Template:Team:Bonn:NetworkData

content.childs=[2, 17];

content.titleLong = "Background";

content.summary= "Introduction to the concept of protein activity regulation";

content.text= "Regulation of protein activity is an important tool in the fields of basic research and synthetic biology.^{<a href=#11>1.1</a>}  The reason for this is that it offers a way to analyze the function of the examined protein. Furthermore the core intent of synthetic biology is the design and production of biological ‘machines’. This is usually accomplished via the modulation of protein activity.^{<a href=#12>1.2</a>} </br></br>An example for this would be the expression of a kill switch that is triggered by the presence of a certain molecule, resulting in cell death.  This example shows that protein regulation generally consists of two parts: the actual method of regulation, and the way this method is induced. In our example, the method of regulation is a rise in cytosolic protein levels and thus an overall increase of activity via an increased rate of expression. The method of induction is the presence of a certain molecule. This molecule could for example only be absent in a controlled environment, so that the organism dies upon leaving this environment.^{<a href=#12>1.2</a>} <div class='content-image'><img src=' https://static.igem.org/mediawiki/2013/4/47/Bonn-Backgroun%28overview%29-1.jpg'></br> Image detailing the aforementioned example.^{<a href=#12>1.2</a>} </div> There are several different methods for both, actual protein regulation and the induction of this regulation. Through combination of these ‘bricks’, several methods for regulation of protein activity can be designed, each with its own advantages and disadvantages.^{<a href=#12>1.2</a>} In order to help the reader understand the thought process we undertook in designing our own approach in regulation of protein activity, we are going to first explain these ‘bricks’ and discuss their pros and cons.</br> <h2>References</h2><a name=11>1.1</a> <a href=’http://www.ncbi.nlm.nih.gov/pubmed/18272963’> Amy B Tyszkiewicz & Tom W Muir: &quot;Activation of protein splicing with light in yeast&quot;. &quot;Nature Methods&quot; | Vol.5 No.4 | 303 (April 2008)</a></br> <a name=12>1.2</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3554958/'> Gerd H. G. Moe-Behrens et al., &quot;Preparing synthetic biology for the world&quot;, Front Microbiol. 2013; 4: 5.</a>";

content.parents=1;  

content.childs=[4,5,9,11,7,10];  

content.titleShort = "Protein regulation";  

content.titleLong = "Protein regulation mechanisms";  

content.summary= "We compare different regulation systems, focused on advantages and disadvanteges for scienticific use";

content.text= "Understanding the role of a specific gene or DNA region is one of the key challenges in modern research. Our system, which allows the <b>fast</b> and <b>convenient</b> elimination of defined proteins, is a new improved technique, with many advantages. The following table compares methods, advantages and disadvantages of several popular regulation systems.</br></br><table><tr><td><i>Regulation system</i></td><td><i>Approach of regulation</i></td><td><i>Activating / Repressing</i></td><td><i>Advantage</i></td><td><i>Disadvantage</i></td></tr><tr><td><b>Knock-In</b></td><td>Insert of DNA</td><td>Activating</td><td>Gain of function; high difference in activity</td><td>No ON/OFF system</td></tr><tr><td><b>Knock-Out</b></td><td>Deletion of DNA</td><td>Deactivating</td><td>0% Protein in organism</td><td>No ON/OFF system</td></tr><tr><td><b>Knock-Down<b></td><td>Inhibition of RNA</td><td>Repressing</td><td>Inducible</td><td>Expensive; low difference in activity</td></tr><tr><td><b>Riboswitches</b></td><td>mRNA structure; Transcription & Translation</td><td>Activating/ repressing/ degradation</td><td>Multiple aproaches and effects</td><td>Difficult to modulate<td></tr><tr><td><b>Zymogen-like</b></td><td>Protein structure</td><td>Activating</td><td>Inducible</td><td>No deactivation</td></tr><tr><td><b>Operon</b></td><td>Transcription</td><td>Inductive (substrate)/ repressing (product)</td><td>Self-regulating in organisms</td><td>Not usable for every protein</td></tr><tr><td><b>TALEs</b></td><td>Transcription</td><td>Both</td><td>Can bind several effectors</td><td>Very specific</td></tr><tr><td><b>Zinc finger</b><td>Transcription</td><td>Both</td><td>Can bind several effectors</td><td>Very specific</td></tr><tr><td><b>Direct regulation</b></td><td>Protein affinity</td><td>Both</td><td>Very fast</td><td>Too specific for easy, general use</td></tr><tr><td><b>ClpXP protease system</b></td><td>Protein degradation</td><td>&quot;Repressing&quot;</td><td>Very fast & transferable</td><td> No obvious disadvantage</td></tr></table></break></br>After comparison of various different Protein regulation mechanisms, our team decided to make use of a protein degradation system. The reason was that we wanted to create such a system was that has an immediate effect and can be used to investigate functions of <b>every</b> protein.<h2>References:</h2>see following articles<li><a onclick=showNode(3)>Irreversible Protein degradation </a></li><li><a onclick=showNode(11)> Direct inhibiton and activation</a></li><li><a onclick=showNode(9)> Transcriptional Regulation</a></li><li><a onclick=showNode(10)> Knock-down</a></li><li><a onclick=showNode(7)> Protein degradation systems</a></li>";

content.type="Background";  

content.i = 3;  

content.titleShort = "Irreversible protein regulation";

content.titleLong = "Irreversible protein regulation";

content.summary= "We point out methods of irreversible protein regulaiton and discuss their advantages and disadvantages.";

content.text= "Irreversible regulation methods are usually associated with knock-in or -outs. But also posttranslational modification of protein structure can be classified as in this field. </br>On the one hand Irreversible interventions cause high differences in activity and therefore are favorable for many investigations of protein functions.</br> On the other hand, it is impossible to revert any of changes made, which makes it harder to analyze protein function in one organism. At least two organisms are needed to simultaneously compare the effect of a normal protein concentration with a complete loss of a certain protein.<h2>References:</h2>see following articles<li><a onclick=showNode(4)>Zymogen-like protein procursors</a></li><li><a onclick=showNode(5)>Gene knock-out and knock-in</a></li>";

content.type="Background";  

content.parents=[2]

content.titleShort = "Protein cleavage";

content.titleLong = "Protein cleavage";

content.summary= "Proteins can be activated through cleavage of the precursor protein.";  

content.text= "One approach to regulate protein activity is to use posttranslational modification, especially by splitting off an inactivating part from a translated pro-peptide. A change in conformation leads to the functional protein. The cleaving can be effected by enzymatic proteolysis, autoproteolysis or surrounding conditions (e.g. pH).<sup><a href='#1.1'>1.1</a></br> A simple example is the activation of pepsinogen which is caused by a low pH (fig. 1). </br> It is a powerful system to activate proteins, which is frequently used in nature and science. Unfortunately posttranslational cleaving can usually not be reversed. Once activated the inactive pro-form of the Protein cannot be restored.<h2>References</h2><a name='4.1'>1.1</a> <a href='http://www.ncbi.nlm.nih.gov/books/NBK22589/'> Berg JM, Tymoczko JL, Stryer L.: &quot;Biochemistry. 5th Edition&quot;. New York: W H Freeman 2002.</a>";  

content.i = 5;  

content.parents=[2];

content.titleShort = "Gene knock-out and knock-in";

content.titleLong = "Gene knock-out and knock-in";

content.summary= "Another irreversible method of protein regulation in bacteria is the Gene knock-in or –out. Here the genomic DNA is manipulated to gain or lose a function, in other words whether a protein is produced or not";

content.text= "To insert the gene of interest in the bacteria genome recombineering is often used. Utilising this system, it is possible to insert or delete DNA. It is based on the homologous recombination of small engineered DNA parts with a target gene. Recombination then occurs in the defined region of the genome, resulting in the insertion of the artificial sequence.</br>One possible method is using bacteriophage proteins from bacteriophage lambda, which mediates the insertion of DNA cassettes ^{<a href=#5.1>5.1</a>} ^{<a href=#5.2>5.2</a>}.</br> Gene expression, i.e protein production, after a knock-in can be regulated by a promoter. </br> The disadvantage of this particular way of protein regulation is the time needed from the beginning of the induction until protein levels are reached. </br>A knock out causes a complete loss of function. When a gene sequence is interrupted, it may still be translated, but the resulting protein will be non-functional. In this case it is not possible to regulate the protein activity at all since the protein does not come into effect at all and therefore cannot be regulated. </br></br> <p><a name=5.1>5.1 </a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC107131/ > Kenan Murphy, Use of bacteriophage &lambda recombination functions to promote gene replacement in Escherichia coli (1998) </a> </p> <p> <a name=5.2> 5.2 </a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC148353/> Rapid modification of bacterial artificial chromosomes by ET- recombination (1998) </a> </p>";

content.childs=[12];

content.titleShort = "Protein degradation systems";

content.titleLong = "Protein degradation systems";

content.summary= "Specifically degrading protein offers a reversible method for protein regulation";

content.text= "Another method of protein control is the introduction of a protein degradation system. </br> There are two very important advantages. On the one hand it works much faster than any regulation system on a genetic base. On the other hand it is a very versatile system which can be transferred to any desirable protein.<h2>References:</h2>see following articles<li><a onclick=showNode(12)> Ec. ClpXP system</a></li>";

content.i = 9;  

content.parents=[1,2,6];

content.childs=[71,72,73];

content.titleShort = "Transcriptional regulation";

content.titleLong = "Transcriptional regulation";  

content.summary= "Advantages and disadvantages of transcriptional regulation";  

content.text= "One important regulation point is the transcription of RNA to DNA. There are many different systems which can be used in order to activate or repress the transcription. The greatest advantage are the wide spread possibilities regulation tools. Some popular examples are the operon, zinc finger and TALE. But on the other hand, one important disadvantage is the long time between induction and effect. <h2>References:</h2>see following articles <li><a onclick=showNode(71)>Operon-model</a></li><li><a onclick=showNode(72)>Zinc finger</a></li><li><a onclick=showNode(73)>TALE</a></li>";

content.parents=[2];

content.titleShort = "Knock-down";

content.titleLong = "Genetical knock-down by RNA-inference";

content.summary= "RNA interference enables reversible regulation (Knock-down) of expressed proteins";

content.text= "The genetical &quot;Knock Down&quot; by RNAi does not affect directly transcription or DNA, but it is based on RNA level. Here a DNA construct, which codes for small double-stranded interfering RNAs (siRNA), is inserted into the cell. The siRNA is processed by the RNA-induced silencing complex (RISC), described by Pratt and MacRae in 2009. The siRNA is complementary to the target mRNA and therefore forms a double strand with it and the RISC. After the RISC localizes to the target mRNA, the RNA is cleaved by a ribonuclease. Hence the mRNA of the knockdown gene is degraded and no protein can be produced.^{<a href=#10.1>10.1</a>}</br><p> <a name=10.1>10.1</a> <a href='http://www.jbc.org/content/284/27/17897.long'>The RNA-induced Silencing Complex: A Versatile Gene-silencing Machine, Ashley J. Pratt and Ian J. MacRae1 </a></p>";

