In ten minutes, they presented their topic in a playful and inspirational manner and the audience decided which slammer has managed to get them carried away the most.</p><p>We managed not only to give floor to a large variety of fields by our slammers from physics, medicine, psychology, informatics, politics and engineering – but to make them be heard by a public which was very mixed indeed.</p><p>As a prelude to the event, we acquainted the audience with synthetic biology and our project by showing what synthetic biology can do for each of us. Distributed questionnaires during the evening showed that -as hoped- our presentation helped to leave a positive mark on the field of synthetic biology in peopleŽs heads:</p><p>For example, 97% of the people, who stated they did not know about synthetic biology before have got a proper picture of it during our event. 86% said, they are now familiar with the iGEM competition and everybody stated, they found the idea to combine the presentation of our iGEM project with a science slam “very good” or “good”.</p><p>The fact that our audience liked this topic had been reflected in our voting: Our representative for synthetic biology, Mogan Ramesh won the science slam. He originally comes from engineering but discovered his passion for “superbacteria e.coli” during his studies which he gladly shared with us. In ten minutes, he taught us that e.coli does not only work as “eeew” on our meal, but showed how his group studies on making e.coli work for us, e.g. producing pharmaceuticals.</p><p>How? For example, recombinant DNA technology can be used to modify Escherichia coli to produce human insulin. Mogans works within a group of scientist that investigate the metabolic network in the ‘Escherichia coli’ bacteria cell. Attempts to raise the copy number of plasmid DNA have been successful, and the goal now is to understand how the cell has accomplished this task. Proteomic studies have indicated an expected cellular adaptation that agrees with attempts to raise copy number in the literature; however, the thermodynamic implications of these results have been minimally explored. The “goal” of a cell remains controversial; one explanation is that the cellular metabolism seeks to maximize the entropy (minimize the Gibbs energy) of the system. Using GAMS as an optimization tool, it is explored if the reaction paths that have proven to be the most likely are also the paths that minimize the Gibbs energy of the system.</p><p>All in all, our science slam has been a great success which demanded a reenactment from a lot of sites.</p><p><b> Here are a few impressions from our event:</b></p><p> <img src=https://static.igem.org/mediawiki/2013/0/07/BonnScienceslam1.PNG> <br /><br /><i>Tobias Bald from experimental dermatology (University of Bonn) spoke about the “Dark side of the immune system”.</i></p><p><img src=https://static.igem.org/mediawiki/2013/d/dd/BonnScienceslam2.PNG><br /><br /><i>Prof. Dr. Herbert Dreiner gave us a review of “Fukushima” with full activity.</i></p><p><img src=https://static.igem.org/mediawiki/2013/0/0c/BonnScienceslam3.PNG><br /><br /><i>Our winner Mogan Ramesh shared his passion for “superbacteria e.coli”.</i></p><p><img src=https://static.igem.org/mediawiki/2013/2/24/BonnScienceslam4.PNG>< br /><br /><i>Full house at our “Science Slam”.</i></p><p><img src=https://static.igem.org/mediawiki/2013/d/d5/BonnScienceslam5.PNG><br /><br /><i>Our audience obviously enjoyed the show.</i></p>";

content.titleShort = "Day of Action SynBio";

content.summary= "In cooperation with the iGEM teams of Germany also the team of Bonn organized a day of action for synthetic biology. ";  

content.text= "<div align='right'><img src='https://static.igem.org/mediawiki/2013/b/b8/BonnAktionstag.JPG' height='260' width='350'></div>In cooperation with the iGEM teams of Germany also the team of Bonn organized a day of action for synthetic biology. </br> At the 7th of September ten of our members met in Bonn downtown to inform the interested civilians of our city about the international genetically engineered machine competition as well as synthetic biology in general and particularly about our project of light inducible degradation of proteins. </br> Therefore we prepared an information booth near the market place, distributed informative leaflets, visualized our ideas in terms of several posters and on top created a survey to examine the people's opinion. </br> <div align='left'><img src='https://static.igem.org/mediawiki/2013/thumb/9/97/BonnAktionstag2.jpg/800px-BonnAktionstag2.jpg' height='260' width='350'></div> At 9 o'clock in the morning we started in front of the LIMES-Institute to arrange the installation of the stand. By car all the needed equipment was transferred to the city center and there assembled under the eyes of the curious townspeople. Two hours later everything was settled and the official part of the day could begin: From 11 until 3 o'clock intrigued city dweller in every range of age stopped by to examine our exhibition walls and to ask questions, which we answered with pleasure. In the end we were surprised about the brisk participation and the lively discussions that aroused, which reflects in the results of the survey, so that we bundled up and left satisfied. Here you can see the summary of our questionaire:<br/> </br> 1. Do you know what synthetic biology means? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/b/bd/BonnQuestion_1.png' height='260' width='350'> <br/> </br> </br> 2. Do you know what the iGEM competition is about? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/4/44/BonnQuestion_2.png' height='260' width='350'> <br/> </br> 3. How do you rate the ralation between chance and risk of sythetic biology? </br><div align='center'><img src='https://static.igem.org/mediawiki/2013/5/58/BonnQuestion3.1.png' height='260' width='350'> </br> 4. What are the main reasons for you that speak against the use of synthetic bioloy? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/6/62/BonnQuestion_4.png' height='260' width='350'> </br> </br> 5. Do you think it is important to inform the public better about the topic ' snythetic biology'?</br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/a/a6/Question_5.png' height='260' width='350'> </br> </br> All in all we consider the action as a great success as we were able to reduce prejudices and elucidate people about advantages of synthetic biology.";

