Template:Team:Bonn:NetworkData

content.text= "Regulation of protein activity is an important tool in the fields of basic research and synthetic biology[1.1]. 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[1.2].</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 be missing in a controlled environment, so that if the organism is to leave, it automatically dies[1.2].<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[1.2]</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[1.2]. In order to 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><h3>References</h3><a href=’http://www.ncbi.nlm.nih.gov/pubmed/18272963’> [1.1] Amy B Tyszkiewicz & Tom W Muir: ‘Activation of protein splicing with light in yeast’. ‘Nature Methods’ | Vol.5 No.4 | 303 (April 2008)</a></br><a href=’http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3554958/’>[1.2] Gerd H. G. Moe-Behrens et al., ‘Preparing synthetic biology for the world’, Front Microbiol. 2013; 4: 5.</a>";

content.childs=[3,11,9,10,7];  

content.text= "To understand the role of a specific gene or DNA region is one of the big 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 methods.</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 of activity</td><td>No ON/OFF system</td></tr><tr><td><b>Knock-Out</b></td><td>Deletion of DNA</td><td>desactivating</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>Eypensive; small activity difference</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>harder 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 the comparison of all the different Protein regulation mechanisms, our team decided to make use of a protein degradation system. The reason was that we wanted to create a system that has an immediate effect and can be used to investigate functions of every protein.";

content.text= "Irreversible regulation methods are usually associated with knock-in or -outs. But also posttranslational modification of protein structure can be subordinated in this field. </br>On the one hand Irreversible interventions cause high differences of activity and therefore appear very favorable for many investigations of protein functions.</br> On the other hand, it is impossible to take back any of those investigations, which makes it harder to analyze protein function in one organism. You need at least two organisms to compare the effect of a high protein concentration with a loss of this protein";

content.parents=[3]

content.titleShort = "Zymogen-like protein procursors";

content.titleLong = "Cleaving as posttranslational modification";

content.text= "An approach to regulate protein activity is to use posttranslational modification, especially by cleaving off an inactivating part from a translated pro-peptid. A change of conformation leads to the functional protein. The cleaving can be effected by enzymatic proteolysis, autoproteolysis or surrounding conditions (e.g. pH).</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.";

content.type="Background";

content.parents=[3];

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

content.text= "To insert the gene of interest in the bacteria genome Recombineering is often used. Utilising this system, it is possible to generate inserts or deletion. It is based on the homologous recombination of a small engineered DNA parts with a target gene. Recombination then occurs in the defined region of the genome, resulting in the insertion of this foreign sequence.</br>One way of doing it is by using bacteriophage proteins, from bacteriophage lambda, which mediates insertion of DNA cassettes ^{<a href=#5.1>[5.1]</a>} ^{<a href=#5.2>[5.2]</a>}.</br> Gene expression, so protein production, after a knock in can be regulated by a promoter. </br> The disadvantage of this way of protein regulation is obviously the durance it needs to activate the expression of the gene till the full appearance of the protein. </br>A knock out causes a full loss of function. When a genes sequence is interrupted, it may still be translated, but the resulting protein will be non-functional.Here it is not possible to regulate the protein activity. The protein misses at any time, so a control of activity levels is not possible. </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=[12,8];

content.text= "Another method on Protein level is introducing a protein degradation system. </br> There are two very important advantages. On the one hand it works much faster than any regulation system on genetically base. On the other hand itŽs a very transparent system which can be translated to a regulator for any polypeptide you could think of.";

content.summary= "Advant/td>ages 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.";

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.summary= "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></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></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=[7];

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

content.titleShort = "Ec. ClpXP system";

content.titleLong = "ClpXP Ec";

content.summary= "ClpXP Ex";

content.text= "ClpXP";  

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 degenerate 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='#13.1'>[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 have a better control, we actually 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='#13.2'>[13.2]</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 longer time^{<a href='#13.3'>[13.3]</a>}^{<a href='13.4'>[13.4]</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 id='13.1'>[13.1]</a> ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554</br> <a id='13.2'>[13.2]</a> See above </br> <a id='13.3'>[13.3]</a> See above </br> <a id='13.4'>[13.4]</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.titleShort = "Ec. sspB adaptor"