content.parents=[2];

content.titleShort = "Direct inhibition and activation";

content.titleLong = "Direct inhibition and activation";

content.summary= "Direct binding of small molecules in order to activate or inactivate protein function";

content.text= "Direct binding of small molecules an activate or inactivate protein function";

content.text= "Moreover proteins can be regulated directly in many different ways. Binding of other Proteins or small molecules for example can activate or deactivate the catalytic function of proteins. For example small molecules can have great impact on protein function. They can often influence protein functions by binding to an allosteric center. Small molecules can be activating, as well as repressing, depending on the type of the molecule^{<a href=#11.1>11.1</a>}.</br></br><div class='content-image'> <img src='https://static.igem.org/mediawiki/2013/1/1c/BonnSmallMolecules.jpg' width='550'> </br>Different mechanisms for small-molecule activation of enzymes^{<a href=#11.2>11.2</a>} </div></br></br>The following picture shows the example of the allosteric activation of a glucokinase</br> <div class='content-image'> <img src=https://static.igem.org/mediawiki/2013/f/f4/BonnSmallMolecules2.jpg width='600'></br>&quot;(a) GK bound to the GKA, compound A, and glucose (blue, Protein Data Bank (PDB) ID 1V4S). Compound A binds at a site distal from the active site, which is highlighted by the presence of the substrate, glucose. (b) Structural overlay of GK in the presence of compound A and glucose with an unliganded, inactive GK (pink, PDB ID 1V4T). In the unbound GK, the GKA binding site is occluded. A large shift in the small subunit of GK occurs from the unbound to bound structures (black arrow). Glucose promotes the active conformation, which is hindered from shifting back to the inactive conformation in the presence of compound A. (c) 7 mutations (out of 13)9 identified in GK (pink) that are associated with disease map to the GKA binding site. These mutations highlight an important regulatory site within GK, and could similarly stabilize a closed, active conformation.&quot;^{<a href=#11.2>[11.2]</a>}</div></br></br><p><a name=11.1>11.1</a><a href='http://www.nature.com/nrd/journal/v3/n4/full/nrd1343.html'>Small-molecule inhibitors of protein–protein interactions: progressing towards the dream; Michelle R. Arkin & James A. Wells</a>/p></br><p><a name=11.2>11.2</a><a href='http://www.nature.com/nchembio/journal/v6/n3/full/nchembio.318.html'>Turning enzymes ON with small molecules, Julie A Zorn & James A Wells</a></p>";

content.parents=[2, 7];

content.childs=[13, 14, 15, 16, 37];

content.titleShort = "ClpXP, ssrA, SspB";

content.titleLong = "Short information about ClpXP, ssrA, SspB and how we used it in our projekt";

content.summary= "The ClpXP protease is able to degrade proteins with an ssrA tag. The SspB protein transfers this tagged protein to ClpXP.";

content.text= "We aimed to create a photosensitive protein degradation system which is useable in several types of cells with various proteins. In order to realize our system we decided to use the ClpXP protease, a protein break down system which is native to E. coli and mostly used to remove defective proteins and limit overall protein longevity ^{<a href=#123>12.3</a>} </br> . ClpXP is an AAA+ Protease which consists of two subunits, the ClpP protein and the ClpX protein. The ClpX unit is assembled from a ring of subunits some of which are able to bind ATP. The ClpP protein consists of two rings encircling a central pore which functions as substrate binding site. When a protein is degraded the tagged substrate binds to the ClpX protein. It is consequently unfolded by the subunits through the hydrolysis of ATP. Finally the substrate is passed on to the ClpP unit which cleaves the amino bonds and releases amino acids. ^{<a href=#121>12.1</a>} </br> </br> The ClpXP protease can only break down proteins with a so called ssrA tag. This is a short amino acid sequence marking the protein for degradation by ClpXP. SsrA can be bound by the transporter protein SspB which increases the efficiency of degradation. </br> The ssrA sequence has two functional parts. One part interacts with the ClpX protein of the ClpXP protease and one part is able to interact with SspB. </br> A ssrA labled protein can either bind SspB or ClpXP in order to induce degradation. In normal E. coli strains this process not necessarily dependent on SspB but rather enhanced. A mutated form of ssrA (DAS+4) weakens the native binding between ClpX and ssrA and therefore increases the dependence of SspB. ^{<a href=#122>12.2</a>} </br> </br> The use of sspB induced protein degradation has been demonstrated with GFP ^{<a href=#124>12.4</a>} ,the neurotransmitter Arc ^{<a href=#125>12.5</a>} and Lac1, the Repressor of the lac operon.^{<a href=#126>12.6</a>} </br> </br> <h3> <b>SspB:</b> </h3> SspB translocates the tagged protein to ClpXP and improves the affinity between ssrA and ClpXP and therefore mediates its breakdown.^{<a href=#127>12.7</a>} </br> To control degradation a split version of SspB can be used consisting of two domains: SspB[CORE] and SspB[XB] each of which cannot induce degradation on their own. To regain a functional construct for inducible degradation both were combined with a chemically inducible heterodimerisation system: FRB and FKBP12. In absence of rapamycin both parts are monomers and thus not functional. On addition of rapamycin FRB and FKBP12 form a dimer and consequently both SspB domains are in spatial proximity rendering them functional again.^{<a href=#129>12.9</a>} </br> Since there is a basic expression of SspB in regular E. coli strains a SspB deficient strain was used aiming to minimize constitutive degradation. </br> In our project the SspB split-version was used but instead of rapamycin induced dimerization we used a LOV-ipaA - VinD1 mediated dimer in order to produce a functional split-SspB. </br> <h2> <b>References:</b> </h2> </br> <a name=121>12.1</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209554/> ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554 </a> </br> <a name=122>12.2</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/?term=Altered+Tethering+of+the+SspB+Adaptor+to+the+ClpXP+Protease+Causes+Changes+in+Substrate+Delivery[http://www.ncbi.nlm.nih.gov/pubmed/?term=Altered+Tethering+of+the+SspB+Adaptor+to+the+ClpXP+Protease+Causes+Changes+in+Substrate+Delivery]> Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </a> </br> <a name=123>12.3</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/17291191[http://www.ncbi.nlm.nih.gov/pubmed/17291191]> The tmRNA System for Translational Surveillance and Ribosome Rescue </a> </br> <a name=124>12.4</a> <a href=http://www.sciencedirect.com/science/article/pii/S1097276503002727[http://www.sciencedirect.com/science/article/pii/S1097276503002727]> Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes Delivery Complexes with the AAA ClpXP Protease, Wah et al, Molecular Cell, 2003, PMID: 14536075 </a> </br> <a name=125>12.5</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/17317664[http://www.ncbi.nlm.nih.gov/pubmed/17317664]> Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </a> </br> <a name=126>12.6</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511798/[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511798/]> Temperature dependence of ssrA-tag mediated protein degradation, Purcell et al, 2012, Journal of Biological Engineering, PMID: 22824000 </a> </br> <a name=127>12.7</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/14967151[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 et al, 2004, Molecular Cell, PMID: 14967151 </a> </br> <a name=128>12.8</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/17317664[http://www.ncbi.nlm.nih.gov/pubmed/17317664]> Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </a> </br> </br> <a name=129>12.9</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220803/[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220803/]> Small-Molecule Control of Protein Degradation Using Split Adaptors, J. Davis et al, ACS Chem. Biol. 2011, 6, 1205-1213, PMID: 21866931 </a> </br>";

content.titleShort = "ClpXP protease";

content.titleLong = "ClpXP protease";

content.summary= "The ClpXP protein complex is an AAA+ protease, which means that it uses the energy of ATP hydrolysis to unfold and degenerate marked proteins.";

content.text= "<b> Introduction: </b> </br>The ClpXP protein complex is an AAA+ protease, which means that it uses the energy of ATP hydrolysis to unfold and degrade marked proteins. The genetic code of this complex is highly conserved and can be found in human cells as well as in the bacteria Escherichia coli. The degradation system was discovered in the early 1990's and is now well established ^{<a href=#131>13.1</a>}.In our project we used ClpXP to degrade specific proteins in order to control their amount and effect. Therefor we utilized the common adaptor sspB. This protein recognizes substrates tagged with ssrA . In order to achieve a better control we made use of a sspB split system. For more detailed information about the ClpXP degradation system in our project go to ClpXP general. </br> </br> <b> Structure:  </b> </br> The ClpXP complex consists of two functional and structural different parts. The ClpX protein, an ATPase, is a hexameric ring (Fig.1) with a pore in the center<div align='lef'><img src='https://static.igem.org/mediawiki/2013/9/98/Bonn_Clp_Fig1.jpg' height='348' width='320'>Fig. 1: the hexameric ring of ClpX, each color represents a subunit, from  &quot;ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554&quot;</div> </br> Each subunit contains a N-terminal domain (Fig.2, B), which assumes the adaptor recognition and is stabilized by coordinated zinc atoms.However, the important part of a subunit is the AAA+ module (Fig.2, C), divided in a large and a small domain. <div align='left'><img src='https://static.igem.org/mediawiki/2013/d/d0/BonnClp_Fig2.jpg' height='262' width='499'>Fig. 2: structure of a ClpX subunit, B: the N-terminal domain with brown zinc atoms, C: the AAA+ module, from  &quot;ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophy Acta, 2012, PMCID: PMC3209554&quot;</div> </br> Between these domains the ATP binding site can be found, but not every subunit is able to bind the nucleotide. The arrangement of ATP binding and not-binding subunits in the hexameric ring is essential for the tertiary structure and the conformation changes after hydrolysis. The ClpP protein is a tetradecameric peptidase (Fig. 3, A and B).The subunits are arranged as two heptameric rings, one ring stacked on top of the other, with also a narrow pore in the center. This pore leads into the proteolytic chamber, which is barrel-shaped. Every subunit accommodates a classical Ser-His-Asp catalytic triad and oxyanion hole inside the chamber. Those proteolytic acitve sites (Fig. 3, C) can form several hydrogen bonds to the substrate ^{<a href=#131>13.1</a>}. <div align='left'><img src='https://static.igem.org/mediawiki/2013/0/06/BonnClp_Fig3.jpg' height='401' width='382'>Fig. 3: structure of ClpP, A: side view with stabilizing residues (blue), B: top view with the pore (red), C: active site of a subunit with a bonded substrate, from &quot;ClpXP, an ATP-powered unfolding and protein-degradation machine, Bakeret al, Biochim Bi phys Acta, 2012, PMCID: PMC3209554&quot;</div> </br> </br> </br><b> Functions: </b> </br>The ClpXP complex has three tasks to fulfill: </br> </br>1. Binding: The substrate binding process at the ClpX unit is normally conducted with the aid of an adaptor protein.This protein identifies tagged substrates and delivers them to the complex (Fig. 4, left). In order to transfer the protein, the adaptor also binds to the ClpX unit (Fig. 4, right), so that parts of the tag get approximated to a special binding site on the complex. After the linking between the tag and the binding site has been performed, the unfolding starts.The binding process also works without an adaptor protein, but an adaptor enhances the degratation by improving enzyme-substrate affinity. </br> </br> 2. Unfolding and translocation: The translocation of polypeptids through the ClpX unit to the ClpP chamber is an active process using energy from ATP-binding and -hydrolysing cycles. Therefor are several ATP molecules linked to the ClpXprotein. The separation of one phosphate molecule results in conformation changes, which pulls the linked protein more inside the pore located in the center of ClpX. The remaining ADP has to be replaced by a new ATP molecule before the cycle can start again. Meanwhile the unfolding is driven automatically, because the large tagged protein has to fit into the narrow pore, which forces the three-dimensional structure to become linear. </br> </br> 3. Degradation: The axial pore of the ClpP unit is also very narrow, allowing the entry of only small unfolded peptides into the proteolytic chamber. Inside the chamber, the substrate binds to an active site over several hydrogen bonds. It also can be linked to multiple active sites. In this position, proteins are cleaved in a maximum speed of around 10,000 proteins per minute by ClpP alone. If the ClpX unit is added, the rate is with ~0.2 proteins per minute and 0.3 &my;M substrate much lower, because the unfolding process takes more time ^{<a href=#131>13.1</a>} ^{<a href=132>13.2</a>}. </br> </br> <div align='left'><img src='https://static.igem.org/mediawiki/2013/b/b8/BonnClp_Fig4.jpg' height='311' width='628'>Fig. 4: Model of the degradation process with the sspB adaptor, from &quot;Engineering controllable protein degradation, McGinness et al, Molecular cell, 2006, PMID:16762842'</div> <h2><b> References </b> </h2></br> </br> <p><a name=131>13.1</a> ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554</br> <a name=132>13.2</a> Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes DeliveryComplexes with the AAA ClpXP Protease, Wah et al, 2003, Molecular cell, PMID: 14536075</p></br>";  

