Template:Team:Bonn:NetworkData

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case 44:

content.i = 44; content.parents=[74]; content.childs=[]; content.titleShort = "ssrA"; content.titleLong = "C. crescentus ssrA"; 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.";

<img src="" align=right width=800>Peptide array for

testing ssrA on amino residues relevant for sspBα 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 sspBα [44.1]

Chien et al. [44.1] tested the 14-amino acid peptide ssrA (AANDNFAEEFAVAA, [44.2]) on the residues crucial for binding to sspBα 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

sspBα. 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 sspBα binding site. </br></br>

References

</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.type="Outlook"; break;

case 42:

content.i = 42; content.parents=[74]; content.childs=[43]; content.titleShort = "sspBα"; content.titleLong = "C. crescentus sspBα"; content.summary= "This article deals with the Structure of sspBα and conformational details of its binding to ssrA and ClpXP during tethering.";

structure is stabilized by two α-helices in interaction, as part B of the figure above shows, each of them located at the N-terminus of either

sspBα molecule. The subsequent parts of the protein form a domain consisting of two β-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 sspBα and its E. coli and H. influenzae sspB orthologs, discovering that in

sspBα the α-helices are significantly longer, more twisted and cover a larger cross section area than the other two sspB orthologs. Also

considering that β-sheets are rotated by around 20° 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 sspBα dimer in C. crescentus, while they are parallel in γ-protobacterial

sspB. </br></br>

<img src="" align=left>By measuring GFP fluorescence intensity,

decrease of GFP-^CCssrA concentration (1) without sspBα added, (2) with mutated sspBα(Q74A) added , (3) with wildtype sspBα added

can be visualized. [42.1]

Chien et al. point out that although there are the remarkable differences in protein structure between sspBα and its γ-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

sspBα binding to the protein which needs to be tethered to the ClpXP protease strongly depends on which ssrA ortholog the protein is tagged with.

sspBα 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 sspBα and the compound of sspBα and ^CCssrA, Chien et al. further proved that binding of

sspBα to ^CCssrA does not lead to significant changes of its 3D conformation. </br></br>

References

</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 "; content.type="Outlook"; break;

case 74:

content.i = 74; content.parents=[40]; content.childs=[42,44]; content.titleShort = "ssrA and sspBα"; content.titleLong = "Introduction to C. crescentus ssrA and sspBα"; content.summary= "This article gives a brief overview of the roles of ssrA and sspBα 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 ^{CC>/sup>sspBα, respectively, omitting Ă„A?€L?Ä‚â€a A}

„‚A‹ Ä‚Ë A?‚¬ÄaˇĂ‚A‚¬Ă„A„â€¦Ä‚Ë A?‚¬ÄaLs^CCĂ„A?€L?Ä‚â€a A„‚A‹ Ä‚Ë A?‚¬ÄaˇĂ‚A‚¬Ă„A„…Ă„A?€Ll 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. sspBα 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>

References

</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.type="Outlook"; break;

case 137:

content.i = 137; content.parents=[130]; content.childs=[]; content.titleShort = "Bonn!137"; content.titleLong = "Bonn!137"; content.summary= "Description of Bonn137"; 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. Stadt. City. Ville. Bonn.!137"; content.type="Team"; break;

case 43:

content.i = 43; 
content.parents=[40, 42]; 
content.childs=[]; 
content.titleShort = "SspB Split"; 
content.titleLong = "SspB Split in C. crescentus"; 
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=""> ^{<a href=#[43.1]>[43.1]</a>}

References:

<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.type="project"; 
break;

case 109:

content.i = 109; content.parents=[105]; content.childs=[]; content.titleShort = "Comic"; content.titleLong = "Comic"; content.summary= "Our comic series "LOV Wars" about Obi-Wan E. coli and his adventures in Galaxy Petri."; 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.type="Human Practice"; break;

case 103:

content.i = 103; content.parents=[101]; content.childs=[]; content.titleShort = "biocom kongress Berlin"; content.titleLong = "biocom kongress Berlin"; content.summary= "our day in Berlin. We met important people and other iGEM teams"; 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.type="Human Practice"; break;

case 37:

content.i = 37; content.parents=[]; content.childs=[4]; content.titleShort = "project summary"; content.titleLong = "our project in a nutshell"; content.summary= "We engineer a photoswitachble protein degradation system relying on the ClpXP protease system an a LOV domain from avena sativa"; 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.type="Projekt"; break;

case 32:

content.i = 32; content.parents=[17]; content.childs=[]; content.titleShort = "Chemical"; content.titleLong = "Chemical induction"; content.summary= "this is a summary6"; 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>}

<img src= height=260 width=260>

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.

