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content.titleLong = "Background"; | content.titleLong = "Background"; | ||
content.summary= "Introduction to the concept of protein activity regulation"; | content.summary= "Introduction to the concept of protein activity regulation"; | ||
| - | content.text= "Regulation of protein activity is an important tool in the fields of basic research and synthetic biology | + | content.text= "Regulation of protein activity is an important tool in the fields of basic research and synthetic biology.<sup><a href=#11>1.1</a></sup> The reason for this is that it offers a way to analyze the function of the examined protein. Furthermore the core intent of synthetic biology is the design and production of biological ‘machines’. This is usually accomplished via the modulation of protein activity.<sup><a href=#12>1.2</a></sup> </br></br>An example for this would be the expression of a kill switch that is triggered by the presence of a certain molecule, resulting in cell death. This example shows that protein regulation generally consists of two parts: the actual method of regulation, and the way this method is induced. In our example, the method of regulation is a rise in cytosolic protein levels and thus an overall increase of activity via an increased rate of expression. The method of induction is the presence of a certain molecule. This molecule could for example only be absent in a controlled environment, so that the organism dies upon leaving this environment.<sup><a href=#12>1.2</a></sup> <div class='content-image'><img src=' https://static.igem.org/mediawiki/2013/4/47/Bonn-Backgroun%28overview%29-1.jpg'></br> Image detailing the aforementioned example.<sup><a href=#12>1.2</a></sup> </div> There are several different methods for both, actual protein regulation and the induction of this regulation. Through combination of these ‘bricks’, several methods for regulation of protein activity can be designed, each with its own advantages and disadvantages.<sup><a href=#12>1.2</a></sup> In order to help the reader understand the thought process we undertook in designing our own approach in regulation of protein activity, we are going to first explain these ‘bricks’ and discuss their pros and cons.</br> <h2>References</h2><a name=11>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></br> <a name=12>1.2</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3554958/'> Gerd H. G. Moe-Behrens et al., "Preparing synthetic biology for the world", Front Microbiol. 2013; 4: 5.</a>"; |
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content.titleShort = "Protein regulation"; | content.titleShort = "Protein regulation"; | ||
content.titleLong = "Protein regulation mechanisms"; | content.titleLong = "Protein regulation mechanisms"; | ||
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content.titleLong = "Irreversible protein regulation"; | content.titleLong = "Irreversible protein regulation"; | ||
content.summary= "We point out methods of irreversible protein regulaiton and discuss their advantages and disadvantages."; | content.summary= "We point out methods of irreversible protein regulaiton and discuss their advantages and disadvantages."; | ||
| - | content.text= "Irreversible regulation methods are usually associated with knock-in or -outs. But also posttranslational modification of protein structure can be classified as in this field. </br>On the one hand Irreversible interventions cause high differences in activity and therefore are favorable for many investigations of protein functions.</br> On the other hand, it is impossible to revert any of changes made, which makes it harder to analyze protein function in one organism. At least two organisms are needed to simultaneously compare the effect of a normal protein concentration with a complete loss of a certain protein.<h2>References:</h2>see following articles | + | content.text= "Irreversible regulation methods are usually associated with knock-in or -outs. But also posttranslational modification of protein structure can be classified as in this field. </br>On the one hand Irreversible interventions cause high differences in activity and therefore are favorable for many investigations of protein functions.</br> On the other hand, it is impossible to revert any of changes made, which makes it harder to analyze protein function in one organism. At least two organisms are needed to simultaneously compare the effect of a normal protein concentration with a complete loss of a certain protein.<h2>References:</h2>see following articles<li><a onclick=showNode(4)>Zymogen-like protein procursors</a></li><li><a onclick=showNode(5)>Gene knock-out and knock-in</a></li>"; |
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| - | content.titleShort = " | + | content.titleShort = "Protein cleavage"; |
| - | content.titleLong = " | + | content.titleLong = "Protein cleavage"; |
content.summary= "Proteins can be activated through cleavage of the precursor protein."; | content.summary= "Proteins can be activated through cleavage of the precursor protein."; | ||
| - | content.text= " | + | content.text= "One approach to regulate protein activity is to use posttranslational modification, especially by splitting off an inactivating part from a translated pro-peptide. A change in conformation leads to the functional protein. The cleaving can be effected by enzymatic proteolysis, autoproteolysis or surrounding conditions (e.g. pH).<sup><a href='#1.1'>1.1</a></br> A simple example is the activation of pepsinogen which is caused by a low pH (fig. 1). </br> It is a powerful system to activate proteins, which is frequently used in nature and science. Unfortunately posttranslational cleaving can usually not be reversed. Once activated the inactive pro-form of the Protein cannot be restored.<h2>References</h2><a name='4.1'>1.1</a> <a href='http://www.ncbi.nlm.nih.gov/books/NBK22589/'> Berg JM, Tymoczko JL, Stryer L.: "Biochemistry. 5th Edition". New York: W H Freeman 2002.</a>"; |
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content.titleShort = "Gene knock-out and knock-in"; | content.titleShort = "Gene knock-out and knock-in"; | ||
content.titleLong = "Gene knock-out and knock-in"; | content.titleLong = "Gene knock-out and knock-in"; | ||
| - | content.summary= "Another irreversible method of protein regulation in bacteria is the Gene knock in or out. Here the genomic DNA is manipulated to | + | content.summary= "Another irreversible method of protein regulation in bacteria is the Gene knock-in or out. Here the genomic DNA is manipulated to gain or lose a function, in other words whether a protein is produced or not"; |
| - | content.text= "To insert the gene of interest in the bacteria genome | + | content.text= "To insert the gene of interest in the bacteria genome recombineering is often used. Utilising this system, it is possible to insert or delete DNA. It is based on the homologous recombination of small engineered DNA parts with a target gene. Recombination then occurs in the defined region of the genome, resulting in the insertion of the artificial sequence.</br>One possible method is using bacteriophage proteins from bacteriophage lambda, which mediates the insertion of DNA cassettes <sup> <a href=#5.1>5.1</a> </sup> <sup> <a href=#5.2>5.2</a> </sup>.</br> Gene expression, i.e protein production, after a knock-in can be regulated by a promoter. </br> The disadvantage of this particular way of protein regulation is the time needed from the beginning of the induction until protein levels are reached. </br>A knock out causes a complete loss of function. When a gene sequence is interrupted, it may still be translated, but the resulting protein will be non-functional. In this case it is not possible to regulate the protein activity at all since the protein does not come into effect at all and therefore cannot be regulated. </br></br> <p><a name=5.1>5.1 </a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC107131/ > Kenan Murphy, Use of bacteriophage &lambda recombination functions to promote gene replacement in Escherichia coli (1998) </a> </p> <p> <a name=5.2> 5.2 </a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC148353/> Rapid modification of bacterial artificial chromosomes by ET- recombination (1998) </a> </p>"; |
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content.titleShort = "Protein degradation systems"; | content.titleShort = "Protein degradation systems"; | ||
content.titleLong = "Protein degradation systems"; | content.titleLong = "Protein degradation systems"; | ||
content.summary= "Specifically degrading protein offers a reversible method for protein regulation"; | content.summary= "Specifically degrading protein offers a reversible method for protein regulation"; | ||
| - | content.text= "Another method | + | content.text= "Another method of protein control is the introduction of a protein degradation system. </br> There are two very important advantages. On the one hand it works much faster than any regulation system on a genetic base. On the other hand it is a very versatile system which can be transferred to any desirable protein.<h2>References:</h2>see following articles<li><a onclick=showNode(12)> Ec. ClpXP system</a></li>"; |
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content.titleShort = "Transcriptional regulation"; | content.titleShort = "Transcriptional regulation"; | ||
content.titleLong = "Transcriptional regulation"; | content.titleLong = "Transcriptional regulation"; | ||
| - | content.summary= " | + | content.summary= "Advantages and disadvantages of transcriptional regulation"; |
| - | content.text= "One important regulation point is the transcription of RNA to DNA. There are many different systems which can be used in order to activate or repress the transcription. The greatest advantage are the wide spread possibilities regulation tools. Some popular examples are the operon, zinc finger and TALE. But on the other hand, one important disadvantage is the long time between induction and effect."; | + | content.text= "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. <h2>References:</h2>see following articles <li><a onclick=showNode(71)>Operon-model</a></li><li><a onclick=showNode(72)>Zinc finger</a></li><li><a onclick=showNode(73)>TALE</a></li>"; |
content.type="Background"; | content.type="Background"; | ||
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content.titleLong = "Genetical knock-down by RNA-inference"; | content.titleLong = "Genetical knock-down by RNA-inference"; | ||
content.summary= "RNA interference enables reversible regulation (Knock-down) of expressed proteins"; | content.summary= "RNA interference enables reversible regulation (Knock-down) of expressed proteins"; | ||
| - | content.text= "The genetical "Knock Down" by RNAi does not affect directly transcription or DNA, but it is based on RNA level. Here a DNA construct, which codes for small double-stranded interfering RNAs (siRNA), is inserted into the cell. The siRNA is processed by the RNA-induced silencing complex (RISC), described by Pratt and MacRae in 2009. The siRNA is complementary to the target mRNA and therefore forms a double strand with it and the RISC. After the RISC localizes to the target mRNA, the RNA is cleaved by a ribonuclease. Hence the mRNA of the knockdown gene is degraded and no protein can be produced.<sup> <a href=#10.1> | + | content.text= "The genetical "Knock Down" by RNAi does not affect directly transcription or DNA, but it is based on RNA level. Here a DNA construct, which codes for small double-stranded interfering RNAs (siRNA), is inserted into the cell. The siRNA is processed by the RNA-induced silencing complex (RISC), described by Pratt and MacRae in 2009. The siRNA is complementary to the target mRNA and therefore forms a double strand with it and the RISC. After the RISC localizes to the target mRNA, the RNA is cleaved by a ribonuclease. Hence the mRNA of the knockdown gene is degraded and no protein can be produced.<sup> <a href=#10.1>10.1</a> </sup></br><p> <a name=10.1>10.1</a> <a href='http://www.jbc.org/content/284/27/17897.long'>The RNA-induced Silencing Complex: A Versatile Gene-silencing Machine, Ashley J. Pratt and Ian J. MacRae1 </a></p>"; |
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content.titleShort = "Direct inhibition and activation"; | content.titleShort = "Direct inhibition and activation"; | ||
content.titleLong = "Direct inhibition and activation"; | content.titleLong = "Direct inhibition and activation"; | ||
| - | content.summary= "Direct binding of small molecules an activate or inactivate protein function"; | + | content.summary= "Direct binding of small molecules in order to activate or inactivate protein function"; |
| - | content.text= "Moreover proteins can be regulated directly in many different ways. Binding of other Proteins or small molecules for example can activate or deactivate the catalytic function of proteins. For example small molecules can have great impact on protein function. They can often influence protein functions by binding to an allosteric center. Small molecules can be activating, as well as repressing, depending on the type of the molecule<sup><a href=#11.1> | + | content.text= "Direct binding of small molecules an activate or inactivate protein function"; |
| + | content.text= "Moreover proteins can be regulated directly in many different ways. Binding of other Proteins or small molecules for example can activate or deactivate the catalytic function of proteins. For example small molecules can have great impact on protein function. They can often influence protein functions by binding to an allosteric center. Small molecules can be activating, as well as repressing, depending on the type of the molecule<sup><a href=#11.1>11.1</a></sup>.</br></br><div class='content-image'> <img src='https://static.igem.org/mediawiki/2013/1/1c/BonnSmallMolecules.jpg' width='550'> </br>Different mechanisms for small-molecule activation of enzymes<sup><a href=#11.2>11.2</a></sup> </div></br></br>The following picture shows the example of the allosteric activation of a glucokinase</br> <div class='content-image'> <img src=https://static.igem.org/mediawiki/2013/f/f4/BonnSmallMolecules2.jpg width='600'></br>"(a) GK bound to the GKA, compound A, and glucose (blue, Protein Data Bank (PDB) ID 1V4S). Compound A binds at a site distal from the active site, which is highlighted by the presence of the substrate, glucose. (b) Structural overlay of GK in the presence of compound A and glucose with an unliganded, inactive GK (pink, PDB ID 1V4T). In the unbound GK, the GKA binding site is occluded. A large shift in the small subunit of GK occurs from the unbound to bound structures (black arrow). Glucose promotes the active conformation, which is hindered from shifting back to the inactive conformation in the presence of compound A. (c) 7 mutations (out of 13)9 identified in GK (pink) that are associated with disease map to the GKA binding site. These mutations highlight an important regulatory site within GK, and could similarly stabilize a closed, active conformation."<sup><a href=#11.2>[11.2]</a></sup></div></br></br><p><a name=11.1>11.1</a><a href='http://www.nature.com/nrd/journal/v3/n4/full/nrd1343.html'>Small-molecule inhibitors of proteinprotein interactions: progressing towards the dream; Michelle R. Arkin & James A. Wells</a>/p></br><p><a name=11.2>11.2</a><a href='http://www.nature.com/nchembio/journal/v6/n3/full/nchembio.318.html'>Turning enzymes ON with small molecules, Julie A Zorn & James A Wells</a></p>"; | ||
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content.i = 12; | content.i = 12; | ||
| - | content.parents=[7]; | + | content.parents=[2, 7]; |
| - | content.childs=[13,14,15,16,37]; | + | content.childs=[13, 14, 15, 16, 37]; |
| - | content.titleShort = " | + | content.titleShort = "ClpXP, ssrA, SspB"; |
| - | content.titleLong = "ClpXP | + | content.titleLong = "Short information about ClpXP, ssrA, SspB and how we used it in our projekt"; |
| - | content.summary= "ClpXP | + | content.summary= "The ClpXP protease is able to degrade proteins with an ssrA tag. The SspB protein transfers this tagged protein to ClpXP."; |
| - | content.text= "ClpXP"; | + | content.text= "We aimed to create a photosensitive protein degradation system which is useable in several types of cells with various proteins. In order to realize our system we decided to use the ClpXP protease, a protein break down system which is native to E. coli and mostly used to remove defective proteins and limit overall protein longevity <sup><a href=#123>12.3</a></sup> </br> . ClpXP is an AAA+ Protease which consists of two subunits, the ClpP protein and the ClpX protein. The ClpX unit is assembled from a ring of subunits some of which are able to bind ATP. The ClpP protein consists of two rings encircling a central pore which functions as substrate binding site. When a protein is degraded the tagged substrate binds to the ClpX protein. It is consequently unfolded by the subunits through the hydrolysis of ATP. Finally the substrate is passed on to the ClpP unit which cleaves the amino bonds and releases amino acids. <sup><a href=#121>12.1</a></sup> </br> </br> The ClpXP protease can only break down proteins with a so called ssrA tag. This is a short amino acid sequence marking the protein for degradation by ClpXP. SsrA can be bound by the transporter protein SspB which increases the efficiency of degradation. </br> The ssrA sequence has two functional parts. One part interacts with the ClpX protein of the ClpXP protease and one part is able to interact with SspB. </br> A ssrA labled protein can either bind SspB or ClpXP in order to induce degradation. In normal E. coli strains this process not necessarily dependent on SspB but rather enhanced. A mutated form of ssrA (DAS+4) weakens the native binding between ClpX and ssrA and therefore increases the dependence of SspB. <sup><a href=#122>12.2</a></sup> </br> </br> The use of sspB induced protein degradation has been demonstrated with GFP <sup><a href=#124>12.4</a></sup> ,the neurotransmitter Arc <sup><a href=#125>12.5</a></sup> and Lac1, the Repressor of the lac operon.<sup><a href=#126>12.6</a></sup> </br> </br> <h3> <b>SspB:</b> </h3> SspB translocates the tagged protein to ClpXP and improves the affinity between ssrA and ClpXP and therefore mediates its breakdown.<sup><a href=#127>12.7</a></sup> </br> To control degradation a split version of SspB can be used consisting of two domains: SspB[CORE] and SspB[XB] each of which cannot induce degradation on their own. To regain a functional construct for inducible degradation both were combined with a chemically inducible heterodimerisation system: FRB and FKBP12. In absence of rapamycin both parts are monomers and thus not functional. On addition of rapamycin FRB and FKBP12 form a dimer and consequently both SspB domains are in spatial proximity rendering them functional again.<sup><a href=#129>12.9</a></sup> </br> Since there is a basic expression of SspB in regular E. coli strains a SspB deficient strain was used aiming to minimize constitutive degradation. </br> In our project the SspB split-version was used but instead of rapamycin induced dimerization we used a LOV-ipaA - VinD1 mediated dimer in order to produce a functional split-SspB. </br> <h2> <b>References:</b> </h2> </br> <a name=121>12.1</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209554/> ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554 </a> </br> <a name=122>12.2</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[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, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </a> </br> <a name=123>12.3</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/17291191[http://www.ncbi.nlm.nih.gov/pubmed/17291191]> The tmRNA System for Translational Surveillance and Ribosome Rescue </a> </br> <a name=124>12.4</a> <a href=http://www.sciencedirect.com/science/article/pii/S1097276503002727[http://www.sciencedirect.com/science/article/pii/S1097276503002727]> Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes Delivery Complexes with the AAA ClpXP Protease, Wah et al, Molecular Cell, 2003, PMID: 14536075 </a> </br> <a name=125>12.5</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/17317664[http://www.ncbi.nlm.nih.gov/pubmed/17317664]> 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 </a> </br> <a name=126>12.6</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511798/[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511798/]> Temperature dependence of ssrA-tag mediated protein degradation, Purcell et al, 2012, Journal of Biological Engineering, PMID: 22824000 </a> </br> <a name=127>12.7</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/14967151[http://www.ncbi.nlm.nih.gov/pubmed/14967151]> Bivalent Tethering of SspB to ClpXP Is Required for Efficient Substrate Delivery: A Protein-Design Study, Bolon et al, 2004, Molecular Cell, PMID: 14967151 </a> </br> <a name=128>12.8</a> <a href=http://www.ncbi.nlm.nih.gov/pubmed/17317664[http://www.ncbi.nlm.nih.gov/pubmed/17317664]> 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 </a> </br> </br> <a name=129>12.9</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220803/[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>"; |
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content.titleShort = "ClpXP protease"; | content.titleShort = "ClpXP protease"; | ||
| + | content.titleLong = "ClpXP protease"; | ||
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.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 | + | 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 degrade 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 <sup><a href=#131>13.1</a></sup>.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 achieve a better control we 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 "ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554"</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 "ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophy Acta, 2012, PMCID: PMC3209554"</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 <sup><a href=#131>13.1</a></sup>. <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 "ClpXP, an ATP-powered unfolding and protein-degradation machine, Bakeret al, Biochim Bi phys Acta, 2012, PMCID: PMC3209554"</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 more time <sup><a href=#131>13.1</a></sup> <sup><a href=132>13.2</a></sup>. </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 "Engineering controllable protein degradation, McGinness et al, Molecular cell, 2006, PMID:16762842'</div> <h2><b> References </b> </h2></br> </br> <p><a name=131>13.1</a> ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554</br> <a name=132>13.2</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>"; |