content.type="Background";  

content.i = 14;

content.titleShort = "Ec. SspB adaptor";

content.titleLong = "Ec. SspB adaptor";

content.summary= "The sspB protein is an adaptor responsible for delivering ssrA-tagged substrates to the ClpXP protease in order to enhance their degradation";

content.text= "<b>Introduction </b> </br> The sspB protein is an adaptor responsible for delivering ssrA-tagged substrates to the ClpXP protease in order to enhance their degradation.Thus, bacterias like E.coli or C. crescentus regulate the concentration of marked proteins and also are in control of their quality. Even though degeneration of tagged substrates is possible without sspB, sspB delivering is a common process, because it improves the affinity between ssrA and ClpXP^{<a href='#14.1'>14.1</a>}. </br> In our project, we used a sspB split variant instead of the normal sspB in order to control cleaving rate. The two parts of this version only stay divided until we ray them with light of a special wavelength. After that, the fractions form an unit and can function normal from now on as described below. </br> </br><b>Structure </b> </br>The sspB adaptor is a homomeric dimer (Fig. 1), which means that itconsists of two identical domains. Together the domains form a pore with the ssrA binding sites inside. Each domain also owns a C-terminal tail ending in a ClpX binding module, called XP. The amino-acid sequence of XP is highly conserved so that mutations in it mostly cause extremely decrease in activity, whereas the linker sequence differs from species to species ^{<a href='#14.2'>14.2</a>}^{<a href='#14.3'>14.3</a>}. <div align='left'><img src='https://static.igem.org/mediawiki/2013/5/5c/BonnsspB_Fig2.jpg' height='202' width='403'>Fig. 1: Ribbon diagramm of the sspB dimer with a bound srrA-tagged protein, from 'Versatile modes of peptide recognition by the AAA+ adaptor protein SspB, Levchenko et al, 2005, nature structural and molecular biology, PMID: 15880122</div> </br> </br></br></br><b>Function</b></br> SspB enhances degradation of ssrA-tagged proteins by lowering the K_M. Thus, with a given substrate concentration sspB-mediated cleaving runs faster than without sspB (Fig. 2) </br> <div align='left'><img src='https://static.igem.org/mediawiki/2013/d/d8/BonnSspB_Fig1.jpg' height='232' width='371'>Fig. 2: Diagramm, shows the degradation rate of GFP-ssrA without sspB, with sspB and with two mutations, the given substrate concentration is 0.3 &my;M, from 'Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes Delivery Complexes with the AAA ClpXP Protease, Wah et al, Molecular Cell, 2003, PMID: 14536075' </div></br> Therefor, the sspB dimer contains a pore and while 'AADENY' is linked with the inside, the 'LAA'-domain (respectively 'DAS') faces outwards, free to bind ClpX (Fig. 3).</br> The affinity of this binding amounts around 20 &my;M, which suggests a relative strong  connection. The two extremely flexible ClpX binding tails with XP at the C-terminal end  dock on ClpX. So the 'LAA'- domain lies closely to ClpX's axial pore and can be bound to it. <div align='left'><img src='https://static.igem.org/mediawiki/2013/7/7c/BonnSsra_fig3.jpg' height='151' width='116'>Fig. 3: Model of sspB with a bound ssrA-tagged substrate, from 'Engineering controllable protein degradation, McGinness et al, Molecular cell, 2006, PMID:16762842' </div></br> To sum up, there are three bonds connecting the ssrA-sspB-ClpX-complex and making it relative stable: ssrA with sspB, sspB with ClpX and ssrA with ClpX. Hence follows a lower K_M than the direct binding process has^{<a href='#14.4'>14.4</a>}^{<a href='#14.5'>14.5</a>}. <h2> References </h2> <a id='14.1'>14.1</a>Bivalent Tethering of SspB to ClpXP Is Required for Efficient Substrate Delivery: A Protein-Design Study, Bolon et al, 2004, Molecular Cell, PMID: 14967151 </br><a id='14.2'>14.2</a>see above </br> <a id='14.3'>14.3</a> Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes Delivery Complexes with the AAA ClpXP Protease, Wah et al, Molecular Cell, 2003, PMID: 14536075 </br> <a id='14.4'>14.4</a> see above</br> <a id='14.6'>[14.5]</a> Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </br> <a id='14.4'>14.4</a>  ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554</br>";  

content.i = 16;  

content.titleShort = "SspB-Split";

content.titleLong = "Control of Protein Degradation Using Split Adaptors";  

content.summary= "Using the protein degradation whith help of SspB, we control the function of SspB by splitting it into two parts, each of which cannot induce degradation on its own.";  

content.text= "The ClpXP protein degradation system can be used for inducible protein degradation as described by Davis et al.  They made use of the native ClpXP system in E. coli with a modified ssrA-tag (DAS+4) at the target protein. ^{<a href=#161>16.1</a>}  The modified DAS+4 ssrA cannot bind the ClpXP without SspB. ^{<a href=#165>16.5</a>} Using this dependency of the protein degradation on SspB, they decided to control the function of SspB by splitting it into two parts, each of which cannot induce degradation on its own. ^{<a href=#161>16.1</a>} </br> </br> Splitting of SspB is possible because its tripartite structure. It consists of (1.) a ssrA-tag binding and dimerization domain (SspB [CORE]), (2.) a flexible linker and (3.) a short peptide module that docks with ClpXP (SspB [XB]). ^{<a href=#163>16.3</a>} To test whether the linker length can be varied, the degradation rates of GFP-DAS+4 (0.3μM) with SspB (0.15μM) were tested in vitro with 4 different linker lengths (of 5, 25, 48 and 91 amino acids). The results showed that the 25 amino acids variant triggered the fastest degradation, followed by the 25 variant with 60%, 5 variant with 30% and 91 variant with 20% of the 25 variant rate. But even the 91 amino acids variant showed a 40times faster degradation rate than the degradation system without any SspB. ^{<a href=#166>16.6</a>}  Further experiments (e.g. the split system with FRB-FKBP12 as a linker, see below) demonstrated that even longer linker regions (more than 200 amino acids) are functional). Thus it can be concluded that not only the length of the linker is important but also its structure. ^{<a href=#161>16.1</a>} </br> </br> <img src='https://static.igem.org/mediawiki/2013/1/1f/Bonn-Gfp.jpg'> ^{<a href=#166>16.6</a>} </br> </br> To bring both SspB parts together again for inducible degradation, they were combined with a chemical inducible heterodimerisation system: FRB and FKBP12. ^{<a href=#161>16.1</a>} FKBP12 (FK 06 binding protein, 12 kDa) is a binding protein (108 amino acids ^{<a href=#167>16.7</a>}) for the small molecule rapamycin. FRB (FKBP-rapamycin binding domain) is the FKBP12-rapamycin binding domain (100 amino acids) of the mammalian protein mTor. In the absence of rapamycin, FKBP12 and FRB show no measurable interaction, while in the presence of rapamycin they build a strong FKBP-rapamycin-FRB ternary complex. ^{<a href=#162>16.2</a>} </br> </br> In order to achieve inducible degradation Davis et al. created the fusion proteins SspB[CORE]-FRB and FKBP12-SspB[XB]. SspB[CORE]-FRB interacts with the ssrA-tag of the target protein. FKBP12-SspB[XB] interacts with the ClpXP. In absence of rapamycin the two parts of SspB can only bind there particular targets but can’t interact with each other. Therefore, they don’t work as an adapter between the ssrA-taged protein and the ClpXP. By adding rapamycin FRB and FKBP12 dimerize. Thus the two parts of SspB get in a spatial closeness and function as an adapter. As a consequent the target protein gets degraded. ^{<a href=#161>16.1</a>} </br> </br> <img src='https://static.igem.org/mediawiki/2013/2/21/Bonn-rapa-split.jpg'> ^{<a href=#161>16.1</a>} </br> The efficiency of this system was demonstrated in the following in vitro experiments: GFP-DAS+4 was incubated with ClpXP, FKBP12-SspB[XP], SspB[CORE]-FRB and an ATP-regenerating system. Without rapamycin there was no degradation of GFP. The addition of rapamycin led to a reduction of GFP-ssrA of around 50% in only 360 seconds (degradation rate of 0.58min-1enzyme-1)(figure 2a). ^{<a href=#161>16.1</a>} Furthermore it was tested how long the degradation system needs to assemble and thus to reach the maximal degradation rate in this system. A time of 20 seconds was measured (figure 2b). ^{<a href=#161>16.1</a>} </br> </br> <img src='https://static.igem.org/mediawiki/2013/d/d0/Bonn-GFP-Abbau.jpg'> ^{<a href=#161>16.1</a>} </br> For in vivo testing they introduced the system into an SspB- mutant of E. coli by the plasmid pJD427. This plasmid contains SspB[CORE]-FRB with the weak constitutive promoter proB, FKBP12-SspB[XB] with the strong constitutive promoter proC and a medium-copy p15a origin of replication. For a target protein the lacI transcription repressor was used with a DAS+4-tag recombined to the C-terminus. Usually LacI represses lacZ transcription and thus production of β-galactosidase. Therefore degradation of LacI leads to an increasing β-galactosidase activity. As the assay showed absence of rapamycin results in no change of the β-galactosidase activity. Addition of rapamycin, however, leads to increasing β-galactosidase activity. Hence the system also worked in vivo (in an acceptable time). ^{<a href=#161>16.1</a>} </br> <img src='https://static.igem.org/mediawiki/2013/2/2d/Bonn-rapa-gel.jpg'> ^{<a href=#161>16.1</a>} </br> <h2> <b>References:</b> </h2> <a name=161>16.1</a> <a href=http://'www.ncbi.nlm.nih.gov/pmc/articles/PMC3220803/'> Small-Molecule Control of Protein Degradation Using Split Adaptors, J. Davis et al, ACS Chem. Biol. 2011, 6, 1205-1213, PMID: 21866931 </a> </br> <a name=162>16.2</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/15796538'> Charaterization of the FKBP•Rapamycin•FRB Ternary Complex, L. Banaszynski, C. Liu et al,  J. AM. CHEM. SOC. 2006, 127, 4715-4721, PMID: 15796538 </a> </br> <a name=163>16.3</a> <a href=http://'www.ncbi.nlm.nih.gov/pubmed/14536075'>  Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes Delivery Complexes with the AAA+ ClpXP Protease, D. Wah et al, Molecular Cell, Vol. 12, 355-363, August, 2003, PMID: 14536075  </a> </br> <a name=164>16.4</a> <a href='http://www.sciencedirect.com/science/article/pii/S0969212607003152'> Structure and Substrate Specifity of an SspB Ortholog: Desing Implications for AAA+ Adaptors, P. Chien et al, Cell Press, October 2007, 1296-1305, PMID: 17937918 </a> </br> <a name=165>16.5</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/16762842'> Engineering controllable protein degredation , McGinness et al, Mol Cell. 2006 Jun 9, PMID: 16762842 </a> </br> <a name=166>16.6</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/?term=Altered+Tethering+of+the+SspB+Adaptor+to+the+ClpXP+Protease+Causes+Changes+in+Substrate+Delivery'> Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinness et al, J Biol Chem. 2007 Apr 13; PMID: 17317664 </a> </br> <a name=167>16.7</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed'> http://www.ncbi.nlm.nih.gov/pubmed";