<img src= height=260 width=260>

References:

<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>

<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>

<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>

<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>

<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>

<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>

content.type="Background"; break;

case 17:

content.i = 17; content.parents=[1]; content.childs=[18,32,33,34]; content.titleShort = "Methods of Induction"; content.titleLong = "Methods of Induction"; content.summary= "Introduction into several methods of Induction and their usage."; (Kurzzusammenfassung des Themas) 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>

References:

<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>

<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>

<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>

<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>

<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>

<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>

"; content.type="Background"; break;

case 108:

content.i = 108; content.parents=[105]; - content.childs=[]; content.titleShort = "School Presentation"; content.titleLong = "School Presentation"; content.summary= "We visited Schools to tell pupils about synthetically biology and our project"; content.text= "What do People already know about synthetically biology? How can we improve the knowledge about synthetically biology? That was 2 of the basic questions when we started brainstorming about our human Practice part of the project. We decided to give lectures about synthetically

biology and our project in schools, as one part of the human practice project. We choose schools, because these pupils will possibly be the next generation

of Scientifics one day. Our idea was to inspire them for science and to delete this bad image science and especially research sometimes has. Almost all classes that we visited had a natural scientific focus (e.g. Bio or Chemie Leistungskurs). But even more interesting were our visits to classes

that had no natural scientific background. In addition you can find some inquiry that we asked the pupils to fill in after the lecture.!108"; content.type="Human Practice"; break;

case 138:

content.i = 138; content.parents=[130]; content.childs=[]; content.titleShort = "University"; content.titleLong = "University"; content.summary= "Description of Bonn138"; 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 • School of Protestant Theology • School of Law and Economics • School of Medicine • School of Humanities • School of Mathematics and Science • School of Agricultural Science

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.type="Team"; break;

case 16:

content.i = 16; content.parents=[12]; content.childs=[]; content.titleShort = "sspB-Split";) content.titleLong = "Control of Protein Degradation Using Split Adaptors"; content.summary= "Using the protein degradation whith help of SspB, we control the function of SspB by splitting it into two parts, each of which cannot

induce degradation on its own."; content.text= "The ClpXP protein degradation system can be used for inducible protein degradation as described by Davis et al. They made use of the native

ClpXP system in E. coli with a modified ssrA-tag (DAS+4) at the target protein. ^{<a href=#161>[16.1]</a>} The modified DAS+4 ssrA cannot bind the

ClpXP without SspB. ^{<a href=#165>[16.5]</a>} Using this dependency of the protein degradation on SspB, they decided to control the function of SspB

by splitting it into two parts, each of which cannot induce degradation on its own. ^{<a href=#161>[16.1]</a>} </br> </br> Splitting of SspB is possible because its tripartite structure. It consists of (1.) a ssrA-tag binding and dimerization domain (SspB [CORE]), (2.) a flexible

linker and (3.) a short peptide module that docks with ClpXP (SspB [XB]). ^{<a href=#163>[16.3]</a>} To test whether the linker length can be varied,

the degradation rates of GFP-DAS+4 (0.3μM) with SspB (0.15μM) were tested in vitro with 4 different linker lengths (of 5, 25, 48 and 91 amino acids). The

results showed that the 25 amino acids variant triggered the fastest degradation, followed by the 25 variant with 60%, 5 variant with 30% and 91 variant with

20% of the 25 variant rate. But even the 91 amino acids variant showed a 40times faster degradation rate than the degradation system without any SspB.

^{<a href=#166>[16.6]</a>} Further experiments (e.g. the split system with FRB-FKBP12 as a linker, see below) demonstrated that even longer linker

regions (more than 200 amino acids) are functional). Thus it can be concluded that not only the length of the linker is important but also its structure.

^{<a href=#161>[16.1]</a>} </br> </br> <img src=""> ^{<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 (FK506 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=""> ^{<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=""> ^{<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=""> ^{<a href=#161>[16.1]</a>} </br>

References:

<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.type="Background"; break;

    }
   }