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| - | content.titleShort = "Ec. | + | content.titleShort = "Ec. SspB adaptor"; |
| + | content.titleLong = "Ec. SspB adaptor"; | ||
content.summary= "The sspB protein is an adaptor responsible for delivering ssrA-tagged substrates to the ClpXP protease in order to enhance their degradation"; | content.summary= "The sspB protein is an adaptor responsible for delivering ssrA-tagged substrates to the ClpXP protease in order to enhance their degradation"; | ||
| - | content.text= "<b>Introduction </b> </br> The sspB protein is an adaptor responsible for delivering ssrA-tagged substrates to the ClpXP protease in order to enhance their degradation.Thus, bacterias like E.coli or C.crescentus regulate the concentration of marked proteins and also are in control of their quality. Even though degeneration of tagged substrates is possible without sspB, sspB delivering is a common process, because it improves the affinity between ssrA and ClpXP<sup><a href='#14.1'> | + | content.text= "<b>Introduction </b> </br> The sspB protein is an adaptor responsible for delivering ssrA-tagged substrates to the ClpXP protease in order to enhance their degradation.Thus, bacterias like E.coli or C. crescentus regulate the concentration of marked proteins and also are in control of their quality. Even though degeneration of tagged substrates is possible without sspB, sspB delivering is a common process, because it improves the affinity between ssrA and ClpXP<sup><a href='#14.1'>14.1</a></sup>. </br> In our project, we used a sspB split variant instead of the normal sspB in order to control cleaving rate. The two parts of this version only stay divided until we ray them with light of a special wavelength. After that, the fractions form an unit and can function normal from now on as described below. </br> </br><b>Structure </b> </br>The sspB adaptor is a homomeric dimer (Fig. 1), which means that itconsists of two identical domains. Together the domains form a pore with the ssrA binding sites inside. Each domain also owns a C-terminal tail ending in a ClpX binding module, called XP. The amino-acid sequence of XP is highly conserved so that mutations in it mostly cause extremely decrease in activity, whereas the linker sequence differs from species to species <sup><a href='#14.2'>14.2</a></sup><sup><a href='#14.3'>14.3</a></sup>. <div align='left'><img src='https://static.igem.org/mediawiki/2013/5/5c/BonnsspB_Fig2.jpg' height='202' width='403'>Fig. 1: Ribbon diagramm of the sspB dimer with a bound srrA-tagged protein, from 'Versatile modes of peptide recognition by the AAA+ adaptor protein SspB, Levchenko et al, 2005, nature structural and molecular biology, PMID: 15880122</div> </br> </br></br></br><b>Function</b></br> SspB enhances degradation of ssrA-tagged proteins by lowering the K<sub>M</sub>. Thus, with a given substrate concentration sspB-mediated cleaving runs faster than without sspB (Fig. 2) </br> <div align='left'><img src='https://static.igem.org/mediawiki/2013/d/d8/BonnSspB_Fig1.jpg' height='232' width='371'>Fig. 2: Diagramm, shows the degradation rate of GFP-ssrA without sspB, with sspB and with two mutations, the given substrate concentration is 0.3 &my;M, from 'Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes Delivery Complexes with the AAA ClpXP Protease, Wah et al, Molecular Cell, 2003, PMID: 14536075' </div></br> Therefor, the sspB dimer contains a pore and while 'AADENY' is linked with the inside, the 'LAA'-domain (respectively 'DAS') faces outwards, free to bind ClpX (Fig. 3).</br> The affinity of this binding amounts around 20 &my;M, which suggests a relative strong connection. The two extremely flexible ClpX binding tails with XP at the C-terminal end dock on ClpX. So the 'LAA'- domain lies closely to ClpX's axial pore and can be bound to it. <div align='left'><img src='https://static.igem.org/mediawiki/2013/7/7c/BonnSsra_fig3.jpg' height='151' width='116'>Fig. 3: Model of sspB with a bound ssrA-tagged substrate, from 'Engineering controllable protein degradation, McGinness et al, Molecular cell, 2006, PMID:16762842' </div></br> To sum up, there are three bonds connecting the ssrA-sspB-ClpX-complex and making it relative stable: ssrA with sspB, sspB with ClpX and ssrA with ClpX. Hence follows a lower K<sub>M</sub> than the direct binding process has<sup><a href='#14.4'>14.4</a></sup><sup><a href='#14.5'>14.5</a></sup>. <h2> References </h2> <a id='14.1'>14.1</a>Bivalent Tethering of SspB to ClpXP Is Required for Efficient Substrate Delivery: A Protein-Design Study, Bolon et al, 2004, Molecular Cell, PMID: 14967151 </br><a id='14.2'>14.2</a>see above </br> <a id='14.3'>14.3</a> Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes Delivery Complexes with the AAA ClpXP Protease, Wah et al, Molecular Cell, 2003, PMID: 14536075 </br> <a id='14.4'>14.4</a> see above</br> <a id='14.6'>[14.5]</a> Altered Tethering of the SspB Adaptor to the ClpXP Protease Causes Changes in Substrate Delivery, McGinnes KE et al, The journal of Biological Chemistry, 2007, PMID: 17317664 </br> <a id='14.4'>14.4</a> ClpXP, an ATP-powered unfolding and protein-degradation machine, Baker et al, Biochim Biophys Acta, 2012, PMCID: PMC3209554</br>"; |
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| - | content.titleShort = " | + | content.titleShort = "SsrA tag"; |
| + | content.titleLong = "SsrA tag"; | ||
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.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<sup><a href='#15.1'> | + | 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<sup><a href='#15.1'>15.1</a></sup>. </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 split 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 "Translation control" 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 "AANDENY"-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 "LAA", interacts with the ClpX subunit of the ClpXP protease. The parts are connected over an Alanine molecule<sup><a href='#15.2'>15.2</a></sup>. </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 "Engineering controllable protein degradation, McGinness et al, Molecular cell, 2006, PMID:16762842" </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 α-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<sup><a href='#15.3'>15.3</a></sup>.</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 "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"</div> </br>2. Adaptor-mediated binding: sspB is an adaptor protein with a special binding site for the "AADENY"-domain of ssrA. Therefor, the sspB dimer contains a pore in each subunit and while "AADENY" is linked with the inside, the "LAA"-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 "LAA"-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<sub>M</sub> than the direct binding process has (hab hierzu keine konkreten Daten). This lower K<sub>M</sub> 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<sup><a href='#15.4'>15.4</a></sup><sup><a href='#15.5'>15.5</a></sup>.</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 "Engineering controllable protein degradation, McGinness et al, Molecular cell, 2006, PMID:16762842" </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 "open reading frame" 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<sup><a href='#15.6'>15.6</a></sup>.</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 "The tmRNA System for Translational Surveillance and Ribosome Rescue, Moore SD et al, Annual Reviews Biochemistry, 2007, PMID: 17291191"</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>"; |
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| - | content.titleShort = " | + | content.titleShort = "SspB-Split"; |
content.titleLong = "Control of Protein Degradation Using Split Adaptors"; | 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.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. <sup><a href=#161> | + | 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. <sup><a href=#161>16.1</a></sup> The modified DAS+4 ssrA cannot bind the ClpXP without SspB. <sup><a href=#165>16.5</a></sup> 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. <sup><a href=#161>16.1</a></sup> </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]). <sup><a href=#163>16.3</a></sup> 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. <sup><a href=#166>16.6</a></sup> 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. <sup><a href=#161>16.1</a></sup> </br> </br> <img src='https://static.igem.org/mediawiki/2013/1/1f/Bonn-Gfp.jpg'> <sup><a href=#166>16.6</a></sup> </br> </br> To bring both SspB parts together again for inducible degradation, they were combined with a chemical inducible heterodimerisation system: FRB and FKBP12. <sup><a href=#161>16.1</a></sup> FKBP12 (FK 06 binding protein, 12 kDa) is a binding protein (108 amino acids <sup><a href=#167>16.7</a></sup>) 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. <sup><a href=#162>16.2</a></sup> </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. Thus the two parts of SspB get in a spatial closeness and function as an adapter. As a consequent the target protein gets degraded. <sup><a href=#161>16.1</a></sup> </br> </br> <img src='https://static.igem.org/mediawiki/2013/2/21/Bonn-rapa-split.jpg'> <sup><a href=#161>16.1</a></sup> </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). <sup><a href=#161>16.1</a></sup> 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). <sup><a href=#161>16.1</a></sup> </br> </br> <img src='https://static.igem.org/mediawiki/2013/d/d0/Bonn-GFP-Abbau.jpg'> <sup><a href=#161>16.1</a></sup> </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). <sup><a href=#161>16.1</a></sup> </br> <img src='https://static.igem.org/mediawiki/2013/2/2d/Bonn-rapa-gel.jpg'> <sup><a href=#161>16.1</a></sup> </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"; |
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content.titleLong = "Methods of Induction"; | content.titleLong = "Methods of Induction"; | ||
content.summary= "Introduction into several methods of Induction and their usage."; | 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<sup><a href='# | + | 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<sup><a href='#171'>17.1</a></sup>, 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.<sup><a href='#2'>[2]</a></sup> However there is a multitude of methods available to induce changes in protein structure or expression.<sup><a href='#171'>17.1</a><a href='#173'>17.3</a><a href='#174'>17.4</a><a href='#175'>17.5</a><a href='#176'>17.6</a></sup>Yet each technique has its own assets and drawbacks which are examined more closely in the following paragraphs.</br> </br> </br><table border='1'><tr><td>Induction Method</td><td>Temporal resolution</td><td>Spatial resolution</td><td>Reliability</td><td>Ease of use</td><td>Distinctive advantage</td></tr><tr><td>Light</td><td>Very high <µs</td><td>Very high</td><td>High</td><td>Cloning, genetical engineering required</td><td>Reversibel, in vivo</td></tr><tr><td>Chemical</td><td>Low 10sec - min</td><td>low</td><td>Very high (especially expression)</td><td>Very easy (expression), genetical engineering required (conformational)</td><td>Fine-tune gene expression</td></tr><tr><td>Heat</td><td>Low (expression only)</td><td>Very low (whole organism affected)</td><td>Very high</td><td>Very easy</td><td>Does not change protein conformation</td></tr><tr><td>Electrical</td><td>High <ms</td><td></td><td></td><td>Application can be difficult</td><td>Exact measurements</td></tr></table> <h2>References:</h2> <p><a name=171>17.1.</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> "Nature Methods" | Vol.5 No.4 | 303 (April 2008)</a></p> <p><a name=172>17.2.</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> "Systems and Synthetic Biology"4(3) (Sep 2010)</a></p> <p><a name=173>17.3.</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> "Current Eye Research" Apr;16(4):303-9 (1997)</a></p> <p><a name=174>17.4.</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> "The journal of general physiology" (Feb 2013)</a></p> <p><a name=175>17.5.</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> "Microbiology"145 ( Pt 10) (Oct 1999)</a></p> <p><a name=176>17.6.</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> "Infection and Immunity" 68(5) (May 2000)</a></p>"; |
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content.parents=[17]; | content.parents=[17]; | ||
| - | content.childs=[ | + | content.childs=[36,21,20]; |
content.titleShort = "Light"; | content.titleShort = "Light"; | ||
content.titleLong = "Light as a means of induction"; | content.titleLong = "Light as a means of induction"; | ||
content.summary= "Discussion of light and it's advantages and disadvantages as a means of induction"; | content.summary= "Discussion of light and it's advantages and disadvantages as a means of induction"; | ||
| - | 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 | + | 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 <sup><a href=#181>18.1</a></sup><sup><a href=#182>18.2</a></sup>. 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 <sup><a href=#183>18.3</a></sup>.</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<sup><a href=#183>18.3</a></sup><sup><a href=#184>18.4</a></sup>. Also the conformational changes in proteins induced by light occur in a matter of seconds and are also reversible<sup><a href=#183>18.3</a></sup>. For these reasons light is used as a method of induction in many fields where high specificity and resolution are needed, e.g. optogenetics <sup><a href=#184>18.4</a></sup>.</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 <sup><a href=#183>18.3</a></sup>.<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).: "Lack of short-wavelength light during the school day delays dim light melatonin onset (DLMO) in middle school students". Neuro Endocrinology Letters 31 (1): 92–6. PMID 20150866.</a></br><a href='http://www.ncbi.nlm.nih.gov/pubmed/18952772'><sup><a href=#182>18.2</a></sup>Han, I.-S, W. Eisinger, T.-S. Tseng, and W. R. Briggs, 2008.: "Phytochrome A regulates the intracellular distribution of phototropin1-green fluorescent protein in Arabidopsis thaliana" Plant Cell 20: 2835-2847.</a></br><a href='http://www.ncbi.nlm.nih.gov/pubmed/22520757'><sup><a href=#183>18.3</a></sup>Lungu et al, April 20, 2012: "Designing Photoswitchable Peptides Using the AsLOV2 Domain" Chemistry and Biology 19, 507-517</a></br><a href='http://www.ncbi.nlm.nih.gov/pubmed/17643087'><sup><a href=#184>18.4</a></sup> Zhang F, Aravanis AM, Adamantidis A, de Lecea L, Deisseroth K.: "Circuit-breakers: optical technologies for probing neural signals and systems." Nat Rev Neurosci. 2007 Sep;8(9):732</a></br><a href='http://www.ncbi.nlm.nih.gov/pubmed/18667691'><sup><a href=#185>18.5</a></sup> Devin Strickland et al., "Light-activated DNA binding in a designed allosteric protein", PNAS, August 5, 2008 vol. 105 no. 31</a>"; |
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content.titleShort = "Other systems"; | content.titleShort = "Other systems"; | ||
| - | content.titleLong = " | + | content.titleLong = "Other systems"; |
content.summary= "More systems"; | content.summary= "More systems"; | ||
content.text= "More systems"; | content.text= "More systems"; | ||
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| - | content.titleShort="Chemical"; | + | content.titleShort="Chemical Induction"; |
| - | content.titleLong="Chemical | + | content.titleLong="Chemical Induction"; |
content.summary= "A review on chemical induction and its advantages and disadvantages"; | content.summary= "A review on chemical induction and its advantages and disadvantages"; | ||
| - | content.text= "Chemical induction can be used to provide both expressional and structural changes in proteins.<sup><a href=# | + | content.text= "Chemical induction can be used to provide both expressional and structural changes in proteins.<sup><a href=#321>32.1</a></sup><sup><a href=#322>32.2</a></sup> As an advantage it is highly reliable and tunable which renders it very useful for ensuring constant expression levels.<sup><a href=#321>32.1</a></sup> Several promoters such as pBad which is inducible with arabinose or pLac which is inducible with IPTG are frequently used for such purpose.<sup><a href=#326>32.6</a></sup> 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<sup><a href=#324>32.4</a></sup>) occur much faster i.e. seconds to minutes.<sup><a href=#322>32.2</a></sup> <div class='content-image' align='center'><a href='https://static.igem.org/mediawiki/2013/8/8c/BonnRapamycin3D.jpg'><img src='https://static.igem.org/mediawiki/2013/8/8c/BonnRapamycin3D.jpg' height=260 width=260></a></br><i>A 3D Structure of a Rapamycin induced FKBP-FRB heteromer<sup><a href='#327'>32.7</a></sup></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.<sup><a href=#324>32.4</a></sup> Secondly any chemical can be bioactive and hence interfere with the cells metabolism or other substances.<sup><a href=#321>32.1</a></sup> Also specificity can be a problem especially in vivo, where often several cell types in multicellular organisms are effected. <sup><a href=#325>32.5</a></sup> 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=321>32.1</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> "PNAS" (June 2000)</a></p> <p><a name=322>32.2</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>"Nature chemical biology" 8, 465–470 (2012) </a></p> <p><a name=323>32.3</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3724991/'>Adilson José da Silva, Antonio Carlos Luperni Horta, Ana Maria Velez, Mônica Rosas C Iemma, Cíntia Regina Sargo, Raquel LC Giordano, Maria Teresa M Novo, Roberto C Giordano, and Teresa Cristina Zangirolami: <i>Non-conventional induction strategies for production of subunit swine erysipelas vaccine antigen in rE. coli fed-batch cultures</i> "Springerplus"2, 322 (2013)</a></p> <p><a name=324>32.4</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>"Journal of the American Chemical Society" 133(3) 420-423 (2011)</a></p> <p><a name=325>32.5</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> "Biological Psychatry"(2012)</a></p> <p><a name=326>32.6</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> "Nucleic Acids Research" (2013) </a></p><p><a name='327'>32.7</a><a href='http://www.ncbi.nlm.nih.gov/pubmed/10089303'>Liang J, Choi J, Clardy J.:Refined structure of the FKBP12-rapamycin-FRB ternary complex at 2.2 A resolution."Acta crystallographica"(1999)</a></p>"; |
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content.titleLong = "Using heat as a means of induction"; | content.titleLong = "Using heat as a means of induction"; | ||
content.summary= "Discussion of heat and it's advantages and disadvantages as an induction tool"; | content.summary= "Discussion of heat and it's advantages and disadvantages as an induction tool"; | ||
| - | 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 | + | 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 <sup><a href=#331>33.1</a></sup>.</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 <sup><a href=#332>33.2</a></sup> <sup><a href=#333>33.3</a></sup>. 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 <sup><a href=#332>33.2</a></sup> <sup><a href=#333>33.3</a></sup>.</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 <sup><a href=#333>33.3</a></sup></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 <sup><a href=#333>33.3</a></sup> <h3>References</h3><a name=331>33.1</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/23912482'>Zhang L. et al., "Characterization of four heat-shock protein genes from Nile tilapia (Oreochromis niloticus) and demonstration of the inducible transcriptional activity of Hsp70 promoter." Fish Physiol Biochem. 2013 Aug 4</a></br> <a name=332>33.2</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/6322174'>Bardwell JC, Craig EA. "Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous." Proc Natl Acad Sci U S A. 1984 Feb;81(3):848-52</a></br><a name=333>33.3</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/10739675'>Attila Ádám et al., "Heat-Inducible Expression of a Reporter Gene Detected by Transient Assay in Zebrafish", Experimental Cell Research 256: 282-290 (2000)</a>"; |
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| - | content.titleShort = "Electrical"; | + | content.titleShort = "Electrical Induction"; |
content.titleLong = "Electrical Induction"; | content.titleLong = "Electrical Induction"; | ||
content.summary= "A review on chemical induction and its advantages and disadvantages"; | content.summary= "A review on chemical induction and its advantages and disadvantages"; | ||
| - | content.text= "In nature there are many examples of voltage altering protein conformation such as channel proteins which can open or close<sup><a href= | + | content.text= "In nature there are many examples of voltage altering protein conformation such as channel proteins which can open or close<sup><a href=#341>34.1</a></sup> or enzymes which change their catalytic activity when the membrane potential changes.<sup><a href=#342>34.2</a></sup> Channel proteins require minimal changes in electrical potential and change conformation in submillisecond timescales.<sup><a href=#343>34.3</a></sup> However its usage in synthetic biology is yet to be demonstrated.<h2>References:</h2> <p><a name=341>34.1</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3412939/'> Venkataswarup Tiriveedhi, Melissa Miller, Peter Butko, and Min Li: <i>Autonomous Transmembrane Segment S4 of the Voltage Sensor Domain Partitions into the Lipid Membrane. </i>"Biochimica et Biophysica Acta" (2012)</a></p> <p><a name=342>34.2</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2869078/'> Xiao Tao, Alice Lee, Walrati Limapichat, Dennis A. Dougherty, and Roderick MacKinnon: <i>A Gating Charge Transfer Center in Voltage Sensors. </i>"Science" (2010) Vol. 328 no. 5974 pp. 67-73 <p><a name=343>34.3</a> <a href='http://www.sciencemag.org/content/314/5799/615.long'> Berkefeld H, Sailer CA, Bildl W, Rohde V, Thumfart JO, Eble S, Klugbauer N, Reisinger E, Bischofberger J, Oliver D, Knaus HG, Schulte U, Fakler B: BKCa-Cav channel complexes mediate rapid and localized Ca2+-activated K+ signaling. "Science" (2006)</a></p>"; |
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content.childs=[37]; | content.childs=[37]; | ||
content.titleShort = "LOV-ipaA & VinD1"; | content.titleShort = "LOV-ipaA & VinD1"; | ||
content.titleLong = "LOV-ipaA & VinD1"; | content.titleLong = "LOV-ipaA & VinD1"; | ||
content.summary= "The LOV-ipaA -vinculin system is a combined system for light inducible heterodimerisation. This powerful tool, which allows photocontroled complex formation was establish by Lungu et al. in 2012."; | content.summary= "The LOV-ipaA -vinculin system is a combined system for light inducible heterodimerisation. This powerful tool, which allows photocontroled complex formation was establish by Lungu et al. in 2012."; | ||