case 17:

content.childs=[18,32,33,34];

content.text= "Regulating protein levels and conformation is a basic feature of any living organism, helping to maintain homeostasis and maximize efficiency while also increasing its versatility and adaptability. Thus, it is of great interest for basic research where tools are needed to provide protein regulation artificially. High spatiotemporal control is vital for essays which study protein function^{<a href='#171'>17.1</a>}, since often exact concentration or conformation is needed. In synthetic biology this is of particular importance since biochemical circuits rely on accurate mechanisms of control and oftentimes employ multiple means of induction.^{<a href='#2'>[2]</a>} However there is a multitude of methods available to induce changes in protein structure or expression.^{<a href='#171'>17.1</a><a href='#173'>17.3</a><a href='#174'>17.4</a><a href='#175'>17.5</a><a href='#176'>17.6</a>}Yet each technique has its own assets and drawbacks which are examined more closely in the following paragraphs.</br> </br> </br><table border='1'><tr><td>Induction Method</td><td>Temporal resolution</td><td>Spatial resolution</td><td>Reliability</td><td>Ease of use</td><td>Distinctive advantage</td></tr><tr><td>Light</td><td>Very high <µs</td><td>Very high</td><td>High</td><td>Cloning, genetical engineering required</td><td>Reversibel, in vivo</td></tr><tr><td>Chemical</td><td>Low 10sec - min</td><td>low</td><td>Very high (especially expression)</td><td>Very easy (expression), genetical engineering required (conformational)</td><td>Fine-tune gene expression</td></tr><tr><td>Heat</td><td>Low (expression only)</td><td>Very low (whole organism affected)</td><td>Very high</td><td>Very easy</td><td>Does not change protein conformation</td></tr><tr><td>Electrical</td><td>High <ms</td><td></td><td></td><td>Application can be difficult</td><td>Exact measurements</td></tr></table>  <h2>References:</h2> <p><a name=171>17.1.</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/18272963'> Amy B Tyszkiewicz & Tom W Muir: <i>Activation of protein splicing with light in yeast.</i> &quot;Nature Methods&quot; | Vol.5 No.4 | 303 (April 2008)</a></p> <p><a name=172>17.2.</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2955201'>X. Gu, M. Trybilo, S. Ramsay,M. Jensen, R. Fulton, S. Rosser, and D. Gilbert <i>Engineering a novel self-powering electrochemical biosensor.</i> &quot;Systems and Synthetic Biology&quot;4(3) (Sep 2010)</a></p> <p><a name=173>17.3.</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/?term=Heat-induced%20conformational%20change%20and%20increased%20chaperone%20activity%20of%20lens%20alpha-crystallin'> Das BK, Liang JJ, Chakrabarti B. <i>Heat-induced conformational change and increased chaperone activity of lens alpha-crystallin.</i> &quot;Current Eye Research&quot;  Apr;16(4):303-9  (1997)</a></p> <p><a name=174>17.4.</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/23359284'> Yang J, Yang H, Sun X, Delaloye K, Yang X, Moller A, Shi J, Cui J. <i>Interaction between residues in the Mg2+-binding site regulates BK channel activation.</i> &quot;The journal of general physiology&quot; (Feb 2013)</a></p> <p><a name=175>17.5.</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/10537212> Richard DJ, Sawers G, Sargent F, McWalter L, Boxer DH. <i>Transcriptional regulation in response to oxygen and nitrate of the operons encoding the [NiFe] hydrogenases 1 and 2 of Escherichia coli.</i> &quot;Microbiology&quot;145 ( Pt 10)  (Oct 1999)</a></p> <p><a name=176>17.6.</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC97448/'> Maen Qa&quot;Dan, Lea M. Spyres, and Jimmy D. Ballard <i>pH-Induced Conformational Changes in Clostridium difficile Toxin B.</i> &quot;Infection and Immunity&quot; 68(5) (May 2000)</a></p>";

content.childs=[36,21,20];

content.titleLong = "Light as a means of induction";

content.summary= "Discussion of light and it's advantages and disadvantages as a means of induction";

content.text= "Using light as a means of regulation is common in nature, as it is for example used to regulate the circadian rhythm of an organism and plays a key role in the phototropism of plants ^{<a href=#181>18.1</a>}^{<a href=#182>18.2</a>}. Through the linkage of peptides with photosensitive domains, regulation of gene expression and the induction of conformational changes in proteins via light can be achieved ^{<a href=#183>18.3</a>}.</br><div class='content-image'><img src='https://static.igem.org/mediawiki/2013/4/44/Bonn-Light_1_-18.3-.jpg'> </br>The image gives and example of how linkage of a photosensitive domain with a desired protein can result in an inducible change of conformation and thus activity[18.3]</div> The use of light has many advantages. It brings with it a high spatiotemporal resolution and specificity, as it only interacts with the photosensitive domains^{<a href=#183>18.3</a>}^{<a href=#184>18.4</a>}. Also the conformational changes in proteins induced by light occur in a matter of seconds and are also reversible^{<a href=#183>18.3</a>}. For these reasons light is used as a method of induction in many fields where high specificity and resolution are needed, e.g. optogenetics ^{<a href=#184>18.4</a>}.</br></br>In bigger organisms like mammals, the lighting of the intended cells can prove to be quite problematic, as there may be several layers of tissue the light has to penetrate. Furthermore, using light requires the modification of the amino acid sequence of the targeted peptide. These changes make the process of designing and building a functioning construct quite difficult and complex, as can be seen in the following diagram ^{<a href=#183>18.3</a>}.<div class='content-image'><img src='https://static.igem.org/mediawiki/2013/7/71/Bonn-Light-2.JPG'> </br>The diagram shows how several constructs were designed, yet only one construct had the desired activity [18.5]</div></br><h3>References</h3><a href='http://www.ncbi.nlm.nih.gov/pubmed/20150866'>[18.1] Figueiro, M.G.; Rea, M.S. (February 2010).: &quot;Lack of short-wavelength light during the school day delays dim light melatonin onset (DLMO) in middle school students&quot;. Neuro Endocrinology Letters 31 (1): 92–6. PMID 20150866.</a></br><a href='http://www.ncbi.nlm.nih.gov/pubmed/18952772'>^{<a href=#182>18.2</a>}Han, I.-S, W. Eisinger, T.-S. Tseng, and W. R. Briggs, 2008.: &quot;Phytochrome A regulates the intracellular distribution of phototropin1-green fluorescent protein in Arabidopsis thaliana&quot; Plant Cell 20: 2835-2847.</a></br><a href='http://www.ncbi.nlm.nih.gov/pubmed/22520757'>^{<a href=#183>18.3</a>}Lungu et al, April 20, 2012: &quot;Designing Photoswitchable Peptides Using the AsLOV2 Domain&quot; Chemistry and Biology 19, 507-517</a></br><a href='http://www.ncbi.nlm.nih.gov/pubmed/17643087'>^{<a href=#184>18.4</a>} Zhang F, Aravanis AM, Adamantidis A, de Lecea L, Deisseroth K.: &quot;Circuit-breakers: optical technologies for probing neural signals and systems.&quot; Nat Rev Neurosci. 2007 Sep;8(9):732</a></br><a href='http://www.ncbi.nlm.nih.gov/pubmed/18667691'>^{<a href=#185>18.5</a>} Devin Strickland et al., &quot;Light-activated DNA binding in a designed allosteric protein&quot;, PNAS, August 5, 2008 vol. 105 no. 31</a>";

content.titleLong = "LOV";

content.summary= "LOV";

content.text= "LOV";  

content.childs=[];

content.titleLong = "Other systems";

content.summary= "More systems";

content.text= "More systems";  

content.childs=[37];

content.titleLong = "pDawn system";  

content.summary= "The plasmid pDawn was designed by Ohlendorf et al.^{<a href=#ref21.1>21.1</a>} in 2012 together with its counter plasmid pDusk. Both plasmids are single plasmid systems, which allow the activation (pDawn) or repression (pDusk) of gene expression by blue light. They are easy to implement in the laboratory and lead to up to 460-fold activity change upon ilumination.";