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.titleShort = "ssrA tag";

content.text= "<b> Introduction </b> </br> For cells it is important to have a steady control over their own functions and reactions. During evolution many regulation systems evolved with controlling protein concentrations amongst them. In fact, increasing or decreasing protein amount is an effective way to manipulate cell activities. Therefore proteins can be marked with special tag sequences, which allows proteolytic enzymes to degrade them. One of those tags is called ssrA and relates to proteases like the ClpXP  complex in E.coli. The tag also allows adaptor proteins such as sspB binding and delivering substrates to the proteases in order to make the process more efficient^{<a href='#15.1'>[15.1]</a>}. </br> For our project, the ssrA tag was very important, because we synthetically marked proteins with it to degrade them by placing the ssrA gen-code next to the protein code and letting ribosomes translate  the new sequence. We also used sspB and the adaptor-mediated variant as described below, whereas the direct binding pathway wasn't an opinion for us. The reason is that we needed to control degradation level and therefor we set in a splitted version of sspB, which we could reunite through light radiation. For further information about the ClpXP degradation system in our project go to ClpXP general. Although it was not part of our project, the information in chapter &quot;Translation control&quot; exhiit another important functional aspect of ssrA tags.</br> </br><b>Structure </b></br>The ssrA tag is a short sequence consisting of eleven amino-acids and is translated with the associated protein simultaneously. The sequence can be divided into two functional parts (Fig. 1). The &quot;AANDENY&quot;-part, which is directly connected with the C-terminal end of the protein, is responsible for the binding to the sspB adaptor. Each letter in the part name stands for another amino-acid, A for example means Alanine. The other part, called &quot;LAA&quot;, interacts with the ClpX subunit of the ClpXP protease. The parts are connected over an Alanine molecule^{<a href='#15.2'>[15.2]</a>}. </br> </br><div align='left'><img src='https://static.igem.org/mediawiki/2013/1/18/BonnSsra_fig1.jpg' height='76' width='344'>Fig. 1: amino-acid sequence of ssrA, from &quot;Engineering controllable protein degradation, McGinness et al, Molecular cell, 2006, PMID:16762842&quot; </div></br><b>Function </b> </br>The ssrA tag works as a degrading signal for proteases like ClpXP. Therefor ClpX owns a ssrA binding site at its axial pore. According to the availability of sspB adaptors, there are two different binding pathways. </br> </br>1. Direct binding: If a tagged protein and the ClpX subunit incidentally bump into each other in correct orientation, they develop a binding. The binding site of ClpX is made out of several loops and ssrA can be crosslinked to them. The determinant factor for this binding is the negative charged &alpha;-COOH group on the terminal alanine of ssrA, because the loops are positive charged. Using this way, ClpXP reaches a maximum degradation rate of around 4 proteins per minute^{<a href='#15.3'>[15.3]</a>}.