| - | content.text= "The LOV-ipaA -vinculin system is a combined system for light inducible heterodimerisation. It consists out of a LOV domain, which undergoes conformational changes upon irradiation with blue light, and the ipaA-vinculin hybridization system. This two building blocks have be combined and described by Lungu et al. in 2012.</br></br> Lungu et al. (2008) where able to measure a 49-fold difference in target binding upon irradiation as compared to the dark state. However, they further modified the system by mutations of the LOV-ipaA construct and successfully weakend the baseline affinity for vinculin (initial design: 3.5 nM to 69 nM; mutant: 2.4 nM to >40µM affinity for vinculin) to reduce the dark state activity. </br></br> Lov-ipaA-VinD1 is a powerful tool which allows photocontroled complex formation. To establish this system Lungu et al. (2012)<sup><a href=# | + | content.text= "The LOV-ipaA -vinculin system is a combined system for light inducible heterodimerisation. It consists out of a LOV domain, which undergoes conformational changes upon irradiation with blue light, and the ipaA-vinculin hybridization system. This two building blocks have be combined and described by Lungu et al. in 2012.</br></br> Lungu et al. (2008) where able to measure a 49-fold difference in target binding upon irradiation as compared to the dark state. However, they further modified the system by mutations of the LOV-ipaA construct and successfully weakend the baseline affinity for vinculin (initial design: 3.5 nM to 69 nM; mutant: 2.4 nM to >40µM affinity for vinculin) to reduce the dark state activity. </br></br> Lov-ipaA-VinD1 is a powerful tool which allows photocontroled complex formation. To establish this system Lungu et al. (2012)<sup><a href=#361>36.1</a></sup> fused the Ja helix of the LOV Domain to ipaA.</br>To be more precise they used the LOV2 domain from Avena sativa photopropin 1 (AsLOV2), which – as previously shown – can be used to photomodulate the affinity of peptides for their binding partners (see Figure 1). </br><div class=contant-image><img src=https://static.igem.org/mediawiki/2013/0/02/Bonn_MS_Figure1_LOV.jpg></br>Figure 1: General design of AsLOV2 fusion proteins (Lungu et al. 2012)<sup><a href=#361>36.1</a></sup></div> </br>In other studies had been shown that the LOV domain can be fused to entire protein domains, allowing photomodulation of the protein binding. However, they stated that it might be of high importance to bring the LOV domain closer to ipaA, in order to allow photomodulation in this case, because ipaA is only a peptide and thus more flexible than folded domains.</br></br>Therefore, Lungu et al. (2012)<sup><a href=361>36.1</a></sup> identified similar amino acid sequences in the ipaA peptide and the Ja helix of the LOV Domain and used this combined with molecular modeling to create photomodulateable AsLOV2-ipaA (see Figure 2). </br><div class=contant-image><img src=https://static.igem.org/mediawiki/2013/5/54/Bonn_MS_Figure2_LOV-ipaA.jpg></br>Figure 2: Light-inducible LOV-ipaA construct (Lungu et al. 2012)<sup><a href=361>36.1</a></sup></div></br>They were able to proof the functionality of the AsLOV2-ipaA system by heterodimerization in yeast (yeast two-hybrid system or Y2H) The yeast two-hybrid system can be used to monitor protein–protein interactions between two proteins. The system is based on a transcription factor, which is split into two separate fragments, called the binding domain (BD) and activating domain (AD). Each domain is fused to one protein and thus only if the proteins interact with each other BD and AD are close enough to initiate the transcription of a reporter gene.</br></br>The basic principle of the LOV-ipaA & VinD1 system works as follows. In the dark state the fusion product LOV-ipaA is not able to form a complex with vinculin, because LOV blocks ipaA sterically. However, activation of the LOV domain with blue light induces conformational changes in the fused molecule, which results in a movement of the Ja helix with the ipaA away from LOV. Thereby, ipaA becomes accessible for VinD1 and a Complex is formed.</br></br>The activation is reversible and the entire system can be genetically encoded. This two facts are the main advantages of this system in contrast to other typically used systems, which like for the chemical system for example, are based on in vivo covalently modified peptides, that can be activated by light induced cleavage. Moreover, the protein used are relatively small and thus should interfere as little as possible with the prokaryotic metabolism, the activity change form dark to light state is high, the system is completely genetically encoded and reversible. But, the most important property of this system is that it allows the light-controlled heterodimerisation of the two split variants of sspB, which is necessary for our system.</br></br><h2>References</h2></br><a name=361>36.1</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334866/>Lungu et al. (2012) Designing photoswitchable peptides using the AsLOV2 domain</a>"; |
| - | content.type=" | + | content.type="Background"; |
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content.parents=[36,21,12]; | content.parents=[36,21,12]; | ||
| - | content.childs=[38,45,53,69,52,57,65,100]; | + | content.childs=[38,45,53,69,52,57,65,100,200]; |
| - | content.titleShort = " | + | content.titleShort = "Project summary"; |
| - | content.titleLong = " | + | 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.summary= "We engineer a photoswitachble protein degradation system relying on the ClpXP protease system an a LOV domain from avena sativa"; | ||
| - | content.text= ""; | + | 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=" | + | content.type="Projekt"; |
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content.titleLong = "Application in Eukaryotes"; | content.titleLong = "Application in Eukaryotes"; | ||
content.summary = "The opportunity of a light switchable system in which a tagged protein is degraded by the protease ClpXP in eukaryotes offers various applications. A puplication from Grilly et al. gives information about the practicability of an application in eukaryotes"; | content.summary = "The opportunity of a light switchable system in which a tagged protein is degraded by the protease ClpXP in eukaryotes offers various applications. A puplication from Grilly et al. gives information about the practicability of an application in eukaryotes"; | ||
| - | content.text = "The application of our system in eukaryotes can yield many advantages.</br>As the ClpXP degradation system does not exist in eukaryotes naturally it does not get influenced by the eukaryotic cell. Besides no homologue to the protease ClpXP has been found in yeast | + | content.text = "The application of our system in eukaryotes can yield many advantages.</br>As the ClpXP degradation system does not exist in eukaryotes naturally it does not get influenced by the eukaryotic cell. Besides no homologue to the protease ClpXP has been found in yeast <sup><a href=#391>39.1</a></sup>. An advantage is that in yeast many genes can get expressed quite easier than in prokaryotes. Moreover a functionality of our system in yeast may offer applications in other eukaryotic model organisms.</br>Hence a degradation system in which degradation of a tagged protein by ClpXP is switchable by light in eukaryotes enables to produce several more proteins light-dependently which can lead to various new applications.</br></br>Grilly et al. made it possible to use the ClpXP degradation system in eukaryotes <sup><a href=#391>39.1</a></sup>.</br>Grilly et al. used the <i>E.coli</i> degradation machinery ClpXP to construct a <i>S.cerevisiae</i> strain that enables a tunable degradation of a tagged protein. A ClpXP system was created which degrades tagged proteins specifically and is tunable by changing concentrations of IPTG in the medium.</br></br>The genes ClpX and ClpP which code for the ClpXP protease were integrated into the yeast genome. However the genes ClpX and ClpP had to be modified with ten silent mutations until they worked in the yeast. The genes were integrated each together with an IPTG inducible promoter.</br>Now there was a system in the yeast genome which produces ClpXP only when IPTG was present.</br>The protein which was chose to be degraded by the ClpXP system was the yeast-enhanced green protein yEGFP. As the rate of fluorescence is measurable the yEGFP worked as an reporter. The yEGFP gene was integrated into the genome and tagged with a ssrA tag.</br>The promoter used was GAL1. GAL1 is induced by galactose and repressed by glucose. Thus the yeast produced yEGFP and was fluorescent in the present of galactose and did not produce yEGFP in the present of glucose.</br>The coexpression of the tagged yEGFP and the degradation machinery (IPTG and Galactose in the medium) leaded to an almost complete loss of fluorescence whereas if an untagged yEGFP was expressed the fluorescence did not decreased. This establishes that the degradation machinery works specifically to target proteins.</br>The influence of different concentrations of IPTG on the activity of ClpXP was studied.</br>By increasing the concentration of IPTG the rate of degradation of the tagged protein yEGFP increased, the fluorescence became less in a shorter time (figure 1). Thus the activity of the degradation system was tunable by concentrations of IPTG.</br></br><div class='content-image'><img src='https://static.igem.org/mediawiki/2013/7/74/Bonn.case39-bild2.PNG'[https://static.igem.org/mediawiki/2013/7/74/Bonn.case39-bild2.PNG]></br></br>Figure 1: The decrease of fluorescence of <i>S.cerevisiae</i> cells in time with different concentrations of IPTG in the medium <sup><a href=#391>39.1</a></sup></div></br></br>All in all considering the results of the work of Grilly et al. one can say that the degradation machinery ClpXP works in yeasts with little modifications. An important observation is that ClpXP is not toxic for yeast, the growth rate and morphology of the cells were unchanged <sup><a href=#391>39.1</a></sup>. Additionally it is possible to create systems in eukaryotes that make a tuning of the ClpXP activity possible.</br>In this system the activity of ClpXP is controlled by the concentration of IPTG whereas in our system ClpXP is controlled via sspB. SspB is not used in eukaryotes yet. However, sspB should be expressed without many problems because of it small size of 164 amino acids. Furthermore Grilly et al. state in their outlook that the coexpression of sspB might increase degradation <sup><a href=#391>39.1</a></sup>.</br>The other components used in our system, LOV-ipA and VinD1, have already been used in eukaryotes.</br>Hence a case can be made that our system should also work with little modifications in eukaryotes. Grilly et al. state that the system should function with any protein which can support the short ssrA and that they expect this degradation system to work with many different applications <sup><a href=#391>39.1</a></sup>. </br></br><h3>References<h3></br><p><a name=391>39.1</a> <a href='http://www.nature.com/msb/journal/v3/n1/full/msb4100168.html#f2'> Chris Grilly, Jesse Stricker, Wyming Lee Pang, Matthew R. Bennett, Jeff Hasty (2007), <i>A synthetic gene network for tuning protein degradation in Saccharomyces cerevisiae</i>, University of California San Diego</a></p>"; |
content.type = "Project"; | content.type = "Project"; | ||
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content.childs=[41,74,43]; | content.childs=[41,74,43]; | ||
content.titleShort = "C. crescentus"; | content.titleShort = "C. crescentus"; | ||
| - | content.titleLong = " | + | content.titleLong = "Ortholog ClpXP system in C. crescentus"; |
| - | content.summary= " | + | content.summary= "Using the ortholog ClpXP protein degradation system of C. crescentus in E. coli enables us to induce protein degradation in native E. coli without interfering with their natural system."; |
| - | content.text= "Caulobacter crescentus is a Gram-negative α-protobacterium often found in fresh water lakes or in the sea. Its cell division cycle is a favoured object of study due to its remarkable asymmetry. <sup><a href= | + | content.text= "Caulobacter crescentus is a Gram-negative α-protobacterium often found in fresh water lakes or in the sea. Its cell division cycle is a favoured object of study due to its remarkable asymmetry. <sup><a href=401>40.1</a></sup></br>ClpXP is a protease that degrades proteins tagged with the ssrA peptide. sspBα is an adaptor protein that can accelerate protein degradation by tethering ssrA-tagged proteins towards the ClpX subunit of the ClpXP protease.</br>For further information, please browse the related articles. </br></br><h2>References</h2></br><a href=401>40.1</a><a href=’ http://www.amazon.com/Brock-Biology-Microorganisms-13th-Edition/dp/032164963X’> Brock Microbiology of Microorganisms, Madigan et al., Pearson, German edition Vol. 13, 2012</a>"; |
content.type="Project"; | content.type="Project"; | ||
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content.parents=[40]; | content.parents=[40]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = " | + | content.titleShort = "<sup>Cc</sup>ClpXP"; |
| - | content.titleLong = "C. crescentus ClpXP"; | + | content.titleLong = "C. crescentus: ClpXP"; |
content.summary= "This article is about the ClpXP protease system in C. crescentus and regulation of proteolysis via ClpXP. It also provides information about similarities and differences between C. crescentus and E. coli orthologs of related proteins."; | content.summary= "This article is about the ClpXP protease system in C. crescentus and regulation of proteolysis via ClpXP. It also provides information about similarities and differences between C. crescentus and E. coli orthologs of related proteins."; | ||
| - | content.text= "<div class='content-image'><img src='https://static.igem.org/mediawiki/2013/d/de/Bonn-41-CC-ClpXP.png'>ClpXP mediated proteolysis <sup><a href= | + | content.text= "<div class='content-image'><img src='https://static.igem.org/mediawiki/2013/d/de/Bonn-41-CC-ClpXP.png'>ClpXP mediated proteolysis <sup><a href=#413>41.3</a></sup></div>ClpXP is an AAA+ protease which is of particular importance for proper cell-cycle progression. It consists of a hexameric ClpX subunit, which recognizes and unfolds tagged proteins, while ATP is hydrolyzed, and a ClpP subunit, that contains the actual peptidase domain. <sup><a href=#411>41.1</a>, <a href=#412>41.2</a></sup> Its structure is highly conserved, such that E. coli and C. crescentus orthologs are very similar. </br>Specific activity of the ClpXP protease is mediated by sspB and ssrA. ssrA is a short peptide consisting of fourteen amino acids in C. crescentus. Proteins which need to be degraded, e.g. for regulatory purpose or due to errors in their structure, are tagged with the ssrA peptide that can be recognized by the ClpX subunit. sspB is a dimeric adaptor protein in C. crescentus which tethers ssrA-tagged proteins to the ClpXP protease and, in this way, accelerates protein degradation. </br>Whilst sspB protein structure is well- conserved among many microorganisms, the structure of <sup>CC</sup>sspBα (i.e., the C. crescentus ortholog of sspB) shows significant differences compared to other orthologs. For example, <sup>CC</sup>sspBα and <sup>EC</sup>sspB (i.e., the E. coli ortholog) only show up with sequence identities of 16%, while CCsspB is still able to specifically bind to <sup>EC</sup>ClpXP<sup><a href=#412>41.2</a></sup>. SsrA tags can also be very different among microorganismic species. </br>This makes it possible to establish a protein degradation system in E. coli involving <sup>CC</sup>sspBα and <sup>EC</sup>ClpXP, as long as the ssrA tag can be recognized by both <sup>CC</sup>sspBα and <sup>EC</sup>ClpXP. </br></br><h2>References</h2></br><a name=411>41.1</a> <a href=’ http://www.ncbi.nlm.nih.gov/pubmed/17937918’>Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</a></br><a name=412>41.2</a> <a href=’http://www.ncbi.nlm.nih.gov/pubmed/20014030’>Versatile modes of peptide recognition by the ClpX N domain mediate alternative adaptor-binding specificities in different bacterial species, Chowdhury et al., Protein Science, 2010, PMID: 20014030</a></br><a name=413>41.3</a> <a href='http://www.biochem.umass.edu/faculty/peter-chien'>Peter Chien, Department of Biochemistry and Molecular Biology (official website), http://www.biochem.umass.edu/faculty/peter-chien, picture URL: cchttp://www.biochem.umass.edu/sites/biochem/files/resize/ClpX-300x268.jpg</a>"; |
content.type="Project"; | content.type="Project"; | ||
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content.parents=[74]; | content.parents=[74]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = "sspBα"; | + | content.titleShort = "<sup>Cc</sup>sspBα"; |
| - | content.titleLong = "C. crescentus 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."; | content.summary= "This article deals with the structure of sspBα and conformational details of its binding to ssrA and ClpXP during tethering."; | ||
| - | 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 <sup><a href= | + | 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 <sup><a href=#421>42.1</a></sup></div>The sspBα dimeric 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. <sup><a href=#422>42.2</a></sup> </br>Chien et al. <sup><a href=#421>42.1</a></sup> 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><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-<sup>CC</sup>ssrA concentration (1) without sspBα added, (2) with mutated sspBα(Q74A) added , (3) with wildtype sspBα added can be visualized. <sup><a href=#421>42.1</a></sup></div>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 <sup>CC</sup>ssrA, with an affinity being 175 times as large as for binding to <sup>EC</sup>ssrA (i.e. the E. coli ortholog). By comparing the crystal structures of both sspBα and the compound of sspBα and <sup>CC</sup>ssrA, Chien et al. further proved that binding of sspBα to <sup>CC</sup>ssrA does not lead to significant changes of its 3D conformation.</br></br><h2>References</h2></br><a name=421>42.1</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/17937918'>Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</a></br><a name=422>42.2</a><a href='http://www.ncbi.nlm.nih.gov/pubmed/14967151'> Bivalent tethering of sspB to ClpXP is required for efficient substrate delivery: a protein design study, Bolon DN et al., Mol Cell, 2004, PMID: 14967151</a>"; |
content.type="Project"; | content.type="Project"; | ||
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content.parents=[40]; | content.parents=[40]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = " | + | content.titleShort = "<sup>Cc</sup>sspB Split"; |
| - | content.titleLong = " | + | 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.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 <sup><a href=#431>43.1</a></sup>. Thus ccssrA only binds ccSspB but not E. coli SspB. <sup><a href=#432>43.2</a></sup> <sup><a href=#433>43.3</a></sup> <sup><a href=#434>43.4</a></sup> 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. <sup><a href=#431>43.1</a></sup> </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 <sup><a href=#431>43.1</a></sup>. </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'> <sup><a href=#431>43.1</a></sup> <h2>References:</h2> </br> <a name=431>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=432>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=433>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=434>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"; | content.type="Project"; | ||
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content.parents=[74]; | content.parents=[74]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = " | + | content.titleShort = "<sup>Cc</sup>SsrA"; |
content.titleLong = "C. crescentus ssrA and its application in E. coli"; | content.titleLong = "C. crescentus ssrA and its application in E. coli"; | ||
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.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 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 <sup><a href= | + | 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 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 <sup><a href=#441>44.1</a></sup> </div></br>Chien et al. <sup><a href=#441>44.1</a></sup> tested the 14-amino acid peptide ssrA (AANDNFAEEFAVAA, <sup><a href=#442>44.2</a></sup>) 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>Griffith and Grossman <sup><a href=#443>44.3</a></sup> engineered a protein degradation system in Bacillus subtilis, using the B. subtilis ClpXP protease for degradation of proteins tagged with modified ssrA tags from E. coli or C. crescentus. These ssrA tags will only deliver a protein to the ClpXP protease if E. coli sspB or C. crescentus sspBα, respectively, is present, as <sup>EC</sup>sspB can only detect <sup>EC</sup>ssrA and <sup>CC</sup>sspBα can only detect <sup>CC</sup>ssrA, which makes it possible to specifically regulate protein degradation by using two different promotors for the two sspB gene orthologs. </br>We used a similarly engineered system in E. coli for our project: By expressing <sup>CC</sup>ssrA and <sup>CC</sup>sspBα in E. coli, it became possible for us to specifically establish a light-inducible protein degradation system by connecting <sup>CC</sup>sspBα to the light-sensitive LOV domain.</br></br><h2>References</h2></br><a name=441>44.1</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/17937918'>Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</a></br><a name=442>44.2</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/20014030'>Versatile modes of peptide recognition by the ClpX N domain mediate alternative adaptor-binding specificities in different bacterial species, Chowdhury et al., Protein Science, 2010, PMID: 20014030</a></br><a name=443>44.3</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/18811726'>Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP, Griffith and Grossman, Moleculare Microbiology, 2008, PMID: 18811726 </a>"; |
content.type="Project"; | content.type="Project"; | ||
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content.parents=[45]; | content.parents=[45]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = " | + | content.titleShort = "Speed"; |
| - | content.titleLong = " | + | content.titleLong = "Speed"; |
content.summary= "Due to the use of the protease system protein regulation is fast"; | content.summary= "Due to the use of the protease system protein regulation is fast"; | ||
content.text= ""; | content.text= ""; | ||
