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 only be absent in a controlled environment, so that the organism dies upon leaving this environment.[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 help the reader understand the thought process we undertook in designing our own approach in regulation of protein activity, we are going to first explain these ‘bricks’ and discuss their pros and cons.</br><h2>References</h2><a href=’http://www.ncbi.nlm.nih.gov/pubmed/18272963’> [1.1] Amy B Tyszkiewicz & Tom W Muir: &quot;Activation of protein splicing with light in yeast&quot;. &quot;Nature Methods&quot; | Vol.5 No.4 | 303 (April 2008)</a></br><a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3554958/'>[1.2] Gerd H. G. Moe-Behrens et al., &quot;Preparing synthetic biology for the world&quot;, Front Microbiol. 2013; 4: 5.</a>";

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.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.titleShort = "ClpXP Ec";

content.titleLong = "Ec. ClpXP system";

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.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.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.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.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.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>";