content.text= "The plasmid pDawn was designed by Ohlendorf et al.^{<a href=#ref21.1>21.1</a>} in 2012 together with its counter plasmid pDusk. Both plasmids are single plasmid systems, which allow the activation (pDawn) or repression (pDusk) of gene expression by blue light. They described their system as easy to implement in the laboratory and suitable for both preparative and analytical purposes. Further, they were able to show up to 460-fold induction upon ilumination using their system. </br></br> Because, pDawn (and pDusk) is a system that regulates gene expression and thus acts on DNA level it is an eligible candidate for us to compare the speed of action of our system, which acts on protein level to an DNA regulation system. As an additional benefit, with pDawn, we implement a powerful light-induceable system, with low background expression and high dark/light expression difference in the iGEM database. </br></br> Ohlendorf et al. (2012)^{<a href=#ref21.1>21.1</a>} had previously designed the histidine kinase YF1, which phosphorylates FixJ in the absence of blue light. The phosphorylated FixJ drives robust gene expression from the FixK2 promotor as shown in figure 1 for both pDawn and the pDusk. Upon light absorption, net kinase activity of YF1 and consequently gene expression under control of the FixK2 promotor is greatly reduced. pDusk an pDawn enable light-repressed or light-induced gene expression in Escherichia coli and are easy to implement in the laboratory. Therefore, all components of the YF1/FixJ TCS were assembled on only one medium-copy plasmid in which YF1 and FixJ are constitutively expressed by the Lac^q promotor. Target genes can be introduced via the multiple-cloning site (MCS) under the control of the pFixK2 promotor, allowing light-repressed gene expression. To obtain pDawn the light effect is inverted by the introduction of a gene-inversion cassette into pDusk. This gene-inversion cassette is based on the &lambda; phage repressor cI and the &lambda; phage promotor pR. (see figure 1)</br></br><div class=contant-image><img src=https://static.igem.org/mediawiki/2013/a/a1/Bonn_MS_Figure1_pDusk%26pDawn.jpg></br>Figure 1: Plasmids pDusk an pDawn. YF1 and FixJ are constitutively expressed by the Lacq promotor. Target genes can be introduced via the multiple-cloning site (MCS) under the control of the pFixK2 promotor. (Ohlendorf et al. 2012^{<a href=#ref21.1>21.1</a>})</div></br>Ohlendorf et al. (2012)^{<a href=#ref21.1>21.1</a>} analyzed the system by the insertion of the red-fluorescent reporter protein DsRed Express2. That way they were able to measure up to 460-fold induction upon ilumination. However, they also found that their plasmid, which acts on gene expression level, only allows light-induced perturbations on the timescale of hours.</br></br><h2>References</h2></br><p><a name=ref21.1>21.1</a> <a href=http://www.sciencedirect.com/science/article/pii/S0022283612000113> Ohlendorf et al. (2009) From Dusk till Dawn One-Plasmid Systems for Light-Regulated Gene Expression.</a></p>";

case 32:

 

content.titleShort = "Heat";

content.titleLong = "Using heat as a means of induction";

content.summary= "Discussion of heat and it's advantages and disadvantages as an induction tool";

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 ^{<a href=#331>33.1</a>}.</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 ^{<a href=#332>33.2</a>} ^{<a href=#333>33.3</a>}. 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 ^{<a href=#332>33.2</a>} ^{<a href=#333>33.3</a>}.</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 ^{<a href=#333>33.3</a>}</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 ^{<a href=#333>33.3</a>} <h3>References</h3><a name=331>33.1</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/23912482'>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 name=332>33.2</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/6322174'>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 name=333>33.3</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/10739675'>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>";

case 34:

content.i = 34;

content.parents=[17];

content.childs=[];

content.titleShort = "Electrical Induction";

content.titleLong = "Electrical Induction";

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=#341>34.1</a>} or enzymes which change their catalytic activity when the membrane potential changes.^{<a href=#342>34.2</a>} Channel proteins require minimal changes in electrical potential and change conformation in submillisecond timescales.^{<a href=#343>34.3</a>}  However its usage in synthetic biology is yet to be demonstrated.<h2>References:</h2> <p><a name=341>34.1</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=342>34.2</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=343>34.3</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.parents=[18];

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

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=#361>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=#361>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=361>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=361>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=361>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.i = 39;

content.parents = [38];

 

content.i = 40;

content.parents=[38];  

content.childs=[41,74,43];

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=401>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=401>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.childs=[];  

content.titleShort = "^CcClpXP";

content.titleLong = "C. crescentus: ClpXP";

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=#413>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=#411>41.1</a>, <a href=#412>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=#412>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 name=411>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 name=412>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 name=413>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.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=#441>44.1</a>} </div></br>Chien et al. ^{<a href=#441>44.1</a>} tested the 14-amino acid peptide ssrA (AANDNFAEEFAVAA, ^{<a href=#442>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=#443>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 name=441>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 name=442>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 name=443>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.type="Project";

content.titleLong = "Advantages";

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

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

content.type="Project";

content.titleShort = "Speed";

content.titleLong = "Speed";

content.summary= "Due to the use of the protease system protein regulation is fast";

content.type="Project";

content.titleShort = "General applicability";

content.titleLong = "General applicability";

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

content.type="Project";

content.titleShort = "Low influence";

content.titleLong = "Low influence on proteins";

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

content.type="Project";

content.titleShort = "Temporal control";

content.titleLong = "Temporal control";

content.summary= "Using light easily makes a temporal specific control of activation possible";

content.type="Project";

content.titleShort = "Spatial control";

content.titleLong = "Spatial control";

content.summary= "High spatial control can be achieved easily by local irradation with light";

content.type="Project";

content.titleShort = "Great activity difference";

content.titleLong = "Great activity difference";

content.summary= "Degradation of the protein leads to a great difference of activity";

content.type="Project";

case 52:

content.i =52;

content.parents=[37];

content.titleShort="Achievements";

content.titleLong="Achievements";

content.summary="How far we managed to realize our project idea";

content.text="";

content.type="Project"

content.childs=[55,54];

content.titleLong = "Applications";

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

content.type="Project";

content.childs=[67, 68];

content.titleShort = "Kill-switch for Lab Safety";

content.titleLong = "Light inducible Kill-switch systems using toxin-antitoxin modules";

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.type="Project";

case 55:

content.i =57;

content.parents=[37];

content.titleShort = "Results";

content.titleLong = "Results";

content.summary= "Results";

content.text="";

content.type="Project";

case 65:

content.i = 66;

content.parents=[2];

content.childs=[];

content.titleShort = "Riboswitch";

content.titleLong = "Riboswitch";

content.summary= "";

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=#661>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=#661>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=#661>66.1</a></div> </br></br> <p><a name=661>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.type="Background";

content.i = 67;

content.parents=[54];

content.childs=[];

content.titleShort = "MazEF";

content.titleLong = "The maz toxin-antitoxin system";

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.type="Project";

content.i = 68;

content.parents=[54];

content.childs=[];

content.titleShort = "ccdAB";  

content.titleLong = "The ccd toxin-antitoxin system";

content.summary= "CcdB leads to double strand breaks in bacterial genomic DNA and thus is a potent toxin leading to cell death. ccdA compromises the toxic effect of ccdB.";  

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=#681>68.1</a>}^{<a href=#682>68.2</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=681>68.1</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=682>68.2</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.type="Project";

case 69:

case 71:

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=#711>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=#712>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 is no’t able to inhibit the transcription. If there is enough product, it can bind to the allosteric center of the repressor, which improves it's affinity to the operator DNA. Now it is able to inhibit transcription.^{<a href=#713>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=#714>71.4</a>}</div></br> </br></br><p><a name=711>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=712>71.2</a> <a href='http://geneticsengineering.blogspot.de/'>geneticsengineering.blogspot </a></p><p><a name=713>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=714>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.i = 72;

content.parents=[9];

content.titleShort = "Zinc finger";

content.titleLong = "Zinc finger";

content.summary= "Zinc fingers can be engineered to bind desired DNA sequences";

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 a DNA binding and a dimerization domain. They can be differentiated due to 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. Hence the activating or repressing effect is not defined by the zinc finger itself, but by the effector Protein it is binding to^{<a href=#721>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=#722>72.2</a>}</div></br></br> <p><a name=721>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=722>72.2</a> <a href='http://en.wikipedia.org/wiki/Zinc_finger'>Cartoon representation of the zinc-finger motif of proteins</a></p>";

content.type="Background";

content.i = 73;

content.parents=[9];

content.childs=[];

content.titleShort = "TALE";

content.titleLong = "Transcription activator-like effectors";

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

content.text= "Tales are proteins which 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 be engineered easily. Thus the user can define the domain to bind to. Very similar to the zinc finger they actually do not regulate transcription but bind effector proteins which are able to activate transcription. The great advance, in comparison to the zinc finger domain, is it's easy way of engineering. Scientists can regulate very specificlly 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>";

case 74:

content.i = 101;

content.parents=[100];

content.childs=[102,103];

content.titleShort = "Presentations"

content.titleLong = "Project presentations";

content.summary= "We presented iGEM and our project on two scientific conferences.";

content.text= "";

content.type="Human Practice";  

case 102:

content.i = 104;

content.parents=[101,105];

content.titleShort = "Wiki"

content.titleLong = "Wiki";

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

content.text= "";

content.type="Human Practice";  

case 106:

content.i = 107;

content.parents=[100];

content.childs=[];

content.titleShort = "SynBio Day";

content.titleLong = "SynBio Day";

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.type="Human Practice";

content.parents=[100];  

content.titleShort = "Schools";  