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content.parents=[45]; | content.parents=[45]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = " | + | content.titleShort = "General applicability"; |
| - | content.titleLong = " | + | content.titleLong = "General applicability"; |
content.summary= "Since the system is commonly used and reliable its easily applicable to a wide variety of proteins"; | content.summary= "Since the system is commonly used and reliable its easily applicable to a wide variety of proteins"; | ||
content.text= ""; | content.text= ""; | ||
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content.parents=[45]; | content.parents=[45]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = " | + | content.titleShort = "Low influence"; |
| - | content.titleLong = " | + | content.titleLong = "Low influence on proteins"; |
content.summary= "Fusing only a 15Aa small tag to the desired protein leads to low influence on its native function"; | content.summary= "Fusing only a 15Aa small tag to the desired protein leads to low influence on its native function"; | ||
content.text= ""; | content.text= ""; | ||
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content.parents=[45]; | content.parents=[45]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = " | + | content.titleShort = "Temporal control"; |
| - | content.titleLong = " | + | content.titleLong = "Temporal control"; |
content.summary= "Using light easily makes a temporal specific control of activation possible"; | content.summary= "Using light easily makes a temporal specific control of activation possible"; | ||
content.text= ""; | content.text= ""; | ||
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content.parents=[45]; | content.parents=[45]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = " | + | content.titleShort = "Spatial control"; |
| - | content.titleLong = " | + | content.titleLong = "Spatial control"; |
content.summary= "High spatial control can be achieved easily by local irradation with light"; | content.summary= "High spatial control can be achieved easily by local irradation with light"; | ||
content.text= ""; | content.text= ""; | ||
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content.parents=[45]; | content.parents=[45]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = " | + | content.titleShort = "Great activity difference"; |
| - | content.titleLong = " | + | content.titleLong = "Great activity difference"; |
content.summary= "Degradation of the protein leads to a great difference of activity"; | content.summary= "Degradation of the protein leads to a great difference of activity"; | ||
content.text= ""; | content.text= ""; | ||
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content.i =52; | content.i =52; | ||
content.parents=[37]; | content.parents=[37]; | ||
| - | content.childs=[ | + | content.childs=[]; |
content.titleShort="Achievements"; | content.titleShort="Achievements"; | ||
content.titleLong="Achievements"; | content.titleLong="Achievements"; | ||
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content.titleShort = "Kill-switch for Lab Safety"; | content.titleShort = "Kill-switch for Lab Safety"; | ||
| - | content.titleLong = "Kill-switch systems using | + | content.titleLong = "Light inducible Kill-switch systems using toxin-antitoxin modules"; |
content.summary= "Toxin-antitoxin systems are composed by an antitoxin and a toxin coding gene. Connecting our light inducible protein degradation system to the antitoxin via an ssrA-tag allows light induced cell death, as predominance of the toxin in a bacterium activates a cell death pathway."; | content.summary= "Toxin-antitoxin systems are composed by an antitoxin and a toxin coding gene. Connecting our light inducible protein degradation system to the antitoxin via an ssrA-tag allows light induced cell death, as predominance of the toxin in a bacterium activates a cell death pathway."; | ||
content.text= "Our system of light inducible protein degradation can be utilized to degrade any specific protein and is thus usable in a light induced kill-switch system. For this application a connection between the degradation system and a toxin-antitoxin module like MazEF or ccdA/ccdB is needed. Either the toxin or the antitoxin could be light inducibly degraded by adding an ssrA-tag, which is detected by our degradation system, to its genetical code: <ul><li><b>Using the degradation of the toxin:</b> For that purpose the insertion of a plasmid containing the ssrA-tagged toxin encoding gene is needed. Since the predominance of the toxin activates a cell death pathway in bacteria, a bacterium containing a module that allows light inducible degradation of the toxin would only be viable, when the toxin is degraded. In darkness the toxin overexpression is no longer compensated and aggregation of it leads to cell death. Apart from the use in lab security such a kill-switch system would also be useful for environmental applications of bacteria, since it opens up the possibility of deploying bacteria for only one day and ensures their passing by nightfall. For example bacteria could be used to perform the ecological stabilization of a lake but after one night any genetically modified bacteria would be dead.</li><li><b>Using the degradation of the antitoxin:</b> Two plasmids are needed: The first one to express the toxin and the second one to express the ssra-tagged antitoxin, in such manner that the amounts of the toxin and the antitoxin are in equilibrium. Once light induces the degradation system, the antitoxin is degraded and the predominant toxin will kill the bacterium. </li></ul> Regarding our idea to improve lab security by implementing a kill-switch system, both described ways seem possible. Usage of the former would require cultivating and working with the bacteria under constant blue light, as darkness would kill them. Realization of the latter would require no usage of any blue light in the lab since bacteria which get into touch with daylight our any blue light would be killed. Due to the high light sensivity of our degradation system it can most likely be induced by daylight, which renders the former killswitch system useless. Bacteria which escape from the lab could survive simply through contact with daylight. Consequently we focused on the second system (the degradation of the antitoxin).</br> With MazEF we described a light inducible kill-switch system via the insertion of plasmids into bacteria. However a final kill-switch system would have to be introduced into the genomic DNA since plasmids in bacteria can be ejected, for instance via cell division, whereas a genomic DNA mutation is less likely to occur. Nevertheless the risk of a loss of function cannot be eliminated , which is why a secure system should countain much more than one kill-switch system to compensate the malfunction of a single kill-switch system. Therefore, we consider the MazEF kill-switch system to be part of a much larger security system for genetically engineered bacteria. </br>"; | content.text= "Our system of light inducible protein degradation can be utilized to degrade any specific protein and is thus usable in a light induced kill-switch system. For this application a connection between the degradation system and a toxin-antitoxin module like MazEF or ccdA/ccdB is needed. Either the toxin or the antitoxin could be light inducibly degraded by adding an ssrA-tag, which is detected by our degradation system, to its genetical code: <ul><li><b>Using the degradation of the toxin:</b> For that purpose the insertion of a plasmid containing the ssrA-tagged toxin encoding gene is needed. Since the predominance of the toxin activates a cell death pathway in bacteria, a bacterium containing a module that allows light inducible degradation of the toxin would only be viable, when the toxin is degraded. In darkness the toxin overexpression is no longer compensated and aggregation of it leads to cell death. Apart from the use in lab security such a kill-switch system would also be useful for environmental applications of bacteria, since it opens up the possibility of deploying bacteria for only one day and ensures their passing by nightfall. For example bacteria could be used to perform the ecological stabilization of a lake but after one night any genetically modified bacteria would be dead.</li><li><b>Using the degradation of the antitoxin:</b> Two plasmids are needed: The first one to express the toxin and the second one to express the ssra-tagged antitoxin, in such manner that the amounts of the toxin and the antitoxin are in equilibrium. Once light induces the degradation system, the antitoxin is degraded and the predominant toxin will kill the bacterium. </li></ul> Regarding our idea to improve lab security by implementing a kill-switch system, both described ways seem possible. Usage of the former would require cultivating and working with the bacteria under constant blue light, as darkness would kill them. Realization of the latter would require no usage of any blue light in the lab since bacteria which get into touch with daylight our any blue light would be killed. Due to the high light sensivity of our degradation system it can most likely be induced by daylight, which renders the former killswitch system useless. Bacteria which escape from the lab could survive simply through contact with daylight. Consequently we focused on the second system (the degradation of the antitoxin).</br> With MazEF we described a light inducible kill-switch system via the insertion of plasmids into bacteria. However a final kill-switch system would have to be introduced into the genomic DNA since plasmids in bacteria can be ejected, for instance via cell division, whereas a genomic DNA mutation is less likely to occur. Nevertheless the risk of a loss of function cannot be eliminated , which is why a secure system should countain much more than one kill-switch system to compensate the malfunction of a single kill-switch system. Therefore, we consider the MazEF kill-switch system to be part of a much larger security system for genetically engineered bacteria. </br>"; | ||
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content.titleLong = "Analysis of protein function"; | content.titleLong = "Analysis of protein function"; | ||
content.summary= "To understand the role of a specific gene or DNA region is one of the big challenges in lifescience research. Our system, which allows the fast and convenient elimination of defined proteins, is a new improved technique, with many advantages."; | content.summary= "To understand the role of a specific gene or DNA region is one of the big challenges in lifescience research. Our system, which allows the fast and convenient elimination of defined proteins, is a new improved technique, with many advantages."; | ||
| - | content.text= "To understand the role of a specific gene or DNA region is one of the big challenges in lifescience research. Our system, which allows the fast and convenient elimination of defined proteins, is a new improved technique, with many advantages. It allows the control of protein activity with respect to time and space, is fast, robust, can be used for different proteins and changes the protein sequence only little, because the ssrA tag consist only out of 15 amino acids. Recently technologies like gene knockout or knockdown had been developed, which allow to investigate the role of a particular gene or DNA region by comparing the knockout organism to a wildtype with a similar genetic background. </br></br> A knockout means that a particular gene is deleted from the genome of an organism. This organism might be bacteria or yeast, but also eukaryotic cells, plants or even animals. To create a knockout organism recombinant DNA is inserted into a gene (Bartke, 2006<sup><a href=# | + | content.text= "To understand the role of a specific gene or DNA region is one of the big challenges in lifescience research. Our system, which allows the fast and convenient elimination of defined proteins, is a new improved technique, with many advantages. It allows the control of protein activity with respect to time and space, is fast, robust, can be used for different proteins and changes the protein sequence only little, because the ssrA tag consist only out of 15 amino acids. Recently technologies like gene knockout or knockdown had been developed, which allow to investigate the role of a particular gene or DNA region by comparing the knockout organism to a wildtype with a similar genetic background. </br></br> A knockout means that a particular gene is deleted from the genome of an organism. This organism might be bacteria or yeast, but also eukaryotic cells, plants or even animals. To create a knockout organism recombinant DNA is inserted into a gene (Bartke, 2006<sup><a href=#553>55.3</a></sup>). When a genes sequence is interrupted, it may still be translated, but the resulting protein will be nonfunctional. Moreover, it is possible to knockout the gene only in defined tissues or at defined time points. This technique is called a conditional knockout. </br></br> On the other hand the knockdown, does not eliminate the specific gene on DNA, but on RNA level. Here interfering RNAs (siRNA) are inserted into the cell, leading to the degradation of the genes mRNA (Pratt and MacRae, 2009<sup><a href=#551>55.1</a></sup>) and hence no protein can be produced. </br></br> In contrast to the knockout and knockdown, our system allows the expression of the gene and the translation into functional protein. However, irradiation with blue light leads to the fast elimination of the particular protein. Therefore, one of the major advantages of our system is its speed, not only in comparison to knockout and knockdown, but also in comparison to other protein level systems. Comparing our system, to other protein level systems, like the system developed by Davis et al.<sup><a href=#552>55.2</a></sup> in 2011, which are induced by small molecules our system would still be faster due to the use of light. In small molecule systems it takes some time until the small molecules reach their target in the case of light this happens within milliseconds. Furthermore, with our system it is not necessary to add any kind of activator molecules, which might effect the results, to the cells. This enables researchers to investigate cell activity with and with out the protein in direct comparison, while the only interference is one light puls.</br></br><h2>References</h2></br></br><p><a name=551>55.1</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709356/>Pratt and MacRae (2009) The RNA-induced silencing complex: a versatile gene-silencing machine.</a></p> </br> <p><a name=552 >55.2</a> <a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3220803/>Davis et al. (2011) Small-molecule control of protein degradation using split adaptors.</a></p></br><p><a name=553 >55.3</a> <a href=http://www.sciencedirect.com/science/article/pii/S0531556506002798>Bartke (2006) New findings in transgenic, gene knockout and mutant mice.</a></p>"; |
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content.titleShort = "Results"; | content.titleShort = "Results"; | ||
content.titleLong = "Results"; | content.titleLong = "Results"; | ||
content.summary= "Results"; | content.summary= "Results"; | ||
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| - | content.titleShort = " | + | content.titleShort = "Plasmids"; |
| - | content.titleLong = "generated plasmids"; | + | content.titleLong = "A list of generated plasmids"; |
| - | content.summary= ""; | + | content.summary= "A list of each and every plasmid we designed combined with short summaries of their function"; |
| - | + | content.text="<groupparts></groupparts></br></br><a href='https://static.igem.org/mediawiki/2013/e/ef/Bonn_AraC-pBAD%28D%29-RBS32-mazF-TT_BBa_K1075040.jpg'><img src='https://static.igem.org/mediawiki/2013/e/ef/Bonn_AraC-pBAD%28D%29-RBS32-mazF-TT_BBa_K1075040.jpg' width='700px'></a>MazF is part of the biological toxin-antitoxin system MazEF. This system is derived from the bacterium Escherichia coli, where it serves as a stress inducable killswitch. MazF, a RNA-degrading enzyme, ist he toxin and is normally inhibited by the presence of MazE. Absence of MazE leads to cell death.<sup><a href=http://parts.igem.org/Part:BBa_K1075040>1</a></sup><a href='https://static.igem.org/mediawiki/2013/3/34/Bonn_pLac1-RBS32-mazF-TT_BBa_K1075039.jpg'><img src='https://static.igem.org/mediawiki/2013/3/34/Bonn_pLac1-RBS32-mazF-TT_BBa_K1075039.jpg' width='700px'></a>We used this part to gain an IPTG and Lactose inducible construct of MazF. This part was used to test the toxicity of MazF as part of our proof of concept. <sup><a href=http://parts.igem.org/Part:BBa_K1075039>2</a></sup><a href='https://static.igem.org/mediawiki/2013/8/87/Bonn_pLac2-RBS32-mazE-%28ec%29ssrA%28DAS%2B4%29-TT_BBa_K1075043.jpg'><img src='https://static.igem.org/mediawiki/2013/8/87/Bonn_pLac2-RBS32-mazE-%28ec%29ssrA%28DAS%2B4%29-TT_BBa_K1075043.jpg' width='700px'></a>This part can be used to gain a Lactose and IPTG inducable construct of mazE-(ec)ssrA(DAS+4). We used this construct as part of our proof of concept. <sup><a href=http://parts.igem.org/Part:BBa_K1075043>3</a></sup><a href='https://static.igem.org/mediawiki/2013/c/cf/Bonn_AraC-pBAD-RBS-mazF-TT-pLac2-RBS-mazE-%28ec%29ssrA%28DAS%2B4%29-TT_BBa_K1075048.jpg'><img src='https://static.igem.org/mediawiki/2013/c/cf/Bonn_AraC-pBAD-RBS-mazF-TT-pLac2-RBS-mazE-%28ec%29ssrA%28DAS%2B4%29-TT_BBa_K1075048.jpg' width='700px'></a>This construct can be used to gain IPTG and Lactose inducible expression of mazE(ec)ssrA(DAS+4) and arabinose inducible expression of MazF. Thus, both parts of the toxin-antitoxin system mazEF are inducible and this construct can be used to further examine this system. We used this construct as part of our proof of concept.<sup><a href=http://parts.igem.org/Part:BBa_K1075048>4</a></sup><a href='https://static.igem.org/mediawiki/2013/7/7f/Bonn_AraC-pBAD-RBS-sspB-TT-pLac2-RBS-mCherry-TT_BBa_K1075049.jpg'><img src='https://static.igem.org/mediawiki/2013/7/7f/Bonn_AraC-pBAD-RBS-sspB-TT-pLac2-RBS-mCherry-TT_BBa_K1075049.jpg' width='700px'></a>The sspB protein is an adaptor responsible for delivering ssrA-tagged substrates to the ClpXP protease in order to enhance their degradation. mCherry is a red fluorescent protein with the excitation maximum at 587 nm and the Emission maximum at 610 nm.<sup><a href=http://parts.igem.org/Part:BBa_K1075049>5</a></sup><a href='https://static.igem.org/mediawiki/2013/4/42/Bonn_pDawn_%28Prom_%28const.%29-RBS34-YF1-FixJ-FixK-LambdaC-pC%29_BBa_K1075044.jpg'><img src='https://static.igem.org/mediawiki/2013/4/42/Bonn_pDawn_%28Prom_%28const.%29-RBS34-YF1-FixJ-FixK-LambdaC-pC%29_BBa_K1075044.jpg' width='700px'></a>The plasmid pDawn was designed by Ohlendorf et al. in 2012 together with its counter plasmid pDusk. Both plasmids are single plasmid systems, which allow the activation (pDawn) or repression (pDusk) of gene expression by blue light. They are easy to implement in the laboratory and lead to up to 460-fold activity change upon ilumination. <sup><a href=http://parts.igem.org/Part:BBa_K1075044>6</a></sup><a href='https://static.igem.org/mediawiki/2013/5/50/Bonn_pDawn_%28Prom_%28const.%29-RBS34-YF1-FixJ-FixK-LambdaC-pC%29-RBS32-ccdB-TT_BBa_K1075047.jpg'><img src='https://static.igem.org/mediawiki/2013/5/50/Bonn_pDawn_%28Prom_%28const.%29-RBS34-YF1-FixJ-FixK-LambdaC-pC%29-RBS32-ccdB-TT_BBa_K1075047.jpg' width='700px'></a>pDawn-RBS32-ccdB-TT can be used to photocontrol the expression of the toxin ccdB. In this system pDawn (BBa_K1075044) can be regulated by blue light, while ccdB (BBa_K1075029) acts as a killswitch. The ccd operon of the F plasmid encodes CcdB, a toxin targeting the essential gyrase of Escherichia coli, and CcdA, the unstable antidote that interacts with CcdB to neutralize its toxicity.<sup><a href=http://parts.igem.org/Part:BBa_K1075047>7</a></sup><a href='https://static.igem.org/mediawiki/2013/0/03/Bonn_AraC-pBAD%28D%29-RBS32-SspB%28Core%28-LOV-ipaA-TT_BBa_K1075019.jpg'><img src='https://static.igem.org/mediawiki/2013/0/03/Bonn_AraC-pBAD%28D%29-RBS32-SspB%28Core%28-LOV-ipaA-TT_BBa_K1075019.jpg' width='700px'></a>The part can be used to regulate the dimerization of the two parts of the split sspB. The binding of ipaA to Vincolin is regulated via the light-sensitive LOV domain. Therefore the two sspB parts only come together when the LOV domain was activated with blue light. <sup><a href=http://parts.igem.org/Part:BBa_K1075019>8</a></sup><a href='https://static.igem.org/mediawiki/2013/d/d0/Bonn_J23105-RBS32-SspB%28Core%29-LOV-ipaA-TT_BBa_K1075018.jpg'><img src='https://static.igem.org/mediawiki/2013/d/d0/Bonn_J23105-RBS32-SspB%28Core%29-LOV-ipaA-TT_BBa_K1075018.jpg' width='700px'></a>The part can be used to regulate the dimerization of the two parts of the split sspB. The binding of ipaA to Vincolin is regulated via the light-sensitive LOV domain. Therefore the two sspB parts only come together when the LOV domain was activated with blue light. <sup><a href=http://parts.igem.org/Part:BBa_K1075018>9</a></sup><a href='https://static.igem.org/mediawiki/2013/5/5a/Bonn_AraC-pBAD%28D%29-RBS32-VinD1-sspBXB-TT_BBa_K1075016.jpg'><img src='https://static.igem.org/mediawiki/2013/5/5a/Bonn_AraC-pBAD%28D%29-RBS32-VinD1-sspBXB-TT_BBa_K1075016.jpg' width='700px'></a>The part can be used to regulate the dimerization of the two parts of the split sspB. As Vincolin bins to ipaA, the sspB dimer is stabilized. We used this part to achieve a light-induced dimerization of the sspB parts. <sup><a href=http://parts.igem.org/Part:BBa_K1075016>10</a></sup><a href='https://static.igem.org/mediawiki/2013/9/9c/Bonn_J23118-RBS32-VinD1-SspBXB-TT_BBa_K1075015.jpg'><img src='https://static.igem.org/mediawiki/2013/9/9c/Bonn_J23118-RBS32-VinD1-SspBXB-TT_BBa_K1075015.jpg' width='700px'></a>The part can be used to regulate the dimerization of the two parts of the split sspB. As Vincolin bins to ipaA, the sspB dimer is stabilized. We used this part to achieve a light-induced dimerization of the sspB parts. <sup><a href=http://parts.igem.org/Part:BBa_K1075015>11</a></sup><a href='https://static.igem.org/mediawiki/2013/7/78/Bonn_pLac_pBAD2-RBS32-SspB-TT_BBa_K1075006.jpg'><img src='https://static.igem.org/mediawiki/2013/7/78/Bonn_pLac_pBAD2-RBS32-SspB-TT_BBa_K1075006.jpg' width='700px'></a>The sspB protein is an adaptor responsible for delivering ssrA-tagged substrates to the ClpXP protease in order to enhance their degradation. Prefixed is RBS, the binding site for a ribosome and the pLac promoter. It also includes a double terminator 'TT', which interrupts the translation. <sup><a href=http://parts.igem.org/Part:BBa_K1075006>12</a></sup><a href='https://static.igem.org/mediawiki/2013/8/8f/Bonn_E._coli_ssrA%28DAS%2B4%29-TT_BBa_K1075021.jpg'><img src='https://static.igem.org/mediawiki/2013/8/8f/Bonn_E._coli_ssrA%28DAS%2B4%29-TT_BBa_K1075021.jpg' width='700px'></a>The (ec)ssrA(DAS+4) tag can be fused to proteins so that they will be delivered to the ClpXP protease and degraded.