Template:Team:Bonn:NetworkData

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

content.text= "<b> Introduction: </b> </br>The ClpXP protein complex is an AAA+ protease, which means that it uses the energy of ATP hydrolysis to unfold and 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.summary= "The ssrA tag is a sequence, which allows proteolytic enzymes to degrade them. It relates to proteases like the ClpXP complex in E.coli and it also allows adaptor proteins such as sspB binding and delivering substrates to the proteases in order to make the process more efficient";

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= "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> <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'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 Rapamycin </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, Antônio 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>";

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

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

content.text= "<div class='content-image'><img src='https://static.igem.org/mediawiki/2013/c/c4/Bonn_OutlookCCSsrA.png' align=right width=800>Peptide array for testing ssrA on  amino residues relevant for sspB&alpha; binding; in every row one of the amino acids was replaced by any other amino acid one by one; the columns denote the amino acids put in after replacement; the darker the spot, the more stable is the binding of ssrA to sspB&alpha;  [44.1] </div> Chien et al. [44.1] tested the 14-amino acid peptide ssrA (AANDNFAEEFAVAA, [44.2]) 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></br> <h2>References</h2> </br>[44.1] Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</br>[44.2] 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";  

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 [42.1]</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. [42.2]Chien et al. [42.1] 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. [42.1]</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>[42.1] Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918 </br> [42.2] 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"; <!-- References-Ueberschrift muss an Format des Bildes angepasst werden -->

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

content.text= "ssrA and sspB are peptides that mediate proteolysis via the ClpXP protease system in bacteria. In this article and the related articles, focus is laid on their orthologs in C. crescentus, being referred to as ^CCssrA and <sup>CC>/sup>sspB&alpha;, respectively, omitting <b>????????????????</b> 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.

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. [74.1, 74.2] 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. 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> [74.1] 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 </br> [74.2] Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918";  

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

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

content.text= "On the mission to find new and interesting means to bring across the concepts of synthetic biology, we introduced a hand-drawn comic series consisting of three episodes in the style of the well known Star Wars movies. The readers will find themselves in a world where Galaxies are petri dishes and all the characters are bacteria. Alongside the action-filled story we step by step introduce basic concepts of synthetic biology. The use of light sabers and laser guns also offered a great opportunity to embed our system of light-degradable proteins in the plot. At our presentations at schools and at our information booth it proved to be an ideal eye-catcher for passers-by and led to them wanting to know more about the subject. During the episodes the reader accompanies the hero Obi-Wan E. Coli and his Padawan Plasmida on their journey through the Galaxy of Petri. They are fighting the villain Darth Cherry and his companions, the clones. But we don’t want to spoil the story for you, just read the comic yourself below.";  

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

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

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

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

content.text= "A reliable, yet easily adaptable mechanism for controlling protein activity is key to most areas of life and medical science research. Still, the most common approaches suffer from various flaws. Knocking genes out using homologous

recombination, knocking a gene down with RNA interference or modulating the behaviour of a protein with a chemical stimulus - just to name a few prominent methods - is either restricted to non-lethal genes, does not yield a big difference in activity, or is absolutely inaccurate and thus prone to secondary effects.</br></br> Would it not be great if one could turn off any protein, at any time, with little to no side effects? That is where iGEM Bonn 2013 and their project comes in. We aim to overcome the aforementioned difficulties by engineering a novel tool based on blue-light-inducible degradation of targeted proteins.</br></br>

choosing, and a light sensing LOV (Light, Oxygen and Voltage) domain from Avena sativa.</br></br> The advantages of our approach are obvious: Not only does the usage of light allow for a superior tempero-spatial control, but it is also much less prone to unwanted side effects than any chemical stimulus.</br> Furthermore, as we rely on a direct degradation of the targeted protein, we expect an onset of the desired effect which is much faster and at least as high as in common approaches.</br> Finally, as our system requires only a minor modification of your target protein we expect its function to not be impaired, and the tag to go unnoticed in functional observations.";  

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.text= "The University of Bonn was founded 1818 on behalf of the Prussian king Friedrich Wilhelm III.  Since that time it is a very popular and traditional alma mater academia. Many well known alumni passed their semester of studying, teaching or scientific research there, such as Karl Marx, Heinrich Heine, Friedrich Nietzsche, Ernst Moritz Arndt , Heinrich Hertz and Konrad Adenauer.The University of Bonn contains 7 different departments:School of Catholic Theology </br>School of Protestant Theology  </br>School of Law and Economics </br>School of Medicine  </br>School of Humanities  </br>School of Mathematics and Science </br> School of Agricultural Science  </br> So there is a broad offer of different courses of studies. There are also interdisciplinary programs and initiatives, like LIMES (Life and Medical Science).";  