</br> </br><div align='left'><img src='https://static.igem.org/mediawiki/2013/a/a7/BonnSsra_fig2.jpg' height='281' width='361'>Fig. 2: direct binding of a tagged GFP protein, GFP is an green fluorescence protein, from &quot;Protein unfolding by a AAA+ protease is dependent on ATP-hydrolysis rates and substrate energy landscapes, Martin et al, Nature Structural & Molecular Biology, 2008, PMID: 18223658&quot;</div> </br>2. Adaptor-mediated binding: sspB is an adaptor protein with a special binding site for the &quot;AADENY&quot;-domain of ssrA. Therefor, the sspB dimer contains a pore in each subunit and while &quot;AADENY&quot; is linked with the inside, the &quot;LAA&quot;-domain faces outwards, free to bind ClpX (Fig. 2). The affinity of this binding amounts around 20 &my;M, which suggests a relative strong  connection. The sspB dimer also owns two extremely flexible ClpX binding tails at each C-terminal end. With docking on ClpX, the &quot;LAA&quot;-domain lies closely to ClpX's axial pore and can be bound to it.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 (hab hierzu keine konkreten Daten). This lower K_M means, that a smaller amount of substrate are needed to reach the maximum degradation speed. So actually sspB doesn't increase the maximum speed, but this tempo can be reached with less substrate concentrations^{<a href='#15.4'>[15.4]</a>}^{<a href='#15.5'>[15.5]</a>}.</br> </br><div align='left'><img src='https://static.igem.org/mediawiki/2013/7/7c/BonnSsra_fig3.jpg' height='251' width='216'>Fig. 3: ssrA tag with sspB adaptor and protein substrate, from &quot;Engineering controllable protein degradation, McGinness et al, Molecular cell, 2006, PMID:16762842&quot; </div> </br><b>Translation control</b> </br>Beneath the already mentioned functions, ssrA tags also play a role in quality control during ribosomal translation of genetic codes into protein sequences. In a normal working translation tRNA molecules deliver the amino-acids and a ribosome puts them together in the right order, using a mRNA strand as template. But this complicated process can be afflicted with mistakes, for example premature abruption or missing stop codons. Mistakes mostly result in defect proteins, which can be dangerous for he cell. In order to circumvent this danger, defect proteins are tagged with ssrA for quick degradation by special tmRNA molecules. TmRNA is a mixture of mRNA and tRNA. On the one hand it is formed like a tRNA molecule, is able to bind to a ribosome and delivers one amino-acid, but on the other hand it do not have an anticodon. Instead, an ORF mRNA part can be found. ORF means &quot;open reading frame&quot; and is a coding sequence mostly for degradation tags like ssrA. As shown in figure 5, ssrA assembly is complex process. The tmRNA molecule binds to the A site of a stalled ribosome, takes over the already assembled amino-acid sequence and adds Alanine. Then it swaps the template mRNA for its ORF region and finishes translation with the new template. As a result the defect protein is now tagged and can be degradated by proteases like ClpXP^{<a href='#15.6'>[15.6]</a>}.</br> </br><div align='left'><img src='https://static.igem.org/mediawiki/2013/0/02/BonnSsra_fig4.jpg' height='500' width='325'>Fig. 4: Model for tmRNA-mediated tagging, from &quot;The tmRNA System for Translational Surveillance and Ribosome Rescue, Moore SD et al, Annual Reviews Biochemistry, 2007, PMID: 17291191&quot;</div> </br><h2><b> References </b></h2> </br><a id='15.1'>[15.1]</a> Engineering controllable protein degradation, McGinnes KE et al, Molecular cell, 2006, PMID: 16762842<a id='15.2'>[15.2]</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='15.3'>[15.3]</a> ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554</br><a id='15.4'>[15.4]</a>  See above </br> <a id='15.5'>[15.5]</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='15.6'>[15.6]</a> The tmRNA System for Translational Surveillance and Ribosome Rescue, Moore SD et al, Annual Reviews Biochemistry, 2007, PMID: 17291191</br>";