<sup><a href=http://parts.igem.org/Part:BBa_K1075021>13</a></sup><a href='https://static.igem.org/mediawiki/2013/6/63/Bonn_AraC-pBad-RBS34-mCherry-ecssrA%28DAS%2B4%29-TT_BBa_K1075025.jpg'><img src='https://static.igem.org/mediawiki/2013/6/63/Bonn_AraC-pBad-RBS34-mCherry-ecssrA%28DAS%2B4%29-TT_BBa_K1075025.jpg' width='700px'></a>As we want to control protein degradation by controlling the function of ecSspB, we tagged the red fluorescent protein mCherry with ecssrA(DAS+4) to measure the degradation rate. The part was designed for proof of principle. Application as a bacterial fotographic film might be possible as well. <sup><a href=http://parts.igem.org/Part:BBa_K1075025>14</a></sup><a href='https://static.igem.org/mediawiki/2013/6/63/Bonn_AraC-pBad-RBS34-mCherry-ecssrA%28DAS%2B4%29-TT_BBa_K1075025.jpg'><img src='https://static.igem.org/mediawiki/2013/6/63/Bonn_AraC-pBad-RBS34-mCherry-ecssrA%28DAS%2B4%29-TT_BBa_K1075025.jpg' width='700px'></a>As we want to control protein degradation by controlling the function of ecSspB, we tagged the red fluorescent protein mCherry with ecssrA(DAS+4) to measure the degradation rate. The part was designed for proof of principle. Application as a bacterial fotographic film might be possible as well. <sup><a href=http://parts.igem.org/Part:BBa_K1075025>15</a></sup><a href='https://static.igem.org/mediawiki/2013/c/cd/Bonn_pJD427%28pC-RBS32-FKBP12-EcsspB%28XB%29-Term-pB-RBS32-EcsspB%28Core%29-FRB-Term-Term%29_BBa_K1075010.jpg'><img src='https://static.igem.org/mediawiki/2013/c/cd/Bonn_pJD427%28pC-RBS32-FKBP12-EcsspB%28XB%29-Term-pB-RBS32-EcsspB%28Core%29-FRB-Term-Term%29_BBa_K1075010.jpg' width='700px'></a>The SspB protein is an adaptor responsible for delivering ssrA-tagged substrates to the ClpXP protease in order to enhance their degradation. To control degradation it is reasonable to control the function of SspB. That is made by splitting it into two parts, each of which cannot induce degradation on its own. To bring both SspB parts together again for inducible degradation, they were combined with a chemical inducible heterodimerisation system: FRB and FKBP12. These two parts interact in the presence of rapamycin <sup><a href=http://parts.igem.org/Part:BBa_K1075010>16</a></sup><a href='https://static.igem.org/mediawiki/2013/d/d6/Bonn_%28pJD427%29-pLac-RBS34-mCherry-ecssrA%28DAS%2B4%29-TT_BBa_K1075027.jpg'><img src='https://static.igem.org/mediawiki/2013/d/d6/Bonn_%28pJD427%29-pLac-RBS34-mCherry-ecssrA%28DAS%2B4%29-TT_BBa_K1075027.jpg' width='700px'></a>As we want to control protein degradation by controlling the function of ecSspB, we tag the red fluorescent protein mCherry with ecssrA(DAS+4) to measure the degradation rate. We control ecSspB by splitting it into two parts each of which cannot induce degradation on its own but regains function by addition of rapamycin. As the co-transformation of the two plasmids didn’t work, we combined them to one.<sup><a href=http://parts.igem.org/Part:BBa_K1075027>17</a></sup><a href='https://static.igem.org/mediawiki/2013/b/bc/Bonn_pJD427-pLac-RBS34-mCherry-TT_BBa_K1075028.jpg'><img src='https://static.igem.org/mediawiki/2013/b/bc/Bonn_pJD427-pLac-RBS34-mCherry-TT_BBa_K1075028.jpg' width='700px'></a>This plasmid is a negative control to the pJD427-pLac2-RBS34-mCherry-ecssrA(DAS+4)-TT plasmid. <sup><a href=http://parts.igem.org/Part:BBa_K1075028>18</a></sup><a href='https://static.igem.org/mediawiki/2013/c/cd/Bonn_pJD427%28pC-RBS32-FKBP12-EcsspB%28XB%29-Term-pB-RBS32-EcsspB%28Core%29-FRB-Term-Term%29_BBa_K1075010.jpg'><img src='https://static.igem.org/mediawiki/2013/c/cd/Bonn_pJD427%28pC-RBS32-FKBP12-EcsspB%28XB%29-Term-pB-RBS32-EcsspB%28Core%29-FRB-Term-Term%29_BBa_K1075010.jpg' width='700px'></a>The Plasmid pJD427 contains the fusion proteins SspB[CORE]-FRB and FKBP12-SspB[XB]: 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. <sup><a href=http://parts.igem.org/Part:BBa_K1075010>19</a></sup><a href='https://static.igem.org/mediawiki/2013/e/e1/Bonn_pLac-RBS32-ccdA-ssrA-TT_BBa_K1075035.jpg'><img src='https://static.igem.org/mediawiki/2013/e/e1/Bonn_pLac-RBS32-ccdA-ssrA-TT_BBa_K1075035.jpg' width='700px'></a>CcdA inhibits the toxin ccdB by binding to it and thus represses cell death. The part contains ccdA with a ssrA tag under the control of the Lac operon. <sup><a href=http://parts.igem.org/Part:BBa_K1075035>20</a></sup><a href='https://static.igem.org/mediawiki/2013/8/81/Bonn_AraC-pBAD%28D%29-RBS32-ccdB-TT_BBa_K1075031.jpg'><img src='https://static.igem.org/mediawiki/2013/8/81/Bonn_AraC-pBAD%28D%29-RBS32-ccdB-TT_BBa_K1075031.jpg' width='700px'></a>The part contains a chemically induced kill-switch. When Arabinose is added to the bacteria, the toxin ccdB is expressed and cell death is initiated. <sup><a href=http://parts.igem.org/Part:BBa_K1075031>20</a></sup>"; | |
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content.titleLong = "Riboswitch"; | content.titleLong = "Riboswitch"; | ||
content.summary= ""; | content.summary= ""; | ||
| - | content.text= "Riboswitches can be identified as a subtopic of transcriptional and translational regulation. They are based on self-regulating mRNA, achieved by combination with an aptamer region and a ligand-binding region. Ligands can be sugars, nucleotides, metal ions or other small molecules. This enables riboswitches to bind special metabolisms in order to induce conformational changes. These conformational changes can block or free the ribosomal binding site and therefore inhibit or activate translation of the mRNA into a polypeptide. Moreover it is able to control transcription by sequestering or releasing termination sequences. In addition to that the aptamer structures can mask or unmask ribozyme binding-sites, which enables a regulated RNA-degradation<sup><a href=# | + | content.text= "Riboswitches can be identified as a subtopic of transcriptional and translational regulation. They are based on self-regulating mRNA, achieved by combination with an aptamer region and a ligand-binding region. Ligands can be sugars, nucleotides, metal ions or other small molecules. This enables riboswitches to bind special metabolisms in order to induce conformational changes. These conformational changes can block or free the ribosomal binding site and therefore inhibit or activate translation of the mRNA into a polypeptide. Moreover it is able to control transcription by sequestering or releasing termination sequences. In addition to that the aptamer structures can mask or unmask ribozyme binding-sites, which enables a regulated RNA-degradation<sup><a href=#661>66.1</a></sup>.</br></br> <div class='content-image'><img src='https://static.igem.org/mediawiki/2013/8/8f/Bonn.Riboswitches.jpg'></br>"Diversity of Riboswitches and Mechanisms of Gene Control in BacteriaMechanisms of modulation of gene expression are highly divergent in prokaryotes and involve control of transcription, translation, splicing, and mRNA stability"<sup><a href=#661>66.1</a></sup></div><br/><div class='content-image'><img src='https://static.igem.org/mediawiki/2013/f/ff/Bonn.Riboswitches3.jpg'></br>"Structural Principles of Ligand Recognition by Riboswitches(AC) Schematic representations of a 'straight' junctional fold"<sup><a href=#661>66.1</a></div> </br></br> <p><a name=661>66.1</a> <a href='http://www.sciencedirect.com/science/article/pii/S0092867412015462'> A Decade of Riboswitches Alexander Serganov, Evgeny Nudler, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016, USA;" |
content.type="Background"; | content.type="Background"; | ||
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content.titleShort = "MazEF"; | content.titleShort = "MazEF"; | ||
| - | content.titleLong = " | + | content.titleLong = "The maz toxin-antitoxin system"; |
content.summary= "The toxin-antitoxin system MazEF is composed by an upstream gene <i>mazE</i>, encoding a labile antitoxin, and a downstream gene <i>mazF</i>, that encodes a stable toxin. Connecting our light inducible protein degradation system to the antitoxin MazE allows light inducible cell death, as predominance of MazF in a bacterium activates cell death pathway."; | content.summary= "The toxin-antitoxin system MazEF is composed by an upstream gene <i>mazE</i>, encoding a labile antitoxin, and a downstream gene <i>mazF</i>, that encodes a stable toxin. Connecting our light inducible protein degradation system to the antitoxin MazE allows light inducible cell death, as predominance of MazF in a bacterium activates cell death pathway."; | ||
| - | + | content.text= "Our system of light inducible protein degradation can be utilized to degrade any specific protein and is therefore usable to realize a light inducible kill-switch system. A connection of protein degradation to a cell death pathway is represented by the stress-induced toxin-antitoxin module <i>mazEF</i> in Escherichia coli. <i>mazEF</i> is located on the chromosome in E.coli which is associated with programmed cell death. The toxin-antitoxin system is composed by an upstream gene <i>mazE</i>, encoding a labile antitoxin, and a downstream gene mazF, that encodes a stable toxin. </br>The product of <i>mazF</i> cleaves mRNAs and tmRNAs at a specific site, which leads to an inhibition of translation. MazF shows a specific cleaving mechanism, which is not well understood yet, but shows that there is also protein synthesis which is unaffected by MazF. These proteins are presumably part of a cell death pathway. </br>The effect of <i>mazF</i> is suppressed by the Product of <i>mazE</i> which is degraded by the Protease ClpAP in bacteria. As a result of stressful conditions expression of the chromosomal <i>mazEF</i> module is reduced which leads to an imbalance between the products of <i>mazF</i> and <i>mazE</i>: When expression is lowered the stable toxin of <i>mazF</i> still persists while the labile antitoxin of <i>mazE</i> is degraded and can no longer suppress the effect of <i>mazF</i> leading to acute toxicity and cell death. </br><i>MazEF</i>-mediated cell death in E. coli can be caused by:<ul><li>extreme amino acid starvation<sup><a href = #674>67.4</a></sup><sup><a href = #675>67.5</a></sup></li><li> inhibition of transcription and/or translation by antibiotics such as rifampin, chloramphenicol, and spectinomycin under specific growth conditions<sup><a href = #676>67.6</a></sup></li><li>inhibition of translation by the Doc protein of prophage P1<sup><a href = #677>67.7</a></sup></li><li>DNA damage caused by thymine starvation<sup><a href = #678>67.8</a></sup> as well as by mitomycin C, nalidixic acid, and UV irradiation<sup><a href = #679>67.9</a></sup></li><li>oxidative stress (H2O2)<sup><a href = #679'>67.9</a></sup></li></ul>Amitai et al. tested in 2004 the Hypothesis of Pedersen et al.<sup><a href = #672>67.2</a></sup>, that chromosomal toxin-antitoxin systems may rather cause a state of reversible bacteriostasis than programmed cell death<sup><a href = #671>67.1</a></sup>.Therefore E.coli strain MC4100 Δ<i>mazE</i>F relA1 lacIq was cotransformated with:<ul><li>pBad-<i>mazF</i></li><li>pQE-Δhis-<i>mazE</i></li></ul><i>mazF</i>-expression can be induced by the addition of Arabinose via the pBad promoter of the first plasmid. The transformation of the second plasmid results firstly in the repression of <i>mazE</i> expression, whereas when IPTG is added <i>mazE</i> production is induced.</br><div class='content-image' align='center' height=501 width=410><a href='https://static.igem.org/mediawiki/2013/a/ac/Team_Bonn_MazF_1.png'><img src='https://static.igem.org/mediawiki/2013/a/ac/Team_Bonn_MazF_1.png' height=491 width=400></a></br><i>Ability of E. coli cells that had been ectopically overexpressing MazF in liquid medium to form colonies when ectopically overexpressing MazE on plates. The cultures were grown in LB medium (A) or M9 minimal medium with 0.5% glycerol (B) at 37°C to midlogarithmic phase (OD600, 0.5)<sup><a href = #671>67.1</a></sup>.</i></div>Using these tools, Amitai et al. tested the effect of MazE overproduction on MazF-overproducing bacteria during growth in liquid medium.</br>The E.coli strain was incubated in LB medium. After <i>mazF</i> expression was induced by adding arabinose two samples were taken at several time points. To repress <i>mazF</i> expression to both of them glucose was added. In addition IPTG was added to one culture to induce <i>mazE</i> expression. The two cultures were compared via the level of protein synthesis and OD600.</br>Finally Amitai et al. confirmed the assumption that the overproduction of MazE after until 6h under overproduction of MazF could resuscitate E.coli cells in LB medium (Fig. 1A)<sup><a href = #671>67.1</a></sup>, but the longer MazF was induced the less cells could be resuscitated by MazE.</br>Whereas MazE overproduction can reverse the inhibitory effect of MazF on translation, it cannot reverse the effect of MazF on colony formation, which is shown in figure 2. Only 1h after the induction of MazE expression, the rate of translation was restored to nearly 100% (Fig.2 Aa, Ab, Ac) but the bacteriocidic effect could not be reversed (Fig.2 Ba, Bb, Bc).</br>Additionally, Amtai et al. found out, that in M9 medium MazE was less able to reverse the effects of MazF overexpression than in LB medium (Fig.1B vs. 1A). it was concluded that there is a point of no return, when MazE is inable to resuscitate a MazF damaged cell, which occurs earlier in M9 medium than in LB medium.</br>Based on their results a model of the MazEF mechanism was built: A <i>mazF</i>-mediated cascade leads to a cell death pathway, but can nevertheless be stopped at several intermediary steps by e.g. <i>mazE</i>. When a point of no return is reached, the cascade cannot be stopped anymore.<div class='content-image' align='center' height=827 width=784><a href='https://static.igem.org/mediawiki/2013/9/9e/Team_Bonn_MazF_2.png'><img src='https://static.igem.org/mediawiki/2013/9/9e/Team_Bonn_MazF_2.png' height=817 width=764></a></br><i>Effect of MazE overproduction during growth in liquid medium on the ability of MazF-overproducing E. coli cells to synthesize proteins and form colonies. To induce <i>mazE</i> expression, IPTG was added to the bacterial culture at 1 h (Aa and Ba), 4 h (Ab and Bb), and 6 h (Ac and Bc) after <i>mazF</i> induction at time zero. The effects of the ectopic overexpression of MazE were measured at 1 and 3 h after the induction of <i>mazE</i> expression.<sup><a href = #671>67.1</a></sup>.</i></div></br>Back to our project and to the idea of a light inducible kill-switch system:</br>As we described in the previous paragraph for both of our kill-switch systems using the MazEF module either MazF or MazE could be degraded:<ul><li>Using the degradation of MazF:</b> For that purpose the insertion of a plasmid containing the ssrA-tagged toxin encoding gene is needed. Since the predominance of the toxin activates a cell death pathway in bacteria, a bacterium containing a module that allows light inducible degradation of the MazF would only be viable, when it is degraded. In darkness the MazF overexpression is no longer compensated and aggregation of it leads to cell death. </li><li><b>Using the degradation of the antitoxin:</b> Two plasmids are needed: The first one to express the MazF and the second one to express the ssra-tagged MazE, in such manner that the amounts of the <i>MazF</i> and the <i>MazE</i> are in equilibrium. Once light induces the degradation system, the MazE is degraded and the predominant toxin will kill the bacterium.</li></ul>As we explained in the general kill-switch system text we finally focused on the second system (via the degradation of MazE).</br>With the design of a MazEF kill-switch system the possibility of resuscitating bacteria in the way Amitai et al. showed has to be considered. A predominant MazF could kill a bacterium in LB within about two hours, but it needs to be predominant over a long period (>7h) to induce its death without it being resuscitated by renewed <i>mazE</i> expression (Fig.1A)with more than 50% probability .</br>Certainly these facts seem to be unfavourable for the realization of a kill switch system via MazEF, but fortunately our system of heterodimerization (Lungu et al.) allows long continuous degradation<sup><a href = #673>67.3</a></sup>, due to the high stability of the light induced hererodimer. Therefore likely a short exposure time will result in prolonged protein degradation sufficing for bacterial death. Additionally, Amitai et al. showed that the less nutrition is available for a bacterium, the earlier the point of no return is reached. If a bacterium escapes the lab, it will likely have less nutrition available than in LB medium. It might reach the point of no return earlier.</br>We described a light inducible MazEF kill-switch system via the insertion of plasmids into bacteria. However a final kill-switch system would have to be implemented in the genomic DNA since plasmids in bacteria can be ejected, for instance via cell division, whereas a genomic DNA mutation is less likely to occur. Nevertheless risk of a loss of function cannot be eliminated , which is why a secure system should countain much more than one kill-switch system to compensate the malfunction of a single kill-switch system. Therefore, we consider the MazEF kill-switch system to be part of a much larger security system for genetically engineered bacteria.<h2>References</h2><a name =671>67.1</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC532418/'>MazF-Mediated Cell Death in Escherichia coli: a Point of No Return, Shahar Amitai et al., Journal of Bacteriology Vol. 186, No. 24, 2004, p.8295–8300.</a></br><a name =672>67.2</a> <a href = 'http://www.ncbi.nlm.nih.gov/pubmed/?term=Rapid+induction+and+reversal+of+bacteriostatic+conditions+by+controlled+expression+of+toxins+and+antitoxins'>Rapid induction and reversal of bacteriostatic conditions by controlled expression of toxins and antitoxins, Pedersen et al., Molecular Microbiology 45, 2002, 501–510.</a></br><a name =673>67.3</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334866/'>Designing Photoswitchable Peptides Using the AsLOV2 Domain, Oana I. Lungu et al., Chem Biol. 2012, 19(4):507-17.</a></br><a name =674>67.4</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC39188/'>An Escherichia coli chromosomal addiction module regulated by guanosine 3,5-bispyrophosphate: a model for programmed bacterial cell death, Aizenman, E., H. Engelberg-Kulka, and G. Glaser, Proc. Natl. Acad. Sci., 1996, USA 93:6059-6063.</a></br><a name =675>67.5</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC28068/'>rexB of bacteriophage lambda is an anti-cell death gene. Engelberg-Kulka, H., M. Reches, S. Narasimhan, R. Schoulaker-Schwarz, Y. Klemes, E. Aizenman, and G. Glaser, Proc. Natl. Acad. Sci., 1998, USA 95:15481-15486.</a></br><a name =676>67.6</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC95100/'>Programmed cell death in Escherichia coli: some antibiotics can trigger mazEF lethality, Sat, B., R. Hazan, T. Fisher, H. Khaner, G. Glaser, and H. Engelberg-Kulka, J. Bacteriol. 2001, 183:2041-2045.</a></br><a name =677>67.7</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC95101/'>Postsegregational killing mediated by the P1 phage addiction module Phd-Doc requires the Escherichia coli programmed cell death system mazEF, Hazan, R., B. Sat, M. Reches, and H. Engelberg-Kulka, J. Bacteriol. 2001, 183:2046–2050.</a></br><a name =678>67.8</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC150121/'>The Escherichia coli mazEF suicide module mediates thymineless death, Sat, B., M. Reches, and H. Engelberg-Kulka, J. Bacteriol. 2003, 185:1803–1807.</a></br><a name =679>67.9</a> <a href = 'http://www.ncbi.nlm.nih.gov/pmc/articles/PMC415763/'>Escherichia coli mazEFmediated cell death is triggered by various stressful conditions, Hazan, R., B. Sat, and H. Engelberg-Kulka, J. Bacteriol. 2004, 186:3663–3669.</a>"; | |
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content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = " | + | content.titleShort = "ccdAB"; |
| - | content.titleLong = " | + | content.titleLong = "The ccd toxin-antitoxin system"; |
| - | content.summary= " | + | content.summary= "CcdB leads to double strand breaks in bacterial genomic DNA and thus is a potent toxin leading to cell death. ccdA compromises the toxic effect of ccdB."; |
| - | content.text= "The ccd module is a toxin-antitoxin (TA) system similar to the mazE/mazF system. The module is located on the F Plasmid in Escherichia coli bacteria and essential for their survival. Normally the toxin ccdB is inactivated by the presence of the antitoxin ccdA in the form of a ccdAB complex. If ccdA is no longer available, ccdB inhibits DNA gyrase which leads to cell death. Gyrase is a type IIA topoisomerase and is able to produce negative DNA supercoiling by making a double-strand break in the DNA and religating it. The gyrase enzyme consists of two subunits: the C-terminal GyrA domain that wraps around the DNA strand and the N-terminal GyrB domain that catalyses the ATP-dependant supercoiling of the DNA. CcdB stabilizes the gyrase cleavage complex by binding to the GyrA domain and thus blocks the catalytic function of the gyrase. That means that the gyrase remains bound to the DNA and the cleaved DNA is not religated. DNA- and RNA polymerases can’t copy the DNA anymore and cell proliferation as well as protein biosynthesis is stopped. The double-stranded breaks in the DNA initiate cell death.<p>Because gyrases are specific to bacteria such as E. coli it is also a target for some anti-bacterial medications e.g. ciprofloxacin (CFX). As can be seen in the data below, CcdB proves to be as effective as CFX at inducing DNA cleavage <sup><a href=# | + | content.text= "The ccd module is a toxin-antitoxin (TA) system similar to the mazE/mazF system. The module is located on the F Plasmid in Escherichia coli bacteria and essential for their survival. Normally the toxin ccdB is inactivated by the presence of the antitoxin ccdA in the form of a ccdAB complex. If ccdA is no longer available, ccdB inhibits DNA gyrase which leads to cell death. Gyrase is a type IIA topoisomerase and is able to produce negative DNA supercoiling by making a double-strand break in the DNA and religating it. The gyrase enzyme consists of two subunits: the C-terminal GyrA domain that wraps around the DNA strand and the N-terminal GyrB domain that catalyses the ATP-dependant supercoiling of the DNA. CcdB stabilizes the gyrase cleavage complex by binding to the GyrA domain and thus blocks the catalytic function of the gyrase. That means that the gyrase remains bound to the DNA and the cleaved DNA is not religated. DNA- and RNA polymerases can’t copy the DNA anymore and cell proliferation as well as protein biosynthesis is stopped. The double-stranded breaks in the DNA initiate cell death.<p>Because gyrases are specific to bacteria such as E. coli it is also a target for some anti-bacterial medications e.g. ciprofloxacin (CFX). As can be seen in the data below, CcdB proves to be as effective as CFX at inducing DNA cleavage <sup><a href=#681>68.1</a></sup><sup> <a href=#682>68.2</a></sup></p><div class='content-image'align='center'><img src='https://static.igem.org/mediawiki/2013/2/2f/Sspb_CFX_compared.jpg' width='300'>Comparison of the effect of CcdB and CFX on gyrase activity. N: negatively supercoiled DNA, L: linear DNA, SC: supercoiled DNA. A higher concentration of CcdB/CFX leads to more cleaved (linear) DNA<sup><a href= http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635281/figure/fig2/>[source]</a></sup></div><div class='content-image' align='center'><img src='https://static.igem.org/mediawiki/2013/e/e4/Bonn_Ccdb_and_ccda.jpg' width='300'> A higher concentration of ccdB leads to blocking of gyrase and positively supercoiled DNA is cleaved to linear DNA instead of being processed to negatively supercoiled DNA. <sup><a href=http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460896/figure/pone-0046499-g003/>[source]</a></sup></div><p>We used this system to build a light-induced kill-switch. Therefore we added the ssrA tag to the antitoxin ccdA. When the bacteria a emitted to light, ccdA is degraded and ccdB is set free and can bind to the gyrase. and cell death is initiated. Like in most TA systems, the toxin ccdB is relatively stable, while the antitoxin ccdA is vulnerable to degradation.</p><h2>References</h2><p><a name=681>68.1</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635281/'>A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site, Andrew B. Smith and Anthony Maxwell, Nucleic Acids Res. 2006 October; 34(17): 4667–4676, PMC 1635281</a></p><p><a name=682>68.2</a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460896/'> A Common Origin for the Bacterial Toxin-Antitoxin Systems parD and ccd, Suggested by Analyses of Toxin/Target and Toxin/Antitoxin Interactions, Andew B. Smith et al, PLoS One. 2012; 7(9): e46499, PMCID: PMC3460896</a></p>"; |