content.i = 108;  

content.parents=[100];  

content.childs=[];  

content.titleShort = "School presentations";  

content.titleLong = "School presentations";  

content.summary= "We gave lectures about synthetic biology and our project in schools in order to reach the younger people. ";  

content.text= " What do People already know about synthetic biology? How can we improve the knowledge about synthetic biology? </br> These were two of the basic questions we asked ourselves when we started brainstorming about our human practice part of the project. We decided to give lectures about synthetic biology and our project in schools in order to reach the younger people. During the summer semester we went to 8 schools nearby Bonn and Cologne.</br>We chose schools, because these pupils will possibly become the next generation of scientists one day. Our idea was to enthuse them about science and to clear up the bad image of science and research amongst some people. </br>Almost all classes we visited had a focus on natural sciences (e.g. biological or chemical advanced courses). But even more interesting were our visits to classes that had no natural scientific background. It was surprising for us that there were so many positive and thrilled comments and questions on this topic and our project. The pupils were so amazed about synthetic biology and its possibilities after the lecture that many of them took flyers with them or visited our homepage to play our mini-game and read more about our project. They were also excited about our comics. With the help of these lectures we reached one of our main goals of human practice: to explain people what synthetic biology is and in addition we could also promote our Wiki.</br></br>Below you can find the answers to a survey we handed out to the pupils after the lecture. </br> Most people of the audience had never heard about synthetic biology or iGEM before, but after our presentation many of them were excited about what they had learned. Most of them gave positive feedback about our presentations. </br> <https://static.igem.org/mediawiki/2013/4/41/Bonn_Schule_1.jpg> </br> When we visited the school classes, we soon realized that it´s sometimes really hard to explain such complex contents in an easily understandable way. Especially in classes that do not have a focus on natural science, there was often not enough time to work out everything out in detail. Nevertheless the pupils were all very interested in what we told them and had the impression that they had learned somethin new.</br> <img src=https://static.igem.org/mediawiki/2013/d/d0/Bonn_Schule_2.png> </br> But even if the time was limited we could elate people and we were very pleased to see that a remarkable part of the people could image to work with synthetic biology. </br> <img src=https://static.igem.org/mediawiki/2013/2/2c/Bonn_Schule_3.png> </br> It was also delighting that we could clear up people’s doubts and fears about synthetic biology. We could show them that synthetic biology can be used for our benefit and also how high the risks really are.</br> <img src=https://static.igem.org/mediawiki/2013/0/0e/Bonn_Schule_4.png> </br> <img src=https://static.igem.org/mediawiki/2013/f/f7/Bonn_Schule_5.png> </br> We then developed another questionnaire with similar questions, but with the difference that we compared the answers before and after our presentation. The results showed again that people originally didn´t had never heard about the topic and iGEM, but after our explanation they knew.</br> <img src=https://static.igem.org/mediawiki/2013/d/d5/Bonn_Schule_6.png> </br> <img src=https://static.igem.org/mediawiki/2013/d/dc/Bonn_Schule_7.png> </br> Here we also asked people how they judge the risk-benefit ration of synthetic biology. If we compare their answers from before and after the presentation we see that after our presentation the number of critical opinions about the topic had decreased, as we could convince them that aside from possible risks there are very useful applications for synthetic biology and genetics. </br> <img src=https://static.igem.org/mediawiki/2013/e/eb/Bonn_Schule_8.png> </br> When we compare the arguments people use against synthetic biology we see almost no change between before and after. So we didn’t influence the reasons why people might consider synthetic biology too dangerous or generally objectionable. </br> <img src=https://static.igem.org/mediawiki/2013/2/2e/Bonn_Schule_9.png> </br> Many people told us that they were convinced that events like our lectures in schools are be the most effective way to spread awareness about topics like synthetic biology. </br> <img src=https://static.igem.org/mediawiki/2013/4/4b/Bonn_Schule_10.png> </br>";  

content.type="Human Practice";