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

content.text= "The ClpXP protein degradation system can be used for inducible protein degradation as described by Davis et al.  They made use of the native ClpXP system in E. coli with a modified ssrA-tag (DAS+4) at the target protein. ^{<a href=#161>[16.1]</a>}  The modified DAS+4 ssrA cannot bind the ClpXP without SspB. ^{<a href=#165>[16.5]</a>} Using this dependency of the protein degradation on SspB, they decided to control the function of SspB by splitting it into two parts, each of which cannot induce degradation on its own. ^{<a href=#161>[16.1]</a>} </br> </br> Splitting of SspB is possible because its tripartite structure. It consists of (1.) a ssrA-tag binding and dimerization domain (SspB [CORE]), (2.) a flexible linker and (3.) a short peptide module that docks with ClpXP (SspB [XB]). ^{<a href=#163>[16.3]</a>} To test whether the linker length can be varied, the degradation rates of GFP-DAS+4 (0.3μM) with SspB (0.15μM) were tested in vitro with 4 different linker lengths (of 5, 25, 48 and 91 amino acids). The results showed that the 25 amino acids variant triggered the fastest degradation, followed by the 25 variant with 60%, 5 variant with 30% and 91 variant with 20% of the 25 variant rate. But even the 91 amino acids variant showed a 40times faster degradation rate than the degradation system without any SspB. ^{<a href=#166>[16.6]</a>}  Further experiments (e.g. the split system with FRB-FKBP12 as a linker, see below) demonstrated that even longer linker regions (more than 200 amino acids) are functional). Thus it can be concluded that not only the length of the linker is important but also its structure. ^{<a href=#161>[16.1]</a>} </br> </br> <img src='https://static.igem.org/mediawiki/2013/1/1f/Bonn-Gfp.jpg'> ^{<a href=#166>[16.6]</a>} </br> </br> To bring both SspB parts together again for inducible degradation, they were combined with a chemical inducible heterodimerisation system: FRB and FKBP12. ^{<a href=#161>[16.1]</a>} FKBP12 (FK 06 binding protein, 12 kDa) is a binding protein (108 amino acids ^{<a href=#167>[16.7]</a>}) for the small molecule rapamycin. FRB (FKBP-rapamycin binding domain) is the FKBP12-rapamycin binding domain (100 amino acids) of the mammalian protein mTor. In the absence of rapamycin, FKBP12 and FRB show no measurable interaction, while in the presence of rapamycin they build a strong FKBP-rapamycin-FRB ternary complex. ^{<a href=#162>[16.2]</a>} </br> </br> In order to achieve inducible degradation Davis et al. created the fusion proteins SspB[CORE]-FRB and FKBP12-SspB[XB]. SspB[CORE]-FRB interacts with the ssrA-tag of the target protein. FKBP12-SspB[XB] interacts with the ClpXP. In absence of rapamycin the two parts of SspB can only bind there particular targets but can’t interact with each other. Therefore, they don’t work as an adapter between the ssrA-taged protein and the ClpXP. By adding rapamycin FRB and FKBP12 dimerize. 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= "<div align='right'><img src='https://static.igem.org/mediawiki/2013/b/b8/BonnAktionstag.JPG' height='260' width='350'></div>In cooperation with the iGEM teams of Germany also the team of Bonn organized a day of action for synthetic biology. </br> At the 7th of September ten of our members met in Bonn downtown to inform the interested civilians of our city about the international genetically engineered machine competition as well as synthetic biology in general and particularly about our project of light inducible degradation of proteins. </br> Therefore we prepared an information booth near the market place, distributed informative leaflets and visualized our ideas in terms of several posters. </br> <div align='left'><img src='https://static.igem.org/mediawiki/2013/thumb/9/97/BonnAktionstag2.jpg/800px-BonnAktionstag2.jpg' height='260' width='350'></div> At 9 o'clock in the morning we started in front of the LIMES-Institute to arrange the installation of the stand. By car all the needed equipment was transferred to the city center and there assembled under the eyes of the curious townspeople. Two hours later everything was settled and the official part of the day could begin: From 11 until 3 o'clock intrigued city dweller in every range of age stopped by to examine our exhibition walls and to ask questions, which we answered with pleasure.In the end we were surprised about the brisk participation and the lively discussions that aroused so that we bundled up and left satisfied. </br> All in all we consider the action as a great success as we were able to reduce prejudices and elucidate people about advantages of synthetic biology.";