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content.titleLong = "Operon-model"; | content.titleLong = "Operon-model"; | ||
content.summary= "The operon-model is a very popular one. The operon allows an organism to regulate the expression of specific genes and therefore the production of corresponding proteins, depending on the concentration of a specific substrate ("substrate-induction") or the lack of an important product ("product-repression")."; | content.summary= "The operon-model is a very popular one. The operon allows an organism to regulate the expression of specific genes and therefore the production of corresponding proteins, depending on the concentration of a specific substrate ("substrate-induction") or the lack of an important product ("product-repression")."; | ||
| - | content.text= "In absence of the substrate, a special repressor binds to the operator DNA sequence in order to inhibit the transcription by the DNA polymerase. The substrate can bind to an allosteric center of the repressor, which leads to a change of conformation. Now the repressor lost his affinity to the operator DNA and cannot inhibit the transcription anymore. This is called "substrate induction".<sup> <a href=# | + | content.text= "In absence of the substrate, a special repressor binds to the operator DNA sequence in order to inhibit the transcription by the DNA polymerase. The substrate can bind to an allosteric center of the repressor, which leads to a change of conformation. Now the repressor lost his affinity to the operator DNA and cannot inhibit the transcription anymore. This is called "substrate induction".<sup> <a href=#711>71.1</a> </sup> </br> </br><div class='content-imgage'> <img src=https://static.igem.org/mediawiki/2013/0/0f/BonnLacOperon.jpg></br><i>lac </i>operon<sup><a href=#712>71.2</a></sup> </br> <a href='geneticsengineering.blogspot.com'> </a> </div> </br> </br> "Product repression" works the other way around. As long as there is an excess of substrate and a lack of product, the repressor has an low affinity to the operator DNA and is not able to inhibit the transcription. If there is enough product, it can bind to the allosteric center of the repressor, which improves it's affinity to the operator DNA. Now it is able to inhibit transcription.<sup> <a href=#713>71.3</a> </sup></br> </br><div class='content-image'> <img src='https://static.igem.org/mediawiki/2013/e/ed/BonnTrpOperon.jpg'></br>Tryptophan operon<sup><a href=#714>71.4</a></sup></div></br> </br></br><p><a name=711>71.1 </a> <a href='http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3722110/'>A single mutation in the core domain of the lac repressor reduces leakiness, Pietro Gatti-Lafranconi, Willem P Dijkman, Sean RA Devenish, and Florian Hollfelder corresponding author.</a></p><p><a name=712>71.2</a> <a href='http://geneticsengineering.blogspot.de/'>geneticsengineering.blogspot </a></p><p><a name=713>71.3</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/22713856'> Influence of the feedback loops in the trp operon of B. subtilis on the system dynamic response and noise amplitude. Zamora-Chimal C, Santillán M, Rodríguez-González J.</a></p><p><a name=714>71.4</a> <a href='http://bio1903.nicerweb.com/Locked/media/ch18/18_21bTrpOperon_2.jpg'> bio1903.nicerweb.com/Locked/media/ch18/18_21bTrpOperon_2.jpg </a>"; |
content.type="Background"; | content.type="Background"; | ||
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content.titleLong = "Zinc finger"; | content.titleLong = "Zinc finger"; | ||
content.summary= "Zinc fingers can be engineered to bind desired DNA sequences"; | content.summary= "Zinc fingers can be engineered to bind desired DNA sequences"; | ||
| - | content.text= "The family of the "zinc finger" proteins has a different approach of Transcription regulation. They contain a zinc ion as a cofactor. Zinc finger proteins have DNA binding and dimerization domain. They can be differentiated | + | content.text= "The family of the "zinc finger" proteins has a different approach of Transcription regulation. They contain a zinc ion as a cofactor. Zinc finger proteins have a DNA binding and a dimerization domain. They can be differentiated due to different loops. On the one hand they can bind to almost every part of DNA and on the other hand they can bind to several receptors. Hence the activating or repressing effect is not defined by the zinc finger itself, but by the effector Protein it is binding to<sup> <a href=#721>72.1</a></sup>. </br></br> <div class='content-image'> <img src='https://static.igem.org/mediawiki/2013/4/4b/BonnZincFinger.jpg' width='400' height='400'></br>"The zinc ion (green) is coordinated by two histidine and two cysteine amino acid residues"<sup><a href=#722>72.2</a></sup></div></br></br> <p><a name=721>72.1</a> <a href=http://www.pnas.org/content/early/2013/09/11/1303625110.long> Transcription factor ZBED6 affects gene expression, proliferation, and cell death in pancreatic beta cellsXuan Wang, Lin Jiang,Ola Wallerman, Ulla Engström, Adam Ameur, Rajesh Kumar, Gupt, YuQi, Leif Andersson and Nils Welsh Science for Life Laboratory, Department of Medical Cell Biology, and Science for Life Laboratory, Department of Medical Biochemistry and Microbiology, Uppsala University, SE-75123 Uppsala, Sweden; Ludwig Institute for Cancer Research Ltd., Science for Life Laboratory, Uppsala University, SE-751 24 Uppsala, Sweden; andScience for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, SE-75185 Uppsala, Sweden </a> </p> </br> <p><a name=722>72.2</a> <a href='http://en.wikipedia.org/wiki/Zinc_finger'>Cartoon representation of the zinc-finger motif of proteins</a></p>"; |
content.type="Background"; | content.type="Background"; | ||
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content.childs=[]; | content.childs=[]; | ||
content.titleShort = "TALE"; | content.titleShort = "TALE"; | ||
| - | content.titleLong = " | + | content.titleLong = "Transcription activator-like effectors"; |
content.summary= "TALEs enable an easy and modular assembly of proteins binding specific desired DNA sequences"; | content.summary= "TALEs enable an easy and modular assembly of proteins binding specific desired DNA sequences"; | ||
| - | content.text= " | + | content.text= "Tales are proteins which bind promoter sequences. Their DNA binding domain consists of several tandem repeats that are able to bind specific domains of the DNA. These tandem repeats can be engineered easily. Thus the user can define the domain to bind to. Very similar to the zinc finger they actually do not regulate transcription but bind effector proteins which are able to activate transcription. The great advance, in comparison to the zinc finger domain, is it's easy way of engineering. Scientists can regulate very specificlly transcription by the use of TALEs.</br></br><p> <a href='http://onlinelibrary.wiley.com/doi/10.1111/jipb.12091/abstract'> Site-Specific Gene Targeting Using Transcription Activator-Like Effector (TALE)-Based Nuclease in Brassica oleracea: Zijian Sun,Nianzu Li, Guodong Huang, Junqiang Xu, Yu Pan, Zhimin Wang, Qinglin Tang, Ming Song*, Xiaojia Wang> </a> </p>"; |
| - | + | ||
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content.parents=[40]; | content.parents=[40]; | ||
content.childs=[42,44]; | content.childs=[42,44]; | ||
| - | content.titleShort = "ssrA and | + | content.titleShort = "ssrA and SspBα"; |
| - | content.titleLong = "Introduction to C. crescentus ssrA and | + | 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.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= " | + | content.text= "SsrA and sspB are peptides that mediate proteolysis via the ClpXP protease system in bacteria. In this article and the related articles, the focus is on their orthologs in C. crescentus, being referred to as <sup>Cc</sup>ssrA and <sup>Cc</sup>sspBα, respectively, omitting <sup>Cc</sup> when obvious out of context. The ClpXP protease has an important function in regulation of the cell division cycle by effective proteolysis of short-lived regulatory proteins.</br>A protein which needs to be degraded will be tagged with the amino acid peptide <sup>Cc</sup>ssrA which is added at its C-terminus during translation. <sup><a href=#741>74.1</a>, <a href=#742>74.2</a></sup> The ClpX subunit of the ClpXP protease recognizes the ssrA tag by specific binding and unfolds the tagged protein, in which ATP is hydrolyzed. In C. crescentus, the ssrA tag has a length of 14 amino acids, while the E. coli ortholog is only eleven amino acids long. </br>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><h2>References</h2></br><a name=741>74.1</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/11535833'>Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis, Flynn et al., Proceedings of the National Academy of Sciences of the United States of America, 2001, PMID: 11535833</a></br><a name=742>74.2</a> <a href='http://www.ncbi.nlm.nih.gov/pubmed/17937918'> Structure and substrate specificity of an SspB ortholog: design implications for AAA+ adaptors, Chien et al., Cell Press, 2007, PMID: 17937918</a>"; |
content.type="Project"; | content.type="Project"; | ||
break; | break; | ||
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content.i = 100; | content.i = 100; | ||
content.parents=[37]; | content.parents=[37]; | ||
| - | content.childs=[106,107,108, | + | content.childs=[106,107,108,112,101,110,130]; |
content.titleShort = "Human Practice"; | content.titleShort = "Human Practice"; | ||
content.titleLong = "Human Practice in General"; | content.titleLong = "Human Practice in General"; | ||
| - | content.summary= " | + | content.summary= "For our Human Practice advances our project's design follows the well-known franchise of Star Wars - being inspired by the LOV domain our project name "LOV WARS" was born. The constant references to the Star Wars movies facilitate the access to synthetic biology to a person that is not familiar with the subject. We put great effort into adapting every aspect of our public presence to Star Wars."; |
content.text= "<p>The major goal in Human Practice is to make synthetic biology easily understandable and interesting for everybody. For that purpose our project's design follows the well-known franchise of Star Wars; our project name 'LOV WARS' was born. The constant references to the Star Wars movies facilitate the access to synthetic biology to a person that is not familiar with the subject. With the most famous icon of Star Wars being the laser sword it offers a great opportunity to present our light induction system in an entertaining way. We put great effort into adapting every aspect of our public presence to Star Wars.</p><p>Therefore we introduced a <a onclick=node(110)>flash game</a> and a <a onclick=node(109)>comic series</a> called LOV Wars in which we explain basic concepts of synthetic biology and our project. The comic and the LOV Wars shooter arouse interest in people that would normally never engage with the field. That way we can spread the possibilities and advantages that modern genetically engineered organisms offer and clear possible misunderstandings or prejudices against the subject.</p>For the same purpose we organized multiple events for general public at which we presented the tools used in synthetic biology and our project; over the last six months we <a onclick=node(108)>visited several High Schools</a> in Bonn and nearby and gave presentations. In addition we had an information booth in our city center informing passersby about the risks and benefits of synthetic biology with the aid of several posters and flyers. A big highlight was a <a onclick=node(106)>Science slam</a> we arranged. In front of a big audience six scientists from different fields presented an interesting aspect of their academic field. The Science Slam was a great success in terms of getting people in touch with science and without exception received very good critiques.<br> At every opportunity we handed out questionnaires in which we evaluated the public's opinion about synthetic biology and whether our human practice events were informative and interesting."; | content.text= "<p>The major goal in Human Practice is to make synthetic biology easily understandable and interesting for everybody. For that purpose our project's design follows the well-known franchise of Star Wars; our project name 'LOV WARS' was born. The constant references to the Star Wars movies facilitate the access to synthetic biology to a person that is not familiar with the subject. With the most famous icon of Star Wars being the laser sword it offers a great opportunity to present our light induction system in an entertaining way. We put great effort into adapting every aspect of our public presence to Star Wars.</p><p>Therefore we introduced a <a onclick=node(110)>flash game</a> and a <a onclick=node(109)>comic series</a> called LOV Wars in which we explain basic concepts of synthetic biology and our project. The comic and the LOV Wars shooter arouse interest in people that would normally never engage with the field. That way we can spread the possibilities and advantages that modern genetically engineered organisms offer and clear possible misunderstandings or prejudices against the subject.</p>For the same purpose we organized multiple events for general public at which we presented the tools used in synthetic biology and our project; over the last six months we <a onclick=node(108)>visited several High Schools</a> in Bonn and nearby and gave presentations. In addition we had an information booth in our city center informing passersby about the risks and benefits of synthetic biology with the aid of several posters and flyers. A big highlight was a <a onclick=node(106)>Science slam</a> we arranged. In front of a big audience six scientists from different fields presented an interesting aspect of their academic field. The Science Slam was a great success in terms of getting people in touch with science and without exception received very good critiques.<br> At every opportunity we handed out questionnaires in which we evaluated the public's opinion about synthetic biology and whether our human practice events were informative and interesting."; | ||
content.type="Human Practice"; | content.type="Human Practice"; | ||
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content.parents=[100]; | content.parents=[100]; | ||
content.childs=[102,103]; | content.childs=[102,103]; | ||
| - | content.titleShort = " | + | content.titleShort = "Presentations" |
content.titleLong = "Project presentations"; | content.titleLong = "Project presentations"; | ||
content.summary= "We presented iGEM and our project on two scientific conferences."; | content.summary= "We presented iGEM and our project on two scientific conferences."; | ||
| Line 793: | Line 703: | ||
content.parents=[101]; | content.parents=[101]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = " | + | content.titleShort = "Congress Berlin"; |
| - | content.titleLong = " | + | content.titleLong = "Biocom congress Berlin"; |
content.summary= "our day in Berlin. We met important people and other iGEM teams"; | 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 | + | content.text= "iGEM Bonn 2013 was invited to join the Biocom AG congress "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. <table><tr><th><div class='content-image'><img src='https://2013.igem.org/File:Bonn_Berlin_Niklas.JPG'>Niklas (left) in a discussion with a member of team Darmstadt</div></th><th><div class='content-image'><img src='https://2013.igem.org/File:Bonn_Poster_Berlin.JPG'>Our Poster for the congress</th><th><div class='content-image'><img src='https://2013.igem.org/File:Bonn_Berlin_Poster_Media.JPG'>Our Poster in place</div></th></tr><tr><th><div class='content-image'><img src='https://2013.igem.org/File:Bonn_Berlin_Poster_explanation_Max.JPG'></th><div class='content-image'><img src='https://2013.igem.org/File:Bonn_Berlin_all_the_iGEMs.JPG'></tr></table>"; |
content.type="Human Practice"; | content.type="Human Practice"; | ||
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content.titleLong = "The Science Slam - Science for Everybody"; | content.titleLong = "The Science Slam - Science for Everybody"; | ||
content.summary = "We organized a science slam to offer an experience of science for everyone. And the event met with great approval!"; | content.summary = "We organized a science slam to offer an experience of science for everyone. And the event met with great approval!"; | ||
| - | content.text = "<h1>Science Slam</h1><p>Our project aims to make versatile control of biological machines easily possible for everybody. During conception we wondered how our human practice work could represent that thought and decided to organize a science slam an event which makes science available to everybody!</p><p>We believe that science must not only stay in laboratories and lecture halls but reach lots of people out there. And without a doubt, the walls of the reputed ivory tower couldnt stand throughout the evening of our event!</p><p>Furthermore, the science slam provided a platform for our slammers to share the passion for their project with more than just their colleagues and thus help to popularize their field. In ten minutes, they presented their topic in a playful and inspirational manner and the audience decided which slammer has managed to get them carried away the most.</p><p>We managed not only to give floor to a large variety of fields by our slammers from physics, medicine, psychology, informatics, politics and engineering but to make them be heard by a public which was very mixed indeed.</p><p>As a prelude to the event, we acquainted the audience with synthetic biology and our project by showing what synthetic biology can do for each of us. Distributed questionnaires during the evening showed that -as hoped- our presentation helped to leave a positive mark on the field of synthetic biology in | + | content.text = "<h1>Science Slam</h1><p>Our project aims to make versatile control of biological machines easily possible for everybody. During conception we wondered how our human practice work could represent that thought and decided to organize a science slam an event which makes science available to everybody!</p><p>We believe that science must not only stay in laboratories and lecture halls but reach lots of people out there. And without a doubt, the walls of the reputed ivory tower couldnt stand throughout the evening of our event!</p><p>Furthermore, the science slam provided a platform for our slammers to share the passion for their project with more than just their colleagues and thus help to popularize their field. In ten minutes, they presented their topic in a playful and inspirational manner and the audience decided which slammer has managed to get them carried away the most.</p><p>We managed not only to give floor to a large variety of fields by our slammers from physics, medicine, psychology, informatics, politics and engineering but to make them be heard by a public which was very mixed indeed.</p><p>As a prelude to the event, we acquainted the audience with synthetic biology and our project by showing what synthetic biology can do for each of us. Distributed questionnaires during the evening showed that -as hoped- our presentation helped to leave a positive mark on the field of synthetic biology in people's heads:</p><p>For example, 97% of the people, who stated they did not know about synthetic biology before have got a proper picture of it during our event. 86% said, they are now familiar with the iGEM competition and everybody stated, they found the idea to combine the presentation of our iGEM project with a science slam very good or good. </br> <p> <img src=https://static.igem.org/mediawiki/2013/d/df/Bonn_Science_Slam_1.png> </br> <p> <img src= https://static.igem.org/mediawiki/2013/f/f1/Bonn_Science_Slam_2.png> </br> <p> <img src=https://static.igem.org/mediawiki/2013/5/55/Bonn_Science_Slam_3.png></br> </p><p>The fact that our audience liked this topic had been reflected in our voting: Our representative for synthetic biology, Mogan Ramesh won the science slam. He originally comes from engineering but discovered his passion for superbacteria e.coli during his studies which he gladly shared with us. In ten minutes, he taught us that e.coli does not only work as eeew on our meal, but showed how his group studies on making e.coli work for us, e.g. producing pharmaceuticals.</p><p>How? For example, recombinant DNA technology can be used to modify Escherichia coli to produce human insulin. Mogans works within a group of scientist that investigate the metabolic network in the Escherichia coli bacteria cell. Attempts to raise the copy number of plasmid DNA have been successful, and the goal now is to understand how the cell has accomplished this task. Proteomic studies have indicated an expected cellular adaptation that agrees with attempts to raise copy number in the literature; however, the thermodynamic implications of these results have been minimally explored. The goal of a cell remains controversial; one explanation is that the cellular metabolism seeks to maximize the entropy (minimize the Gibbs energy) of the system. Using GAMS as an optimization tool, it is explored if the reaction paths that have proven to be the most likely are also the paths that minimize the Gibbs energy of the system.</p><p>All in all, our science slam has been a great success which demanded a reenactment from a lot of sites.</p><p><b> Here are a few impressions from our event:</b></p><p> <img src=https://static.igem.org/mediawiki/2013/0/07/BonnScienceslam1.PNG> <br /><br /><i>Tobias Bald from experimental dermatology (University of Bonn) spoke about the Dark side of the immune system.</i></p><p><img src=https://static.igem.org/mediawiki/2013/d/dd/BonnScienceslam2.PNG><br /><br /><i>Prof. Dr. Herbert Dreiner gave us a review of Fukushima with full activity.</i></p><p><img src=https://static.igem.org/mediawiki/2013/0/0c/BonnScienceslam3.PNG><br /><br /><i>Our winner Mogan Ramesh shared his passion for superbacteria e.coli.</i></p><p><img src=https://static.igem.org/mediawiki/2013/2/24/BonnScienceslam4.PNG><br/><br/><i>Full house at our Science Slam.</i></p><p><img src=https://static.igem.org/mediawiki/2013/d/d5/BonnScienceslam5.PNG><br/><br/><i>Our audience obviously enjoyed the show.</i></p>"; |