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.i = 109;

content.parents=[112];

content.childs=[];

content.titleShort = "Comic: About";

content.titleLong = "About the comic";

content.summary= "We introduced a hand drawn comic series called &quot;LOV Wars&quot; about Obi-Wan E. Coli and his adventures in the Galaxy of Petri. Basic concepts of synthetic biology are explained along the way.";  

content.text="On the mission to find new and interesting means to bring across the concepts of synthetic biology, we introduced a comic series consisting of three episodes in the style of the well known Star Wars movies. The readers will find themselves in a world where Galaxies are petri dishes and all the characters are bacteria. Alongside the action-filled story we step by step introduce basic concepts of synthetic biology. The use of light sabers and laser guns also offered a great opportunity to embed our system of light-degradable proteins in the plot. At our presentations at schools and at our information booth it proved to be an ideal eye-catcher for passers-by and led to them wanting to know more about the subject. During the episodes the reader accompanies the hero Obi-Wan E. Coli and his Padawan Plasmida on their journey through the Galaxy of Petri. They are fighting the villain Darth Cherry and his companions, the clones. But we don&apos;t want to spoil the story for you, just read the comic yourself below."; content.type="Human Practice";

case 110:

content.i = 110;

content.parents=[100];

content.titleShort = "LOV-Wars Minigame</br>About";

content.titleLong = "About the LOV-Wars Shooter";

content.summary= "The thoughts and ideas behind our game.";

content.text= "<p>In our effort to present our project in a simple and yet entertaining way, we introduced a mini game in May 2013. We hoped that this new, never before used approach to human practices arouses the interest of people that would normally not deal with synthetic biology. The game takes up different aspects of synthetic biology and our project and embeds it in an entertaining environment.</br>The game&apos;s overall design was adapted to our comic series. Like that the player is able to recognize the characters from the comic in the game and draw a connection between them.</p><p>The game is a mixture between a simple aim-and-shoot game and an adventure game: at certain score levels the player gets the possibility to achieve upgrades that influence the player&apos;s success. The gameplay and the upgrades convey our system of light-induced degradation of proteins.</p><p>In the game the player has to kill the villains, the red &quot;Clones&quot;, with a laser gun in order to gain points. However neutral bacteria, the &quot;Civilians&quot;, will also be killed by the UV light and thus points are deducted. To avoid that, the player is able to upgrade his UV laser to a blue laser, which has got a defined wavelength and affects only the red Clones. As those carry a ssrA-tagged protein, they are rendered harmless by the blue light: the &quot;evil&quot; proteins are degraded, they turn from evil clone warriors to nice and peaceful bacteria. The Civilians however are not harmed by the blue light.</p><p>Every once in a while super villain &quot;Darth Cherry&quot; appears in the game. If you succeed to hit him three times the player is rewarded with extra time and a score bonus. With the upgrade &quot;plasmid of death&quot; Darth Cherry can be terminated with only one hit, which demonstrates the facility to change properties of bacteria by inserting plasmids.</p><p>The other upgrades show the player how to improve our system: You can get a wider and stronger laser so that you can hit more bacteria at a time, the &quot;Falcon of LB&quot; upgrade can be used to attract more bacteria and ice makes all the bacteria slower.</br>As the game&apos;s main intention is to give a simple introduction and to draw interest, we had to generalize and simplify all these aspects in order to be able to embed them in the game and not to overwhelm the player.</br>As of this writing already several hundred people from all over the world have played and enjoyed our game. The shooter has proved to be a great mean of promotion!";

content.i = 111;

content.summary = "Play our great Minigame and fight against Darth Cherry!";

content.text = "Darth Cherry becomes more and more powerful, recruiting an armee of his red-troopers. Defeat the evil empire with the power of blue light! <br/><br/>(You will need the Java7 web plugin to run the game. You can download Java from <a href='http://java.com'>java.com</a>. The game is signed with a key belonging to the iGEM-Team Bonn, which you have to trust manually. Java will ask you for permission.)<br/><br/><br/><small>Hint: Please enter your two-letter counry code! A list of all codes can be <a href='https://en.wikipedia.org/wiki/ISO_3166-1_alpha-2#Officially_assigned_code_elements'>viewed here</a>, usually the code is the first two letters of your countrie's name. For example the code for Germany is DE.</small><br/><br/><br/></div><div align='center' style='padding-bottom: 120px;'><iframe src='http://igem13.uni-bonn.de/shared/game/game-container.html' width='100%' height='636px' frameborder='0' seamless style='margin-top:000px;background:transparent;margin-bottom:-200px;'></iframe></div><br/><br/><br/><br/><br/><br/></div>";

content.i = 112;

content.text = "<img src=https://static.igem.org/mediawiki/2013/c/c1/Bonn_Comic_episode1_page1.jpg width=735px><img src=https://static.igem.org/mediawiki/2013/a/ad/Bonn_Comic_episode1_page2.jpg  width=735px><img src=https://static.igem.org/mediawiki/2013/1/19/Bonn_Comic_episode1_page3.jpg width=735px><img src=https://static.igem.org/mediawiki/2013/8/80/Bonn_Comic_episode1_page4.jpg  width=735px><img src=https://static.igem.org/mediawiki/2013/6/6c/Comic_EN_2.0_%281%29.jpg width=735px><img src=https://static.igem.org/mediawiki/2013/8/84/Comic_EN_2.0_%282%29.jpg  width=735px><img src=https://static.igem.org/mediawiki/2013/9/97/Comic_EN_2.0_%283%29.jpg width=735px><img src=https://static.igem.org/mediawiki/2013/1/19/Comic_EN_2.0_%284%29.jpg  width=735px><img src=https://static.igem.org/mediawiki/2013/8/8d/Bonn_Comic_EN_3.0_%281%29.jpg width=735px><img src=https://static.igem.org/mediawiki/2013/5/58/Bonn_Comic_EN_3.0_%282%29.jpg  width=735px><img src=https://static.igem.org/mediawiki/2013/8/8c/Bonn_Comic_EN_3.0_%283%29.jpg width=735px><img src=https://static.igem.org/mediawiki/2013/b/b4/Bonn_Comic_EN_3.0_%284%29.jpg  width=735px>";

content.i = 130;

content.parents=[100];

content.childs=[131,133,136,137,138];

content.titleShort = "About us";

content.summary= "The Team - Presentation of all team members";

content.summary= "Pictures from any Event hosted by our Team and many more";

content.text= "<table border=0><tr><td colspan='2'><div align='left'><b><h2> Science Slam</h2></div></b></td>          </tr>          <tr>              <td colspan='2'>                <ahref='https://static.igem.org/mediawiki/2013/5/5b/BonnSlamPanorama.jpg'>                <img src='https://static.igem.org/mediawiki/2013/5/5b/BonnSlamPanorama.jpg' width='840' height='200'></a></td>          </tr>          <tr>              <td>                <a href='https://static.igem.org/mediawiki/2013/d/d2/BonnSlam2.JPG'>                <img src='https://static.igem.org/mediawiki/2013/d/d2/BonnSlam2.JPG' width='418' height='350'> </a>  </td><td>                <a href='https://static.igem.org/mediawiki/2013/6/6e/BonnSlam3.JPG'>            <img src='https://static.igem.org/mediawiki/2013/6/6e/BonnSlam3.JPG' width='418' height='350'> </a>  </td></tr>          </table>          <table border=0>          <tr rowspan='2'>              <td colspan='2'><a href='https://static.igem.org/mediawiki/2013/2/2d/BonnSlamGruppenfoto.JPG'>                <img src='https://static.igem.org/mediawiki/2013/2/2d/BonnSlamGruppenfoto.JPG' width='518' height='650'></a>              </td>              <td>                <a href='https://static.igem.org/mediawiki/2013/f/f8/BonnSlam5.JPG'>                <img align='top' src='https://static.igem.org/mediawiki/2013/f/f8/BonnSlam5.JPG' width='318' height='310'> </a>        <a href='https://static.igem.org/mediawiki/2013/5/5f/BonnSlam6.JPG'>                <img style='padding-top:5px' align='top' src='https://static.igem.org/mediawiki/2013/5/5f/BonnSlam6.JPG' width='318' height='335'>              </td>          </tr>              <td colspan='3'>                <a href='https://static.igem.org/mediawiki/2013/e/e5/BonnSlam7.JPG'>                <img src='https://static.igem.org/mediawiki/2013/e/e5/BonnSlam7.JPG' width='840' height='525'> </a>  </td></table>          <table border=0>          <tr>              <td colspan='2'>                            <divalign='left'>              <b><h2> School presentations </h2></b> </div>              </td>        </tr><tr>              <td colspan='2'>                <a href='https://static.igem.org/mediawiki/2013/2/2c/BonnSchule.JPG'>                <img src='https://static.igem.org/mediawiki/2013/2/2c/BonnSchule.JPG' width='840' height='390'> </a>  </td></tr>          <tr>              <td>                <a href='https://static.igem.org/mediawiki/2013/f/fc/BonnFranziSchule.JPG'>                <img align='top' src='https://static.igem.org/mediawiki/2013/f/fc/BonnFranziSchule.JPG' width='448' height='250'> </a></td>              <td rowspan='2'>                <a href='https://static.igem.org/mediawiki/2013/3/30/BonnLinz2.JPG'>                <img src='https://static.igem.org/mediawiki/2013/3/30/BonnLinz2.JPG' width='388' height='590'> </a>  </td></tr>          <tr>              <td>                <a href='https://static.igem.org/mediawiki/2013/a/ac/BonnLinz1.JPG'>                <img align='top' src='https://static.igem.org/mediawiki/2013/a/ac/BonnLinz1.JPG' width='448' height='335'></a>    </td></tr>          </table>          <table border=0>          <tr>              <td colspan='2'>                <div align='left'>              <b><h2>Information booth</h2> </div></b>              </td>          </tr>    <tr><td colspan='2'>                <a href='https://static.igem.org/mediawiki/2013/3/3c/BonnStand3.JPG'><img src='https://static.igem.org/mediawiki/2013/3/3c/BonnStand3.JPG' width='840' height='600'> </a>  </td></tr>          <tr>              <td>                <a href='https://static.igem.org/mediawiki/2013/4/4c/BonnStand2.JPG'>                <img src='https://static.igem.org/mediawiki/2013/4/4c/BonnStand2.JPG' width='523' height='350'> </a>  </td><td>                <a href='https://static.igem.org/mediawiki/2013/a/a4/BonnStand1.JPG'>            <img src='https://static.igem.org/mediawiki/2013/a/a4/BonnStand1.JPG' width='313' height='350'> </a>  </td></tr>          </table>          <table border=0>          <tr>              <td colspan='2'>                <divalign='left'>              <b><h2>Biocom Congress</h2> </div></b>              </td>          </tr><tr>              <td>                <a href='https://static.igem.org/mediawiki/2013/c/c9/BonnBiocomCongress1.JPG'>                <img src='https://static.igem.org/mediawiki/2013/c/c9/BonnBiocomCongress1.JPG' width='313' height='350'></a>              </td>              <td>                <a href='https://static.igem.org/mediawiki/2013/f/f7/BonnBiocomCongress2.png'>                <img src='https://static.igem.org/mediawiki/2013/f/f7/BonnBiocomCongress2.png' width='523' height='350'></a>              </td>          </tr>          </table>          <table border=0>          <tr>  <td><div align='left'>              <b><h2>Bio NRW Video Contest</h2> </div></b>              </td></tr>          <tr>              <td>                <a href='https://static.igem.org/mediawiki/2013/1/1d/BonnVideowettbewerb.JPG'>                <img src='https://static.igem.org/mediawiki/2013/1/1d/BonnVideowettbewerb.JPG' width='840' height='600'></a>              </td>          </tr>          </table>          <table border=0>          <tr>    <td colspan='3'>              <div align='left'>              <b><h2>Labwork</h2> </div></b>              </td></tr>          <tr>              <td rowspan='2'>                <a href='https://static.igem.org/mediawiki/2013/f/f8/BonnLabAnnikaNiklas.jpg'>                <img src='https://static.igem.org/mediawiki/2013/f/f8/BonnLabAnnikaNiklas.jpg' width='322' height='450'></a>              </td>              <td>                <a href='https://static.igem.org/mediawiki/2013/2/27/BonnLBKulturen.jpg'>                <img src='https://static.igem.org/mediawiki/2013/2/27/BonnLBKulturen.jpg' width='255' height='225'> </a></td>              <td>                <a href='https://static.igem.org/mediawiki/2013/8/88/BonnZentrifuge.jpg'>                <img src='https://static.igem.org/mediawiki/2013/8/88/BonnZentrifuge.jpg' width='255' height='225'> </a></td>          </tr>          <tr>              <td>                <a href='https://static.igem.org/mediawiki/2013/9/96/BonnCherryKulturen.jpg'>                <img src='https://static.igem.org/mediawiki/2013/9/96/BonnCherryKulturen.jpg' width='255' height='220'></a>              </td>              <td>                <a href='https://static.igem.org/mediawiki/2013/1/11/BonnPlatten.jpg'>                <img src='https://static.igem.org/mediawiki/2013/1/11/BonnPlatten.jpg' width='255' height='220'> </a>  </td></tr>          </table>          <table border=0>          <tr>              <td>                <ahref='https://static.igem.org/mediawiki/2013/c/ca/BonnPellets.jpg'>                <imgsrc='https://static.igem.org/mediawiki/2013/c/ca/BonnPellets.jpg' width='418' height='350'> </a></td>              <td>                <a href='https://static.igem.org/mediawiki/2013/a/ac/BonnLabNiklas.jpg'>                <img src='https://static.igem.org/mediawiki/2013/a/ac/BonnLabNiklas.jpg' width='418' height='350'> </a></td>          </tr>          </table>";