content.titleShort = "sspB-Split";

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. As a consequence 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";

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='#1'>[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='#1'>[1]</a><a href='#3'>[3]</a><a href='#4'>[4]</a><a href='#5'>[5]</a><a href='#6'>[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=1>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=2>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=3>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=4>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=5>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=6>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=[19,21,20];

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 [18.1][18.2]. 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 [18.3].</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[18.3][18.4]. Also the conformational changes in proteins induced by light occur in a matter of seconds and are also reversible[18.3]. For these reasons light is used as a method of induction in many fields where high specificity and resolution are needed, e.g. optogenetics [18.4].</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 [18.3].<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'>[18.2] 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'>[18.3] 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'>[18.4] 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'>[18.5] 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 = "other systems";

 

content.titleShort="Chemical";

content.titleLong="Chemical induction";

content.text= "Chemical induction can be used to provide both expressional and structural changes in proteins.^{<a href=#1>[1]</a>}^{<a href=#2>[2]</a>} As an advantage it is highly reliable and tunable which renders it very useful for ensuring constant expression levels.^{<a href=#1>[1]</a>} Several promoters such as pBad which is inducible with arabinose or pLac which is inducible with IPTG are frequently used for such purpose.^{<a href=#6>[6]</a>} Yet changes in protein expression require large timescales i.e. tens of minutes to hours, whereas structural changes such as dimerization (for example rapamycin induced dimerization of FRB and FKBP12^{<a href=#4>[4]</a>}) occur much faster i.e. seconds to minutes.^{<a href=#2>[2]</a>} <div class='content-image' align='center'><a href='https://static.igem.org/mediawiki/2013/8/8c/BonnRapamycin3D.jpg'><img src='https://static.igem.org/mediawiki/2013/8/8c/BonnRapamycin3D.jpg' height=260 width=260></a></br><i>A 3D Structure of a Rapamycin induced FKBP-FRB heteromer^{<a href='#7'>[7]</a>}</i></div> However compared to other methods of induction such temporal resolution is inferior. Additionally there are several problems arising from the use of chemical agents. Firstly to come into effect any molecule has to penetrate the cell membrane thus either being actively ingested by the cell or diffusing passively through it, which becomes a severe hindrance when none of these requirements are met.^{<a href=#4>[4]</a>}  Secondly any chemical can be bioactive and hence interfere with the cells metabolism or other substances.^{<a href=#1>[1]</a>}  Also specificity can be a problem especially in vivo, where often several cell types in multicellular organisms are effected. ^{<a href=#5>[5]</a>} Sub cellular spatial resolution can be difficult to achieve since molecules are subject to diffusion. It can be concluded that spatiotemporal resolution is low in chemically induced systems. <div class='content-image' align='center'><a href=https://static.igem.org/mediawiki/2013/0/0f/BonnLacOperon.jpg><img src='https://static.igem.org/mediawiki/2013/0/0f/BonnLacOperon.jpg' height=260 width=260></a></br><i>The Lac Operon: Origin of the Lac1 Promoter</i></div> <h2>References:</h2> <p><a name=1>1.</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC16554/'>J. Keith Joung, Elizabeth I. Ramm, and Carl O. Pabo: <i>A bacterial two-hybrid selection system for studying protein–DNA and protein–protein interactions.</i> &quot;PNAS&quot; (June 2000)</a></p> <p><a name=2>2.</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368803/'>Takafumi Miyamoto, Robert DeRose, Allison Suarez, Tasuku Ueno, Melinda Chen, Tai-ping Sun, Michael J. Wolfgang, Chandrani Mukherjee, David J. Meyers, and Takanari Inoue: <i>Rapid and Orthogonal Logic Gating with a Gibberellin-induced Dimerization System. </i>&quot;Nature chemical biology&quot; 8, 465–470 (2012) </a></p> <p><a name=3>3.</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724991/'>Adilson José da Silva, Antonio Carlos Luperni Horta, Ana Maria Velez, Mônica Rosas C Iemma, Cíntia Regina Sargo, Raquel LC Giordano, Maria Teresa M Novo, Roberto C Giordano, and Teresa Cristina Zangirolami: <i>Non-conventional induction strategies for production of subunit swine erysipelas vaccine antigen in rE. coli fed-batch cultures</i> &quot;Springerplus&quot;2, 322 (2013)</a></p> <p><a name=4>4.</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3133816/'>Andrei V. Karginov, Yan Zou, David Shirvanyants, Pradeep Kota, Nikolay V. Dokholyan, Douglas D. Young, Klaus M. Hahn, and Alexander Deiters: <i>Light-regulation of protein dimerization and kinase activity in living cells using photocaged rapamycin and engineered FKBP </i>&quot;Journal of the American Chemical Society&quot; 133(3) 420-423 (2011)</a></p> <p><a name=5>5.</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3529099/'>Yuan Mei and Feng Zhang:<i>Molecular Tools and Approaches for Optogenetics</i> &quot;Biological Psychatry&quot;(2012)</a></p> <p><a name=6>6.</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711423/'>Jarno Mäkelä, Meenakshisundaram Kandhavelu, Samuel M. D. Oliveira, Jerome G. Chandraseelan, Jason Lloyd-Price, Juha Peltonen, Olli Yli-Harja and Andre S. Ribeiro:<i> In vivo single-molecule kinetics of activation and subsequent activity of the arabinose promoter</i> &quot;Nucleic Acids Research&quot; (2013) </a></p><p><a name='7'>7.</a><a href='http://www.ncbi.nlm.nih.gov/pubmed/10089303'>Liang J, Choi J, Clardy J.:Refined structure of the FKBP12-rapamycin-FRB ternary complex at 2.2 A resolution.&quot;Acta crystallographica&quot;(1999)</a></p>";

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

content.titleShort = "Electrical";

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

content.parents=[19];

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

content.childs=[38,45,53,69,52,57,65,100];

content.titleShort = "project summary";  

content.titleLong = "our project in a nutshell";

content.text= "";  

content.type="Project";  

content.i = 38;

content.parents=[37];

content.childs=[40,39];

content.titleShort = "Outlook";

content.titleLong = "Outlook";

content.summary= "Outlook";

content.text= "Outlook";  

content.type="Project";

content.titleShort = "Eucaryotes";

content.titleLong = "Applications in Eucaryotes";