content.type = "Human Practice"; | content.type = "Human Practice"; | ||
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case 107: | case 107: | ||
content.i = 107; | content.i = 107; | ||
| - | content.parents=[ | + | content.parents=[100]; |
| - | content.titleShort = " | + | content.childs=[]; |
| + | content.titleShort = "SynBio Day"; | ||
| + | content.titleLong = "SynBio Day"; | ||
content.summary= "In cooperation with the iGEM teams of Germany also the team of Bonn organized a day of action for synthetic biology. "; | content.summary= "In cooperation with the iGEM teams of Germany also the team of Bonn organized a day of action for synthetic biology. "; | ||
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, visualized our ideas in terms of several posters and on top created a survey to examine the people's opinion. </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, which reflects in the results of the survey, so that we bundled up and left satisfied. Here you can see the summary of our questionaire:<br/> </br> 1. Do you know what synthetic biology means? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/b/bd/BonnQuestion_1.png' height='260' width='350'> <br/> </br> </br> 2. Do you know what the iGEM competition is about? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/4/44/BonnQuestion_2.png' height='260' width='350'> <br/> </br> 3. How do you rate the ralation between chance and risk of sythetic biology? </br><div align='center'><img src='https://static.igem.org/mediawiki/2013/5/58/BonnQuestion3.1.png' height='260' width='350'> </br> 4. What are the main reasons for you that speak against the use of synthetic bioloy? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/6/62/BonnQuestion_4.png' height='260' width='350'> </br> </br> 5. Do you think it is important to inform the public better about the topic ' snythetic biology'?</br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/a/a6/Question_5.png' height='260' width='350'> </br> </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."; | 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, visualized our ideas in terms of several posters and on top created a survey to examine the people's opinion. </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, which reflects in the results of the survey, so that we bundled up and left satisfied. Here you can see the summary of our questionaire:<br/> </br> 1. Do you know what synthetic biology means? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/b/bd/BonnQuestion_1.png' height='260' width='350'> <br/> </br> </br> 2. Do you know what the iGEM competition is about? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/4/44/BonnQuestion_2.png' height='260' width='350'> <br/> </br> 3. How do you rate the ralation between chance and risk of sythetic biology? </br><div align='center'><img src='https://static.igem.org/mediawiki/2013/5/58/BonnQuestion3.1.png' height='260' width='350'> </br> 4. What are the main reasons for you that speak against the use of synthetic bioloy? </br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/6/62/BonnQuestion_4.png' height='260' width='350'> </br> </br> 5. Do you think it is important to inform the public better about the topic ' snythetic biology'?</br> <div align='center'><img src='https://static.igem.org/mediawiki/2013/a/a6/Question_5.png' height='260' width='350'> </br> </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."; | ||
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content.parents=[100]; | content.parents=[100]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = " | + | content.titleShort = "Schools"; |
content.titleLong = "School presentations"; | content.titleLong = "School presentations"; | ||
content.summary= "We gave lectures about synthetic biology and our project in schools in order to reach the younger people. "; | content.summary= "We gave lectures about synthetic biology and our project in schools in order to reach the younger people. "; | ||
| Line 845: | Line 757: | ||
case 109: | case 109: | ||
content.i = 109; | content.i = 109; | ||
| - | content.parents=[ | + | content.parents=[112]; |
| - | content.childs=[ | + | content.childs=[]; |
| - | content.titleShort = "About | + | content.titleShort = "Comic: About"; |
content.titleLong = "About the comic"; | content.titleLong = "About the comic"; | ||
| - | content.summary= "On the mission to find new and interesting means to bring across the concepts of synthetic biology, we introduced a comic series consisting of three episodes in the style of the well known Star Wars movies. The readers will find themselves in a world where Galaxies are petri dishes and all the characters are bacteria. Alongside the action-filled story we step by step introduce basic concepts of synthetic biology. The use of light sabers and laser guns also offered a great opportunity to embed our system of light-degradable proteins in the plot. At our presentations at schools and at our information booth it proved to be an ideal eye-catcher for passers-by and led to them wanting to know more about the subject. | + | content.summary= "We introduced a hand drawn comic series called "LOV Wars" about Obi-Wan E. Coli and his adventures in the Galaxy of Petri. Basic concepts of synthetic biology are explained along the way."; |
| - | content.type="Human Practice"; | + | content.text="On the mission to find new and interesting means to bring across the concepts of synthetic biology, we introduced a comic series consisting of three episodes in the style of the well known Star Wars movies. The readers will find themselves in a world where Galaxies are petri dishes and all the characters are bacteria. Alongside the action-filled story we step by step introduce basic concepts of synthetic biology. The use of light sabers and laser guns also offered a great opportunity to embed our system of light-degradable proteins in the plot. At our presentations at schools and at our information booth it proved to be an ideal eye-catcher for passers-by and led to them wanting to know more about the subject. 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; | break; | ||
| Line 857: | Line 769: | ||
content.parents=[100]; | content.parents=[100]; | ||
content.childs=[]; | content.childs=[]; | ||
| - | content.titleShort = " | + | content.titleShort = "LOV-Wars Minigame</br>About"; |
content.titleLong = "About the LOV-Wars Shooter"; | content.titleLong = "About the LOV-Wars Shooter"; | ||
content.summary= "The thoughts and ideas behind our game."; | content.summary= "The thoughts and ideas behind our game."; | ||
| Line 874: | Line 786: | ||
content.type = "Human Practice"; | content.type = "Human Practice"; | ||
break; | break; | ||
| + | |||
case 112: | case 112: | ||
content.i = 112; | content.i = 112; | ||
| - | content.parents = [100 | + | content.parents = [100]; |
| - | content.childs = []; | + | content.childs = [109]; |
content.titleShort = "Comic"; | content.titleShort = "Comic"; | ||
content.titleLong = "Comic – The adventures of Obi Wan E.Coli"; | content.titleLong = "Comic – The adventures of Obi Wan E.Coli"; | ||
content.summary = "Read about the adventures of Obi Wan E.Coli"; | content.summary = "Read about the adventures of Obi Wan E.Coli"; | ||
| - | content.text = "<img src=https://static.igem.org/mediawiki/2013/c/c1/Bonn_Comic_episode1_page1.jpg width= | + | content.text = "<img src=https://static.igem.org/mediawiki/2013/c/c1/Bonn_Comic_episode1_page1.jpg width=735px><img src=https://static.igem.org/mediawiki/2013/a/ad/Bonn_Comic_episode1_page2.jpg width=735px><img src=https://static.igem.org/mediawiki/2013/1/19/Bonn_Comic_episode1_page3.jpg width=735px><img src=https://static.igem.org/mediawiki/2013/8/80/Bonn_Comic_episode1_page4.jpg width=735px><img src=https://static.igem.org/mediawiki/2013/6/6c/Comic_EN_2.0_%281%29.jpg width=735px><img src=https://static.igem.org/mediawiki/2013/8/84/Comic_EN_2.0_%282%29.jpg width=735px><img src=https://static.igem.org/mediawiki/2013/9/97/Comic_EN_2.0_%283%29.jpg width=735px><img src=https://static.igem.org/mediawiki/2013/1/19/Comic_EN_2.0_%284%29.jpg width=735px><img src=https://static.igem.org/mediawiki/2013/8/8d/Bonn_Comic_EN_3.0_%281%29.jpg width=735px><img src=https://static.igem.org/mediawiki/2013/5/58/Bonn_Comic_EN_3.0_%282%29.jpg width=735px><img src=https://static.igem.org/mediawiki/2013/8/8c/Bonn_Comic_EN_3.0_%283%29.jpg width=735px><img src=https://static.igem.org/mediawiki/2013/b/b4/Bonn_Comic_EN_3.0_%284%29.jpg width=735px>"; |
content.type = "Human Practice"; | content.type = "Human Practice"; | ||
break; | break; | ||
| + | |||
| + | |||
| + | |||
| + | case 130: | ||
| + | content.i = 130; | ||
| + | content.parents=[100]; | ||
| + | content.childs=[131,133,136,137,138]; | ||
| + | content.titleShort = "About us"; | ||
| + | content.titleLong = "About us"; | ||
| + | content.summary= "The Team - Presentation of all team members"; | ||
| + | content.text= "<div><div class='subpage-text'><div align='center'><div width:870px; height:485px; id='aboutus-group' style='position:relative;left:-90px'><img src='https://static.igem.org/mediawiki/2013/e/e9/Teammod.jpg' width='870px' style='z-index:1;position:relative;'><div id='aboutus-group-kristina' onmouseover=showMemberDetails('Kristina') onmouseout=hideMemberDetails() style='position:absolute;top:160px;left:57px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-dustin' onmouseover=showMemberDetails('Dustin') onmouseout=hideMemberDetails() style='position:absolute;top:153px;left:130px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-marc' onmouseover=showMemberDetails('Marc') onmouseout=hideMemberDetails() style='position:absolute;top:275px;left:124px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-niklas' onmouseover=showMemberDetails('Niklas') onmouseout=hideMemberDetails() style='position:absolute;top:139px;left:174px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-ben' onmouseover=showMemberDetails('Ben') onmouseout=hideMemberDetails() style='position:absolute;top:158px;left:197px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-annika' onmouseover=showMemberDetails('Annika') onmouseout=hideMemberDetails() style='position:absolute;top:278px;left:220px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-matthias' onmouseover=showMemberDetails('Matthias') onmouseout=hideMemberDetails() style='position:absolute;top:153px;left:252px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-max' onmouseover=showMemberDetails('Max') onmouseout=hideMemberDetails() style='position:absolute;top:159px;left:282px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-maria' onmouseover=showMemberDetails('Maria') onmouseout=hideMemberDetails() style='position:absolute;top:273px;left:289px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-sadrija' onmouseover=showMemberDetails('Sadrija') onmouseout=hideMemberDetails() style='position:absolute;top:160px;left:358px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-franzi' onmouseover=showMemberDetails('Franzi') onmouseout=hideMemberDetails() style='position:absolute;top:277px;left:355px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-cori' onmouseover=showMemberDetails('Cori') onmouseout=hideMemberDetails() style='position:absolute;top:172px;left:420px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-pauline' onmouseover=showMemberDetails('Pauline') onmouseout=hideMemberDetails() style='position:absolute;top:282px;left:421px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-olli' onmouseover=showMemberDetails('Olli') onmouseout=hideMemberDetails() style='position:absolute;top:163px;left:472px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-nina' onmouseover=showMemberDetails('Nina') onmouseout=hideMemberDetails() style='position:absolute;top:277px;left:489px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-miriam' onmouseover=showMemberDetails('Miriam') onmouseout=hideMemberDetails() style='position:absolute;top:163px;left:506px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-thomas' onmouseover=showMemberDetails('Thomas') onmouseout=hideMemberDetails() style='position:absolute;top:140px;left:535px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-katharina' onmouseover=showMemberDetails('Katharina') onmouseout=hideMemberDetails() style='position:absolute;top:276px;left:557px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-philipp' onmouseover=showMemberDetails('Philipp') onmouseout=hideMemberDetails() style='position:absolute;top:147px;left:591px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-martina' onmouseover=showMemberDetails('MartinaB') onmouseout=hideMemberDetails() style='position:absolute;top:162px;left:622px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-sebastian' onmouseover=showMemberDetails('Sebastian') onmouseout=hideMemberDetails() style='position:absolute;top:153px;left:664px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-annikag' onmouseover=showMemberDetails('AnnikaG') onmouseout=hideMemberDetails() style='position:absolute;top:150px;left:703px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-jan' onmouseover=showMemberDetails('Jan') onmouseout=hideMemberDetails() style='position:absolute;top:136px;left:769px;width:30px;height:30px;z-index:5;'></div><div id='aboutus-group-details' style='background-color:black;opacity:0.8;position:absolute;height:80px;width=870px;top:0px;left:0px;right:-24px;z-index:2;'><div id='aboutus-group-details-name' style='color:white'></div><div id='aboutus-group-details-course' style='color:white'></div><div id='aboutus-group-details-semester' style='color:white'></div><div id='aboutus-group-details-tasks' style='color:white'></div></div><div id='aboutus-group-move' style='background-color:black;opacity:0.8;position:absolute;height:20px;width=870px;top:0px;left:0px;right:-24px;z-index:2;'><div id='aboutus-group-about' style='color:white'>Hover over faces to show group member details.</div></div></div></div></div><div id='team-members'><div class='team-members-row'><div class='team-member'><div class='team-member-name'>Thomas Berger</div><div class='team-member-field'>Bachelor Molecular Biomedicine</div><div class='team-member-sem'>2nd semester</div><div class='job'>labwork, jamboree presentation, school presentations</div></div><div class='team-member'><div class='team-member-name'>Sadrija Cukoski</div><div class='team-member-field'>Bachelor Molecular Biomedicine</div><div class='team-member-sem'>4th semester</div><div class='job'>science slam</div></div><div class='team-member'><div class='team-member-name'>Dustin Dankelmann</div><div class='team-member-field'>Bachelor Biology</div><div class='team-member-sem'>2nd semester</div><div class='job'>labwork, financing</div></div></div><div class='team-members-row'><div class='team-member'><div class='team-member-name'>Katharina Düker</div><div class='team-member-field'>Bachelor Molecular Biomedicine</div><div class='team-member-sem'>4th semester</div><div class='job'>startup advice, skills course, school presentation</div></div><div class='team-member'><div class='team-member-name'>Maria Gädeke</div><div class='team-member-field'>Bachelor Biology</div><div class='team-member-sem'>2nd semester</div><div class='job'>labwork</div></div><div class='team-member'><div class='team-member-name'>Jan Hansen</div><div class='team-member-field'>Bachelor Molecular Biomedicine</div><div class='team-member-sem'>2nd semester</div><div class='job'>labwork, webdesign, lov-wars shooter, design, comic, school presentations, pictures</div></div></div><div class='team-members-row'><div class='team-member'><div class='team-member-name'>Kristina Klein</div><div class='team-member-field'>Bachelor Molecular Biomedicine</div><div class='team-member-sem'>2nd semester</div><div class='job'>labwork</div></div><div class='team-member'><div class='team-member-name'>Matthias Klumpp</div><div class='team-member-field'>Bachelor Molecular Biomedicine</div><div class='team-member-sem'>4th semester</div><div class='job'>webmaster</div></div><div class='team-member'><div class='team-member-name'>Franziska Kohl</div><div class='team-member-field'>Bachelor Molecular Biomedicine</div><div class='team-member-sem'>4th semester</div><div class='job'>survey and evaluation, school presentations</div></div></div><div class='team-members-row'><div class='team-member'><div class='team-member-name'>Sebastian Martin</div><div class='team-member-field'>Bachelor Molecular Biomedicine</div><div class='team-member-sem'>4th semester</div><div class='job'>safety</div></div><div class='team-member'><div class='team-member-name'>Miriam Melake</div><div class='team-member-field'>Bachelor Molecular Biomedicine</div><div class='team-member-sem'>2nd semester</div><div class='job'>science slam</div></div><div class='team-member'><div class='team-member-name'>Nina Offermann</div><div class='team-member-field'>Bachelor Molecular Biomedicine</div><div class='team-member-sem'>2nd semester</div><div class='job'>labwork, comic, school presentations</div></div></div><div class='team-members-row'><div class='team-member'><div class='team-member-name'>Oliver Rippel</div><div class='team-member-field'>Bachelor Molecular Biomedicine</div><div class='team-member-sem'>2nd semester</div><div class='job'>labwork, school presentations</div></div><div class='team-member'><div class='team-member-name'>Philipp Sander</div><div class='team-member-field'>Bachelor Molecular Biomedicine</div><div class='team-member-sem'>4th semester</div><div class='job'>startup advice, financing, skills course, school presentation</div></div><div class='team-member'><div class='team-member-name'>Florian Schäfer</div><div class='team-member-field'>Bachelor Mathematics</div><div class='team-member-sem'>6th semester</div><div class='job'>modelling</div></div></div><div class='team-members-row'><div class='team-member'><div class='team-member-name'>Max Schelski</div><div class='team-member-field'>Bachelor Molecular Biomedicine</div><div class='team-member-sem'>4th semester</div><div class='job'>management, labwork, jamboree presentation, jamboree poster, webdesign, school presentations, skills course</div></div><div class='team-member'><div class='team-member-name'>Niklas Schmacke</div><div class='team-member-field'>Bachelor Molecular Biomedicine</div><div class='team-member-sem'>4th semester</div><div class='job'>labwork, jamboree presentation, jamboree poster webdesign, design</div></div><div class='team-member'><div class='team-member-name'>Corinna Schmalohr</div><div class='team-member-field'>Bachelor Molecular Biomedicine</div><div class='team-member-sem'>2nd semester</div><div class='job'>labwork, comic, school presentation, design</div></div></div><div class='team-members-row'><div class='team-member'><div class='team-member-name'>Annika Schneider</div><div class='team-member-field'>Bachelor Molecular Biomedicine</div><div class='team-member-sem'>2nd semester</div><div class='job'>labwork, jamboree presentation, meetings, financing</div></div><div class='team-member'><div class='team-member-name'>Marc Schulte</div><div class='team-member-field'>Master Molecular Biotechnology</div><div class='team-member-sem'>4th semester</div><div class='job'>labwork, design</div></div><div class='team-member'><div class='team-member-name'>Benjamin Syllwasschy</div><div class='team-member-field'>Bachelor Molecular Biomedicine</div><div class='team-member-sem'>2nd semester</div><div class='job'>labwork, school presentations</div></div></div><div class='team-members-row'><div class='team-member'><div class='team-member-name'>Pauline Ulmke</div><div class='team-member-field'>Bachelor Applied Biology</div><div class='team-member-sem'>2nd semester</div><div class='job'>labwork, school presentation</div></div><div class='team-member'><div class='team-member-name'>Leonie von Berlin</div><div class='team-member-field'>Bachelor Biology</div><div class='team-member-sem'>2nd semester</div><div class='job'>labwork</div></div></div></div></div></div>"; | ||
| + | content.type="Team"; | ||
| + | break; | ||
| + | |||
| + | case 131: | ||
| + | content.i = 131; | ||
| + | content.parents=[130]; | ||
| + | content.childs=[]; | ||
| + | content.titleShort = "Picture Gallery"; | ||
| + | content.titleLong = "Picture Gallery"; | ||
| + | content.summary= "Pictures from any Event hosted by our Team and many more"; | ||