content.i = 133;

content.parents=[130];

content.i = 136;

content.text = "<h1>History</h1><p>Coming off a successful first competition entry and an amazing first jamboree in Amsterdam for our team, our team dove head-first into a off-season video production jaunt, winning first prize in a video competition organized by our state’s ministry of science, research and innovation. In our video, we addressed the contest’s theme of 'Biotechnology Today' by creating an animated short titled 'Synthetic Biology - A Simply Explained'. In addition to an invitation to the prize ceremony held at a cinema museum in nearby Düsseldorf, our winnings included 1500 Euros, which we put to good use as seed money for 2013’s iGEM competition!</p><p>In continuing our practice of recruiting first-year students for our team, we held a well-attended introductory presentation in november and managed to attract over 40 interested students and faculty from our University. After our initial warm-up iGEM team meetings, in which we  introduced our new team members to the competition, presenting some successful projects and innovative human practice advances in the last year as well as brain-storming both wetlab and outreach ideas, we signed up 23 full members and developed both an ambitious wetlab project as well as a full schedule of human practice activities.</p><p>During our mid-winter break in February and March, our experienced and veteran team members took to Powerpoint and the teaching lab as we provided our new members with a two-week seminar- and practical-based crash course on molecular biology and its corresponding lab techniques, offering the very first biology lab experience for quite a few of our Freshmen. It is our philosophy that we try to provide early hands-on research experience as well as an eye-opener into the world of synthetic biology for interested students from any university department.</p><p>In March, our whole team, advisors and instructors in tow, crashed a departmental seminar talk at the nearby CAESAR institute held by Prof. Dr. Andreas Möglich, who has done extensive research on light-sensitive proteins. As a team that has been working with those very same light-sensitive LOV proteins for the last two years, it was a great opportunity to discuss the many pros and cons surrounding LOV proteins with an expert in the field! Based on Prof. Dr. Möglich’s recommendations, we tweaked our project outline, incorporating a novel light-sensitive protein switch in our proposed protein degradation system.</p><p>Following our introductory lab sessions focussing on mastering that arcane technique of pipetting Bradford Assays (to measure and improve pipetting accuracy, not to humilate the pipetter with low R2 values!), we started preliminary project-related work, gathering and cloning required genes from labs all over the world.</p><p>And thus it began!</p>";

content.type = "Team";

content.text= "Bonn has a long and eventful history. The city was founded by the Romans in the year 12 before Christ and continued to grow ever since. Bonn also was the birthplace of the famous musician and composer Beethoven and even was the Capitol of West Germany for 41 years. Today Bonn has around 300 000 habitants and has become the cultural of the region. It is set on the river Rhine very close to Cologne. Everybody gets smitten with its charm and unique mixture of tradition and modern lifestyle. The city&apos;s historic center and the setting close to the river Rhine turn it into a wonderful place to live and study.<p style='text-align:center;'>the city&apos;s slogan:</br><i>&quot;Stadt. City. Ville. Bonn&quot;</i></p></br><p><a href=https://static.igem.org/mediawiki/2013/0/0b/Bonn_by_night.JPG><div class='content-image'align='center'><img src='https://static.igem.org/mediawiki/2013/0/0b/Bonn_by_night.JPG' width='600'></a>Bonn by night ^{&copy;Jan Hansen}</div></p><p><div class='content-image'align='center'><a href=https://static.igem.org/mediawiki/2013/a/a6/Bonn_poppelsdorfer_schloss.JPG><img src='https://static.igem.org/mediawiki/2013/a/a6/Bonn_poppelsdorfer_schloss.JPG' width='600'></a>The university ^{&copy;Jan Hansen}</div><p><a href=https://static.igem.org/mediawiki/2013/0/0b/Bonn_by_night.JPG><div class='content-image'align='center'><img src='https://static.igem.org/mediawiki/2013/7/75/Bonn_beethoven.JPG' width='600'></a>The famous Beethoven statue in the city center ^{<a href=http://www.bone-diagenesis.org/moziloCMS/gallery.php?gal=Bonn-nbsp~Impressions>[source]</a>}</div></p><h2>References</h2><p><a name='1'>1.</a> <a href='http://www.bonn.de'>http://www.bonn.de</a></p><p><a name='2'>2.</a> <a href='http://de.wikipedia.org/wiki/Bonn'>http://de.wikipedia.org/wiki/Bonn</a></p>";

content.text= "The University of Bonn was founded 1818 on behalf of the Prussian king Friedrich Wilhelm III.  Since then it has been a very popular and traditional alma mater academia. Many well known scientists such as Karl Marx, Heinrich Heine, Friedrich Nietzsche, Ernst Moritz Arndt, Heinrich Hertz and Konrad Adenauer studied, teached or researched here.</br></br>The University of Bonn contains 7 different departments:<li>School of Mathematics and Science</li><li>School of Law and Economics</li><li>School of Medicine</li><li>School of Humanities</li><li>School of Agricultural Science</li><li>School of Catholic Theology</li><li>School of Protestant Theology</li><p>So there is a broad offer of different courses of studies. There are also interdisciplinary programs and initiatives, e. g. the LIMES (LIfe and MEdical Science) institute.</p><p><a href='https://static.igem.org/mediawiki/2013/b/bc/Bonn_Hauptgeb%C3%A4ude_Mudda.JPG'><div class='content-image'><img width='550' align='center' src='https://static.igem.org/mediawiki/2013/b/bc/Bonn_Hauptgeb%C3%A4ude_Mudda.JPG'></a><p align=center>The university^{&copy;Jan Hansen}</p></img></div><p><a href='https://static.igem.org/mediawiki/2013/a/a6/Bonn_poppelsdorfer_schloss.JPG'><div class='content-image'><img width='550' align='center' src='https://static.igem.org/mediawiki/2013/a/a6/Bonn_poppelsdorfer_schloss.JPG'></a><p align=center>Poppelsdorfer Schloss^{&copy;Jan Hansen}</p></img></div>";

content.text= "Since the first International Genetically Engineered Machine competition (iGEM) in 2003 an increasing number of teams participates every year in this exiting competition. </br></br>However, our personal impression was, that even in the scientific community synthetic biology and iGEM are only poorly known. Therefore, we wanted to further inform the scientific community about this event, the idea behind it and the our individual project. In order to do so, we decided to participate in scientific events and join the discussion with other researchers. Depending on the even we had the opportunity to give a Presentation or present a Poster about our project.</br></br><div class=contant-image><img src='https://static.igem.org/mediawiki/2013/0/0a/Bonn_MS_Presentations1.jpg'></br>Figure 1: Pictures from the different scientific conventions</div></br>Moreover, such events are very beneficial for us as iGEM team, because we usually get all lot of good ideas and input form other scientists regarding our project. Further, we used this events to meet with other iGEM teams.</br></br>Nonetheless, we were aware that we cannot know how many scientists are already well informed about this topics or if our approach is suitable to inform other scientists. For this reason we came up with the idea to evaluate this question by the use of questionnaires, to check if this impression is true and how effective our initiative in this area is.";

