Template:Team:Bonn:NetworkData

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>

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 ccSspB?10-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.";  

content.text= "iGEM Bonn 2013 was invited to join the Biocom AG Kongress 'Biotechnologie 2020+' (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.";  

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>

Our system relies on two key components: A tiny (just 15 amino acids!) tag that is fused to the C-Terminus of a protein of your

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= "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 align='center'><img src=https://static.igem.org/mediawiki/2013/8/8c/BonnRapamycin3D.jpg height=260 width=260> </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 align='center'><img src=https://static.igem.org/mediawiki/2013/0/0f/BonnLacOperon.jpg height=260 width=260></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: A bacterial two-hybrid selection system for studying protein–DNA and protein–protein interactions. “PNAS” (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: Rapid and Orthogonal Logic Gating with a Gibberellin-induced Dimerization System. “Nature chemical biology” 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: Non-conventional induction strategies for production of subunit swine erysipelas vaccine antigen in rE. coli fed-batch cultures “Springerplus”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: Light-regulation of protein dimerization and kinase activity in living cells using photocaged rapamycin and engineered FKBP “Journal of the American Chemical Society” 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:Molecular Tools and Approaches for Optogenetics “Biological Psychatry”(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:In vivo single-molecule kinetics of activation and subsequent activity of the arabinose promoter “Nucleic Acids Research” (2013)"; </a></p>

content.summary= "Introduction into several methods of Induction and their usage.';

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: Activation of protein splicing with light in yeast. “Nature Methods” | 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 Engineering a novel self-powering electrochemical biosensor. “Systems and Synthetic Biology”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. Heat-induced conformational change and increased chaperone activity of lens alpha-crystallin. “Current Eye Research”  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. Interaction between residues in the Mg2+-binding site regulates BK channel activation. “The journal of general physiology” (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. Transcriptional regulation in response to oxygen and nitrate of the operons encoding the [NiFe] hydrogenases 1 and 2 of Escherichia coli. “Microbiology”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 pH-Induced Conformational Changes in Clostridium difficile Toxin B. “Infection and Immunity” 68(5) (May 2000)</a></p>