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

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

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

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

content.titleShort = "C. crescentus ClpXP";

content.titleLong = "C. crescentus ClpXP";

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

content.titleShort = "sspB&alpha;";

content.titleLong = "C. crescentus sspB&alpha;";

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

content.titleShort = "SspB Split";  

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

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

content.titleShort = "ssrA";

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

content.titleShort = "speed";

content.titleLong = "speed";

content.titleShort = "general applicability";

content.titleLong = "general applicability";

content.titleShort = "low influence";

content.titleLong = "low influence on proteins";

content.titleShort = "temporal control";

content.titleLong = "temporal control";

content.titleShort = "spatial control";

content.titleLong = "spatial control";

content.titleShort = "great activity difference";

content.titleLong = "great activity difference";

content.childs=[70];

content.childs=[55,56,54];

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

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

content.childs=[58,59,60,61,62,63,64];

content.titleShort = "generated plasmids";

content.titleLong = "generated plasmids";

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=#66.1>[66.1]</a>}.</br></br> <div class='content-image'><img src='https://static.igem.org/mediawiki/2013/8/8f/Bonn.Riboswitches.jpg'></br>&quot;Diversity of Riboswitches and Mechanisms of Gene Control in BacteriaMechanisms of modulation of gene expression are highly divergent in prokaryotes and involve control of transcription, translation, splicing, and mRNA stability&quot;^{<a href=#66.1>[66.1]</a>}</div><br/><div class='content-image'><img src='https://static.igem.org/mediawiki/2013/f/ff/Bonn.Riboswitches3.jpg'></br>&quot;Structural Principles of Ligand Recognition by Riboswitches(A–C) Schematic representations of a 'straight' junctional fold&quot;<sup><a href=#66.1>[66.1]</a></div> </br></br> <p><a name=66.1>[66.1]</a> <a href='http://www.sciencedirect.com/science/article/pii/S0092867412015462'> A Decade of Riboswitches Alexander Serganov, Evgeny Nudler, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA;"

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

content.titleShort = "ccdA/ccdB";  

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

content.summary= "this is a summary6";  

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

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

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

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

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

content.type="Background";

content.titleShort = "ssrA and sspB&alpha;";

content.titleLong = "Introduction to C. crescentus ssrA and sspB&alpha;";

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

content.childs=[106,107,108,109,101,110,130];

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

content.titleShort = "Project presentations"

content.titleShort = "biocom kongress Berlin";

content.titleLong = "biocom kongress Berlin";

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

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

content.parents=[105];

content.titleShort = "Day of Action SynBio";   

content.titleShort = "School Presentation";  

content.titleLong = "School Presentation";  

content.summary= "We visited Schools to tell pupils about synthetically biology and our project";  

content.text= "What do People already know about synthetically biology? How can we improve the knowledge about synthetically biology? That was 2 of the basic questions when we started brainstorming about our human Practice part of the project. We decided to give lectures about synthetically biology and our project in schools, as one part of the human practice project. We choose schools, because these pupils will possibly be the next generation of Scientifics one day. Our idea was to inspire them for science and to delete this bad image science and especially research sometimes has. Almost all classes that we visited had a natural scientific focus (e.g. Bio or Chemie Leistungskurs). But even more interesting were our visits to classes that had no natural scientific background. In addition you can find some inquiry that we asked the pupils to fill in after the lecture.";  

content.parents=[105];

content.titleShort = "Comic";

content.titleLong = "Comic";

content.summary= "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.</br>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’t want to spoil the story for you, just read the comic yourself below.";  

content.type="Human Practice";

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

content.titleLong = "Bonn";  

content.summary= "Description of Bonn";  

content.text= "Founded by the Romans in the year 12 before Christ, birthplace of Beethoven, once Capitol of Germany- now: Bonn is a vivid place to life and study. Next to Cologne, Bonn is also set on the river Rhine. Everybody gets smitten with it´s charm- such a unique mixture of tradition and modern lifestyle.</br>Stadt. City. Ville. Bonn.";  

content.summary= "this is a summary6"; (Kurzzusammenfassung des Themas)

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> <img src='https://static.igem.org/mediawiki/2013/a/a6/Bonn_poppelsdorfer_schloss.JPG' width='600'>The university ^{&copy;Jan Hansen}</div>

"; (kompletter Text!)

content.type=""; (Human PRactice, Projekt, Background, Team)