content.text= "<b><a href='#1'>1. 3A – Assembly</a></b></br></br><b><a href='#2'>2. Agarose preparation</a></b></br></br><b><a href='#3'>3.Agarose gel casting</a></b></br></br><b><a href='#4'>4.loading the agarose gel and starting electrophoresis</a></b></br></br><b><a href='#5'>5. Preparation of Antibiotic stocks</a></b></br></br><b><a href='#6'>6. Colony PCR</a></b></br></br><b><a href='#7'>7. Preparation of Glycerol Stocks (iGEM) </a></b></br></br><b><a href='#8'>8.Plasmid Preparation </a></b></br></br><b><a href='#8a'>8a. Midi-Prep (Promega)</a></b></br></br><b><a href='#8b'>8b. Mini-Prep (Promega)</a></b></br></br><b><a href='#9'>9.Preparation of LB agar plates</a></b></br></br><b><a href='#10'>10. PCR - Clean-Up (Macherey und Nagel)</a></b></br></br><b><a href='#11'>11. Preparation of chemocompetent DH5-alpha cells</a></b></br></br><b><a href='#12'>12. Re-transformation of BioBricks</a></b></br></br><b><a href='#13'>13. Strand directed Mutagenesis PCR</a></b></br></br><b><a href='#14'>14. Transformation using Ligation product (in DH5alpha or XL1Blue) </a></b></br></br></br></br><h2><a name='1'>1. 3A – Assembly</a></h2><hr><i>NOTE: Enzymes and buffers were provided by Promega </i></br></br><b>Restriction (50 &micro;l Reaction)</b></br>- 25 &micro;l Mastermix Restriction-Enzyme Buffer (2x, with BSA)</br>- add 1 &micro;l from every restriction enzyme to 500ng backbone equimolar DNA</br>- fill up to 50 &micro;l dest. water</br>- incubate for 1.5h-3h at 37&ordm;C </br>- inactivate for 20min at 70&ordm;C (no clean-up, if directly used for ligation) </br></br><b>Ligation (20 &micro;l Reaction)</b></br>- 2,0 &micro;l equimolare restriction samples (inserts)</br>- 20ng/1.5 &micro;l backbone</br>- fill up to 17.7  &micro;l with dest. water</br>- incubate 5min at 37&ordm;C</br>- add 2 &micro;l Ligation buffer (10x) and 0.3 &micro;l T4 DNA Ligase </br>- incubate for 3h RT or 15&ordm;C over night </br>- inactivate for 10min at 70&ordm;C</br></br></br><h2><a name='2'>2.Agarose preparation</a></h2><hr><b>Materials:</b></br>- Agarose </br>- 500ml bottle</br>- TBE (1x)</br></br><b>Procedure:</b></br>- dissolve 1g agarose in 100ml TBE (1x), resulting in 1% agarose</br>- heat for 2 minutes in a microwave at maximal power</br>- mix</br>- cook until it boils (1 min)</br>- mix carefully</br><i>2 clues for a successful boiling: 1. no cords, 2. boiling retardation</i></br>- store in 65&ordm;C incubator</br></br></br><h2><a name='3'>3.Agarose gel casting</a></h2><hr>- assemble the gel chamber (chamber + 2 fences + 1 - 2 gel combs ) under the ethidiumbromid hood</br>- prepare 50ml falcon tube (label it!)</br>- fill 40ml warm agarose in the falcon tube</br>- add 4 &micro;l  ethidiumbromid (1:10.000) under the ethidiumbromid hood</br>- mix by inverting 2-3 times</br>- fill agarose in the gel chamber</br>- wait until the gel becomes solid (about 20minutes)</br></br></br><h2><a name='4'>4.loading the agarose gel and starting electrophoresis</a></h2><hr>- add LoadingDye (1:6) to your sample</br>- remove comb and fence from the gel</br>- place agarose gel in the electrophoresis chamber</br>- pipette samples carefully in the pockets (small pockets: up to 20 &micro;l, big pockets: up to 50 &micro;l)</br>- Place lid on the electrophoresis chamber and connect the electrodes to it.</br>- set parameters (high resolution: 120V, 20-30minutes, low resolution: 130V, 15min)</br>- evaluate gel under UV-light</br></br></br><h2><a name='5'>5. Preparation of Antibiotic stocks</a></h2><hr><b>Ampicillin:</b></br>- dissolve 100 mg ampicillin in 1 ml dest. water</br>- store at 20 &ordm;C</br></br><b>Chloramphenicol:</b></br>- dissolve 18 mg chloramphenicol in 1 ml ethanol</br>- store at 20 &ordm;C</br></br></br><h2><a name='6'>6. Colony PCR</a></h2><hr>- inoculate 10 &micro;l dest. water with colony.</br>- use 1  &micro;l of this water for one reaction:</br><img src='https://static.igem.org/mediawiki/2013/9/91/Bonn_MS_Methods1.png' width='550px'></br></br></br><h2><a name='7'>7.Preparation of Glycerol Stocks (iGEM)</a></h2><hr>- autoclave glycerol (60%)</br>- add 0,5 ml Glycerol to 1,5 ml cell culture in a cryo tube</br>- mix</br>- shock freeze in liquid nitrogen</br>- store at -80 &ordm;C</br></br></br><h2><a name='8'>8. Plasmid Preparation</a></h2><hr><h2><a name='8a'>8a. Midi-Prep (Promega)</a></h2><hr>- centrifuge 50 ml of liquid cell culture for 10min at 5000g </br>- decant supernatant</br>- resuspend with 3 ml resuspension solution </br>- add 3 ml cell lysis solution and incubate for maximal 3 min at room temperature </br>- add 5 ml neutralization solution </br>- centrifuge for 20 min at 20 &ordm;C, 5000g </br>- vacuum pump lysat through cleaning column into binding column </br>- abolish cleaning column</br>- vacuum pump with 10 ml endotoxin removal wash solution</br>- vacuum pump with 20 ml column wash solution</br>- dry membrane by vacuum</br>- add 600  &micro;l nuclease free water on membrane</br>- centrifuge for 5 min at 1750 g into a fresh tube</br></br></br><h2><a name='8b'>8b. Mini-Prep (Promega)</a></h2><hr>- fill 1,5 ml overnight-culture in a new tube</br>- centrifuge for 30 seconds at maximal speed </br>- decant supernatant</br>- Repeat previous steps 2-5 times (depending on growth density)</br>- resuspend with 600  &micro;l dest water </br>- add 100  &micro;l cell lysis buffer </br>- after 1min (maximum 2min) add 350  &micro;l of neutralization buffer </br>- centrifuge 3min at maximal speed</br>- place mini column in a collection tube and transfer supernatant into PureYield^TM Mini column</br>- centrifuge for 15 sec at maximal speed</br>- add 200  &micro;l Endotoxin Removal Wash</br>- centrifuge for 15 sec at maximal speed</br>- add 400  &micro;l column Wash solution</br>- centrifuge for 30 sec at maximal speed</br>- place mini column in a new tube</br>- add 30  &micro;l elution buffer to the mini column, incubation at RT for 1 min</br>- centrifuge for 15 seconds at maximal speed </br>- store DNA at -20 &ordm;C</br></br></br><h2><a name='9'>9. Preparation of LB agar plates</a></h2><hr>1l LB agar will result in approximately 30 plates</br></br>- dissolve 15g agar and 20g LB in 1l dest. Water</br>- autoclave</br>- cool down to 60-70&ordm;C </br>- add antibiotics (1:1000) under the laminar airflow cabinet</br>- mix</br>- cast plates (approximately  20ml / plate)</br>- dry for 2h by room temperature</br>- store at 4&ordm;C</br></br></br><h2><a name='10'>10. PCR - Clean-Up (Macherey und Nagel) </a></h2><hr><b>Gel Extraction: </b></br>1. add double amount NTI to gel</br>2. Incubate 3-7minutes at 50&ordm;C and at 1000rpm (until gel is dissolved)</br>- continue with regular Clean-up Protocol (from step 3.) </br></br><b>Cleanup: </b></br>1. fill up sample with dest. water to 50 &micro;l, if necessary</br>2. add double amount NTI to the sample</br>3. place column in a collection and add transfer solution to the column</br>4. centrifuge 30 seconds at 11.000g and discard flow through </br>5. add 700 &micro;l NT3 </br>6. centrifuge 30 seconds at 11.000g and discard flow through </br>7. repeat step 5) and 6)</br>8. centrifuge 1minute at 11.000g</br>9. place column in a new tube and dry column at 70&ordm;C for 5min</br></br>small parts (<1000bp): </br>9a. place column in a new tube and add 30 &micro;l Elution buffer </br>9b. incubate 1min at room temperature</br>9c. Centrifuge 1minute at 11.000g </br></br>Bigger Parts: (>1000bp) </br>9A. place column in a new tube and add 20 &micro;l Elution buffer </br>9B. incubate at 70&ordm;C for 5minutes </br>9C. centrifuge at 50g for 1minute</br>9D. centrifuge at 11.000g for 1minute</br>9E. repeat step 9A. to 9D.</br></br></br><h2><a name='11'>11. Preparation of chemocompetent DH5-alpha cells</a></h2><hr>- Start with 200 ml Overnight culture with OD₆₀₀ of 0,6-0,8</br>- centrifuge at 4 &ordm;C, 4500 g, 10 minutes </br>- decant supernatant</br>- resuspend with 40 ml inoune transformation buffer </br>- centrifuge at 4 &ordm;C, 4500 g, 10 minutes </br>- decant supernatant</br>- resuspend in 20 ml inoune transformation buffer </br>- add 1,5 ml DMSO</br>- incubate 10 minutes on ice</br>- transfer 100 – 200 &micro;l into precooled tubes</br>- shock freeze in liquid nitrogen</br></br></br><h2><a name='12'> 12. Re-transformation of Bio Bricks</a></h2><hr>- add 10  &micro;l sterile dest water to DNA on plate </br>- incubate for 10 min at RT</br>- take 2  &micro;l, leave rest on plate </br>- store plates at -20 &ordm;C </br>- add the 2  &micro;l DNA solution to 5  &micro;l competent DH5-alpha</br>- incubate for 30 min on ice </br>- heat shock for 45 s at 42 &ordm;C </br>- incubate 3 min on ice </br>- add 250  &micro;l LB medium at 37 &ordm;C </br>- incubate for 45 min at 37 &ordm;C, 800 rpm </br>- plate 300 &micro;l on Agar-plate with appropriate antibiotic </br>- dry 15min at RT</br>- incubate at 37&ordm;C over night</br></br></br><h2><a name='13'> 13. Strand directed Mutagenesis PCR</a></h2><hr>Prepare master mix and add template as follows: </br><img src='https://static.igem.org/mediawiki/2013/b/b6/Bonn_MS_Methods2.png' width='550px'></br>- start PCR-Program: </br>1. initial denaturation 94&ordm;C for 120 seconds </br>2. Denaturating 94&ordm;C for 30 seconds</br>3. Annealing 94&ordm;C for 30 seconds</br>4. Elongation 68 for 720 seconds</br>5. Repeat step 2) to 4) 12x </br></br></br><h2><a name='14'>14. Transformation using Ligation product (in DH5alpha or XL1Blue) </a></h2><hr>- thaw bacteria on ice </br>- add 2-4 &micro;l Ligation mixture to 50 &micro;l bacteria</br>- incubate 30minutes on ice </br>- heat shock 30-45 seconds (XL1Blue preferably 35 seconds) at 42&ordm;C</br>- incubate 6min on ice</br>- add 250 &micro;l LB medium at 37&ordm;C</br>- incubate for 45minutes at 37&ordm;C, 800rpm </br>- plate 300 &micro;l on appropriate antibiotic</br>- dry 15 minutes at RT</br>- incubate at 37&ordm;C over night</br>";

content.type="Project";

content.summary= " On of the most important things in biotechnology is to create and maintain optimal environmental conditions for the microorganisms. Therefore temperature, pressure, pH, nutrition, gasexchange and other factors need to be adjusted and controlled in a Bioreactor";

content.text= "Microorganisms can be used to produce a variety of different products. Especially, when using genetic engineering it is possible to produce complex products like antibiotics or drugs, but also food additives like amino acids.</br></br>However, with the use of microorganisms for the production of desired products certain difficulties arise. On of the most important things in biotechnology is to create and maintain optimal environmental conditions for the microorganisms. Therefore temperature, pressure, pH, nutrition, gasexchange and other factors need to be adjusted and controlled. Therefore, a bioreactor is not only a device to grow cells or tissues, but in most cases a high-tech device, with real-time control of the relevant parameters.</br></br>Bioreactors can be classified as batch, fed batch or continuous.";

content.i = 112;