| + | content.text= "<table border=0><tr><td colspan='2'><div align='left'><b><h2> Science Slam</h2></div></b></td> </tr> <tr> <td colspan='2'> <ahref='https://static.igem.org/mediawiki/2013/5/5b/BonnSlamPanorama.jpg'> <img src='https://static.igem.org/mediawiki/2013/5/5b/BonnSlamPanorama.jpg' width='840' height='200'></a></td> </tr> <tr> <td> <a href='https://static.igem.org/mediawiki/2013/d/d2/BonnSlam2.JPG'> <img src='https://static.igem.org/mediawiki/2013/d/d2/BonnSlam2.JPG' width='418' height='350'> </a> </td><td> <a href='https://static.igem.org/mediawiki/2013/6/6e/BonnSlam3.JPG'> <img src='https://static.igem.org/mediawiki/2013/6/6e/BonnSlam3.JPG' width='418' height='350'> </a> </td></tr> </table> <table border=0> <tr rowspan='2'> <td colspan='2'><a href='https://static.igem.org/mediawiki/2013/2/2d/BonnSlamGruppenfoto.JPG'> <img src='https://static.igem.org/mediawiki/2013/2/2d/BonnSlamGruppenfoto.JPG' width='518' height='650'></a> </td> <td> <a href='https://static.igem.org/mediawiki/2013/f/f8/BonnSlam5.JPG'> <img align='top' src='https://static.igem.org/mediawiki/2013/f/f8/BonnSlam5.JPG' width='318' height='310'> </a> <a href='https://static.igem.org/mediawiki/2013/5/5f/BonnSlam6.JPG'> <img style='padding-top:5px' align='top' src='https://static.igem.org/mediawiki/2013/5/5f/BonnSlam6.JPG' width='318' height='335'> </td> </tr> <td colspan='3'> <a href='https://static.igem.org/mediawiki/2013/e/e5/BonnSlam7.JPG'> <img src='https://static.igem.org/mediawiki/2013/e/e5/BonnSlam7.JPG' width='840' height='525'> </a> </td></table> <table border=0> <tr> <td colspan='2'> <divalign='left'> <b><h2> School presentations </h2></b> </div> </td> </tr><tr> <td colspan='2'> <a href='https://static.igem.org/mediawiki/2013/2/2c/BonnSchule.JPG'> <img src='https://static.igem.org/mediawiki/2013/2/2c/BonnSchule.JPG' width='840' height='390'> </a> </td></tr> <tr> <td> <a href='https://static.igem.org/mediawiki/2013/f/fc/BonnFranziSchule.JPG'> <img align='top' src='https://static.igem.org/mediawiki/2013/f/fc/BonnFranziSchule.JPG' width='448' height='250'> </a></td> <td rowspan='2'> <a href='https://static.igem.org/mediawiki/2013/3/30/BonnLinz2.JPG'> <img src='https://static.igem.org/mediawiki/2013/3/30/BonnLinz2.JPG' width='388' height='590'> </a> </td></tr> <tr> <td> <a href='https://static.igem.org/mediawiki/2013/a/ac/BonnLinz1.JPG'> <img align='top' src='https://static.igem.org/mediawiki/2013/a/ac/BonnLinz1.JPG' width='448' height='335'></a> </td></tr> </table> <table border=0> <tr> <td colspan='2'> <div align='left'> <b><h2>Information booth</h2> </div></b> </td> </tr> <tr><td colspan='2'> <a href='https://static.igem.org/mediawiki/2013/3/3c/BonnStand3.JPG'><img src='https://static.igem.org/mediawiki/2013/3/3c/BonnStand3.JPG' width='840' height='600'> </a> </td></tr> <tr> <td> <a href='https://static.igem.org/mediawiki/2013/4/4c/BonnStand2.JPG'> <img src='https://static.igem.org/mediawiki/2013/4/4c/BonnStand2.JPG' width='523' height='350'> </a> </td><td> <a href='https://static.igem.org/mediawiki/2013/a/a4/BonnStand1.JPG'> <img src='https://static.igem.org/mediawiki/2013/a/a4/BonnStand1.JPG' width='313' height='350'> </a> </td></tr> </table> <table border=0> <tr> <td colspan='2'> <divalign='left'> <b><h2>Biocom Congress</h2> </div></b> </td> </tr><tr> <td> <a href='https://static.igem.org/mediawiki/2013/c/c9/BonnBiocomCongress1.JPG'> <img src='https://static.igem.org/mediawiki/2013/c/c9/BonnBiocomCongress1.JPG' width='313' height='350'></a> </td> <td> <a href='https://static.igem.org/mediawiki/2013/f/f7/BonnBiocomCongress2.png'> <img src='https://static.igem.org/mediawiki/2013/f/f7/BonnBiocomCongress2.png' width='523' height='350'></a> </td> </tr> </table> <table border=0> <tr> <td><div align='left'> <b><h2>Bio NRW Video Contest</h2> </div></b> </td></tr> <tr> <td> <a href='https://static.igem.org/mediawiki/2013/1/1d/BonnVideowettbewerb.JPG'> <img src='https://static.igem.org/mediawiki/2013/1/1d/BonnVideowettbewerb.JPG' width='840' height='600'></a> </td> </tr> </table> <table border=0> <tr> <td colspan='3'> <div align='left'> <b><h2>Labwork</h2> </div></b> </td></tr> <tr> <td rowspan='2'> <a href='https://static.igem.org/mediawiki/2013/f/f8/BonnLabAnnikaNiklas.jpg'> <img src='https://static.igem.org/mediawiki/2013/f/f8/BonnLabAnnikaNiklas.jpg' width='322' height='450'></a> </td> <td> <a href='https://static.igem.org/mediawiki/2013/2/27/BonnLBKulturen.jpg'> <img src='https://static.igem.org/mediawiki/2013/2/27/BonnLBKulturen.jpg' width='255' height='225'> </a></td> <td> <a href='https://static.igem.org/mediawiki/2013/8/88/BonnZentrifuge.jpg'> <img src='https://static.igem.org/mediawiki/2013/8/88/BonnZentrifuge.jpg' width='255' height='225'> </a></td> </tr> <tr> <td> <a href='https://static.igem.org/mediawiki/2013/9/96/BonnCherryKulturen.jpg'> <img src='https://static.igem.org/mediawiki/2013/9/96/BonnCherryKulturen.jpg' width='255' height='220'></a> </td> <td> <a href='https://static.igem.org/mediawiki/2013/1/11/BonnPlatten.jpg'> <img src='https://static.igem.org/mediawiki/2013/1/11/BonnPlatten.jpg' width='255' height='220'> </a> </td></tr> </table> <table border=0> <tr> <td> <ahref='https://static.igem.org/mediawiki/2013/c/ca/BonnPellets.jpg'> <imgsrc='https://static.igem.org/mediawiki/2013/c/ca/BonnPellets.jpg' width='418' height='350'> </a></td> <td> <a href='https://static.igem.org/mediawiki/2013/a/ac/BonnLabNiklas.jpg'> <img src='https://static.igem.org/mediawiki/2013/a/ac/BonnLabNiklas.jpg' width='418' height='350'> </a></td> </tr> </table>"; | ||
| + | content.type="Team"; | ||
| + | break; | ||
case 133: | case 133: | ||
| Line 893: | Line 830: | ||
content.titleLong = "Sponsors"; | content.titleLong = "Sponsors"; | ||
content.summary= "Many sponsors made our work possible."; | content.summary= "Many sponsors made our work possible."; | ||
| - | + | content.text= "<div class=subpage-text> <table class=subpage-sponsors style=margin-left:-50px;background-color:transparent><tr><td colspan=2><h2>Our Partners</h2></td></tr><tr class=subpage-sponsor style=border-style:solid;border-width:5px;border-color:grey;> <td colspan=2 class=subpage-sponsor> <h2>BIO.NRW Cluster Biotechnology North Rhine-Westphalia</h2> <img src=https://static.igem.org/mediawiki/2013/9/90/Bonn_sponsor_BioNRW.png class=bottom-sponsor width=500px id=sponsor-limes style=margin-bottom:-50px></td><tr><td colspan=2></br>North Rhine-Westphalia is situated at Europe’s geographic and economic center. It is the largest of Germany’s 16 federal states, and the number one exporter. If classified as an independent exporting nation, NRW would rank 18th in the world, on a par with the Netherlands.</br>North Rhine-Westphalia’s state government has established a number of technology clusters to systematically improve NRW’s strengths and talents in established industries and up-and-coming fields like biotechnology. Goal of the “ExcellenceNRW” cluster strategy is to create a favourable climate for innovation, as that is the best way to sustain the competitive edge and stimulate growth and employment in the companies that call the state their home.North Rhine-Westphalia’s biotechnology cluster BIO.NRW is a central catalyst for the sustainable development of the state’s biotech sector. It activates cooperation between business, research, investors and policy-makers. The cluster also promotes the strengths and achievements of biotechnology in the state.</br>To support young as well as already established biotech companies, BIO.NRW offers the following core competencies:</br>– Individual matchmaking for collaborations and partners</br>– Overview on all up-to-date R&D activities in industry and academia</br>– Profound knowledge on financing possibilities</br>– International promotion and marketing for NRW as biotech location</br>– Direct contacts to decision makers</br><h3>Our services include</h3><bold>Technology Transfer</bold></br>Tech transfer support is a key contribution from BIO.NRW. We organize events, working platforms and meetings to promote the dialogue between all stakeholders in the field of biotechnology and to encourage cooperation.</br><bold>Biotech Business & Sciences</bold></br>BIO.NRW compiles comprehensive and current online databases of the academic institutions and companies active in the life sciences in NRW. Free to access and easy-to-use, these resources are valuable tools for identifying prospective business partners. More information on <a href=http://www.bio.nrw.de>www.bio.nrw.de</a></br><bold>Fairs, Exhibitions and Conferences</bold></br>Companies and academic institutions can generate awareness of their activities locally, nationally and internationally by being a part of the BIO.NRW common stands on fairs, exhibitions and conferences. BIO.NRW also organizes a series of workshops and symposia, called BIO.NRW.academy.</br><bold>Support of Young Professionals</bold></br>BIO.NRW takes a special interest in supporting young professionals in biotechnology. For example, we organize conventions where graduates meet representatives from industry and academic science. The ‘Business Angel Network – BIO.NRW’ helps financing and funding biotech start-ups. In addition, a forum that brings together investment institutions, private investors and business angels and developers provides information about the current NRW biotech scene. These meetings are a valuable opportunity for start-up companies to receive coaching and financing.</br></br>To learn more about the Cluster BIO.NRW and to stay informed about the latest biotech developments in NRW please visit <a href=http://www.bio.nrw.de>www.bio.nrw.de</a>.</td> </tr><tr><td colspan=2><h2>Our Sponsors</h2></td></tr><tr class=subpage-sponsor style=border-style:solid;border-width:5px;border-color:grey;> <td class=subpage-sponsor> <img src=https://static.igem.org/mediawiki/2013/3/36/Bonn_sponsor_promega.png class=bottom-sponsor width=250px id=sponsor-limes> </td> <td> <h2>Promega</h2> </br> Promega is one of the five biggest worldwide acting Life Science Research company. It was founded in Madison, WI (USA) and produces products and system solutions for gen-, protein- and cell-analysis. With the help of these products biological systems can be explored easily. Promega-products can be used in basic research, development of medicaments, molecular diagnostic and identification of human genetic constitution. </td> </tr> <tr class=subpage-sponsor> <td> <img src=https://static.igem.org/mediawiki/2013/3/36/Bonn_sponsors_eppendorf.jpg class=bottom-sponsor width=250px id=sponsor-eppendorf> </td> <td> <h2>Eppendorf</h2> </br> Eppendorf is a biotechnical company that develops, produces and sells systems for life science research for laboratories all over the world. The assortment of goods contains pipettes, dispensers, centrifuges, reaction tubes and pipette tips. Moreover Eppendorf offers instruments and systems to manipulate cells, automated machines for Liquid Handling and for DNA- Amplification, as well as Biochips. </td> </tr> <tr class=subpage-sponsor> <td> <img src=https://static.igem.org/mediawiki/2013/5/5a/Bonn_sponsor_IKA.png class=bottom-sponsor width=250px id=sponsor-ika> </td> <td><h2> IKA </h2> </br> In 1910 the companys history of IKA began, China had not been invented yet Peoples Republic and the word globalization. Today, the IKA group about 800 employees at eight locations on four continents and is pleased with clients such as BASF, Bayer and Procter & Gamble. In most product groups, we are sovereign world market leader and a symbol of development and growth. Or as our new slogan: "IKA - Designed to work perfectly." </td> </tr> <tr class=subpage-sponsor> <td> <img src=https://static.igem.org/mediawiki/2013/b/bf/Bonn_sponsors_Ella.jpg class=bottom-sponsor width=250px'id=sponsor-ella> </td> <td> <h2>Ella Biotech</h2> </br> ELLA is an independent, privately owned company founded in October 2004. ELLA offers creative services for the production of oligonucleotides driven by the goal of continually improving our production strategies. ELLA offers tangible advantages to its customers and partners through its validated technology platform, its experienced interdisciplinary team, and its resolute attitude towards the highest quality in our products. </td> </tr> <tr class=subpage-sponsor> <td> <img src=https://static.igem.org/mediawiki/2013/c/c0/Bonn_sponsor_genscript.jpg class=bottom-sponsor width=370px id=sponsor-genscript> </td> <td> <h2> Gen Script </h2> </br> GenScript is a leading biology CRO focusing exclusively on early drug discovery and development services. Built on our assembly-line mode, one-stop solution, continuous improvement, and stringent IP protection, GenScript provides a comprehensive portfolio of services that include Bio-Reagent, Bio-Assay, Lead Optimization, and Antibody Drug Development which can be effectively integrated into your value chain and your operations. We strive with competence and confidence to meet your demand for developing pre-clinical drug candidates time-efficiently and cost-effectively. With track performance record, GenScript is your ideal and reliable innovation partner in drug discovery. </td> </tr> <tr class=subpage-sponsor> <td> <img src=https://static.igem.org/mediawiki/2013/1/13/Bonn_sponsor_idt.jpg class=bottom-sponsor width=250px id=sponsor-idt> </td> <td><h2> IDT </h2> </br> Genetically engineered vaccines and pharmaceuticals for the global marketplace. The IDT Biologicals is an innovative medium-sized company that is involved in more than 90 years with its products and services to the health maintenance of humans and animals. Genetically engineered vaccines and pharmaceuticals for the domestic and international market are manufactured. In fiscal year 2012, the IDT biologics had a turnover of around 151 million euros. In the IDT biologics around 1,100 people are currently employed. IDT Biologics is an independent company of the Klocke Group, which developed and implemented at five production sites innovative packaging solutions for the pharmaceutical, cosmetic, food and chemical-technical industry.</br> Decades of research and development of vaccines</br> Since the beginning of the IDT successfully fought the various pathogens in animals with complex vaccine development from the laboratory to production and sales in one location. From research and development to manufacturing and testing and approval, national and international marketing of the range of tasks. The IDT Animal Health operates its own modern research complex for the development of animal vaccines.</br> Integrated biopharmaceutical services. </br> Since its founding in 1921, the IDT has biologics developed into a center for the pharmaceutical and biotechnology with the divisions Animal Health, human vaccines and pharmaceuticals. More than 250 million euros have been invested since privatization in 1993 in the continuous expansion of an integrated biopharmaceutical site and thus not only created excellent conditions of production, but also highly modern workplaces. </td> </tr> <tr class=subpage-sponsor> <td> <img src=https://static.igem.org/mediawiki/2013/3/3a/Bonn_sponsor_neb.jpg class=bottom-sponsor width=250px id=sponsor-neb> </td> <td> <h2> New England Biolabs </h2> </br> Never before in the history of science, the demands on the molecular biology industry have been as high as today: The researcher asks for the absolute best and most reliable products - there is no room for compromise. New England Biolabs fulfills this requirement. For 35 years we have been a leader in the development and production of enzymes for molecular biology and other reagents in the "life sciences" such as for proteomics and drug discovery. Our expertise in enzyme technology based on our strategic cloning and expression of DNA-Restriktions-/Modifikationssystemen program. So we have for years set the standards in terms of quality and price. </td> </tr> <tr class=subpage-sponsor> <td> <img src=https://static.igem.org/mediawiki/2013/3/34/Bonn_sponsors_MN.png class=bottom-sponsor width=250px id=sponsor-mn> </td> <td> <h2> Macherey und Nagel </h2> </br> MACHEREY-NAGEL is a family-run concern in the fourth generation. The comprehensive portfolio includes the areas of filtration, rapid tests, water analysis, chromatography and bioanalysis. MACHEREY-NAGEL employs more than 470 highly skilled employees in sales, production as well as research and development, including 10% post-doctoral researchers. They all guarantee an exceptional service. </td> </tr> <tr class=subpage-sponsor> <td> <img src=https://static.igem.org/mediawiki/2013/3/37/Bonn_Sponsor_roche.jpg class=bottom-sponsor width=250px id=sponsor-roche> </td> <td> <h2> Roche </h2> </br> Roche Headquartered in Basel, Switzerland, is a leading research-focused healthcare company with the pharmaceuticals and diagnostics businesses. As the worlds largest biotech company developing clinically differentiated medicines in oncology, virology, inflammation, metabolism and central nervous system. Roche, a pioneer in diabetes management, is also the world leader in in-vitro diagnostics, tissue-based cancer diagnostics. Medicines and diagnostic tools that enable tangible improvements in the health, quality of life and survival of patients is the strategic goal of personalized medicine from Roche. This concept is based on new molecular insights and molecular diagnostic tests that allow a more precise tuning of therapy and better control of the disease. Therapies are tailored to patient groups that have similarities in their disease. The only way to improve the efficacy of drugs targeted and maintain quality of life. </td> </tr> <tr class=subpage-sponsor> <td> <img src=https://static.igem.org/mediawiki/2013/d/d2/Bonn_sponsor_geneious.gif class=bottom-sponsor width=250px id=sponsor-geneious> </td> <td> <h2> Geneious </h2> </br> In Good Company</br> First released in 2005, Geneious is one the worlds leading bioinformatics software platforms, used by over 2500 universities and institutes and commercial companies in more than 65 countries. Geneious is used by all 20 of the top 20 Universities globally (Times Higher Education, 2012) and by seven of the 10 largest pharmaceutical companies.</br> Dedicated to excellence</br> Our software has won a number of prestigious awards, including the Computerworld Excellence Awards from Innovative Use of ICT and the United Nations World Summer Awards and Winner in the e-Science Category in 2007, the Recruit IT Innovative Software Product Award at the PriceWaterhouseCoopers Hi-Tech Awards in 2009 and a Global Finalist in the IT and Informatics category at the Bio-IT World Awards in Boston in 2009. </td> </tr> <tr class=subpage-sponsor> <td> <img src=https://static.igem.org/mediawiki/2013/5/55/Bonn_Sponsor_vwr.jpg class=bottom-sponsor width=250px id=sponsor-vwr> </td> <td> <h2> VWR </h2> </br> VWR is science for the advancement of the worlds most important research through the distribution of a wide range of products and services to a variety of important companies in the pharmaceutical, biotechnology, and healthcare industries as well as government agencies, universities and schools. We offer our customers all the resources they need to be successful, ie an extensive range of the best products in the areas of chemicals, furniture, appliances, instruments, apparel and consumables, from a wide variety of leaders in the field of science manufacturers. With 160 years of experience in this industry, VWR further supports its customers through a combination of strength, vision, innovation and a well-established distribution network that reaches thousands of specialized labs and facilities across the planet. VWR is not just a product supplier - it keeps the most important research in the world in motion. VWRs expertise in the areas of supply chain and logistics services enables customers to fully concentrate on their areas of expertise. Of the management of procurement processes to the integration of supply chains: VWR helps specialized research facilities and laboratories to work with maximum efficiency. VWR has over 8,000 employees in 30 countries with direct offices throughout the world working to streamline the way, as researchers from North America, Europe and the Asia-Pacific region supply and maintain their labs. In addition, VWR further supports its customers by providing onsite services, storeroom management, product procurement, supply chain systems integration and technical services. We are expanding our global presence and adhere to the principle that customers benefit from the availability and expertise of our local sales teams.</br> In todays economy VWR helps its customers to focus on increasing productivity and reducing costs and optimizing procurement processes. </br> Headquartered in Radnor, PA (USA), earned VWR International, LLC, in 2012 global sales of more than 4.1 billion U.S. dollars. </td> </tr> <tr class=subpage-sponsor> <td> <img src=https://static.igem.org/mediawiki/2013/0/0b/Bonn_Sponsors_Roth.png class=bottom-sponsor width=170px id=sponsor-roth> </td> <td> <h2> ROTH – A COMPANY WITH TRADITION </h2> </br> 1879 </br> Carl ROTH founded in Karlsruhe, a "material, Colonial and dye business and Droguerie".</br>1899</br> The first sales and mail order catalog is published.</br> 1956</br> Publication of the first issue of the publication a "Rarea " Natural Products</br> 1990</br> First ROTH general catalog. Complete with the areas of laboratory, life sciences and chemicals in one.</br> 2005</br> Completion of a modern establishment for the production and storage of laboratory chemicals and reagents in Karlsruhe Rhine port area. </td> </tr> <tr class=subpage-sponsor> <td> <img src=https://static.igem.org/mediawiki/2013/7/79/Bonn_starlab.jpg class=bottom-sponsor width=250px id=sponsor-starlab> </td> <td> <h2> Starlab </h2> </br> STARLAB is a company specializing in liquid handling technology group. With subsidiaries in Germany, France, Britain and Italy is available in the direct sales an extensive range of products available. Plus, you get our products to many countries around the world via our international trading partners. Our success is based on many years of experience in manufacturing and marketing of liquid handling disposable products - with TipOne we have established ourselves as a leading supplier of pipette tips systems worldwide. This quality, price and service come first. </td> </tr> <tr class=subpage-sponsor> <td> <img src=https://static.igem.org/mediawiki/2013/b/bf/Bonn_caesar.gif class=bottom-sponsor width=200px id=sponsor-casar> </td> <td> <h2> Caesar </h2> </br> The center of advanced european studies and research (caesar) is an institute of the Max Planck Society, which is located at the boundaries between neuroscience, cell biology and biophysics. The focus of the research is the particular cellular and neural signal processing. </br> Caesar works with modern photonic, molecular biological, chemical and micro-technological methods. The focus of kinetic, spectroscopic and microscopic methods are research and control of cellular activity. </td> </tr> <tr class=subpage-sponsor> <td> <img src=https://static.igem.org/mediawiki/2013/6/6e/Bonn_Eurofins.png class=bottom-sponsor width=250px id=sponsor-eurofins> </td> <td> <h2> Eurofins MWG operon </h2> </br> Discover our fascination about the world of the four bases.</br> We are fascinated about the power of DNA and how it is incorporated in everything we do, work and live. Being passionate about our strong customer orientation, our service and our quality standards, we continuously challenge ourselves to stay ahead and remain one of the leading genomics service providers worldwide. Eurofins MWG Operon is globally known for its innovative and customised technologies in the life science industries and academic research institutions. With the combined power of an international network of Eurofins companies in the field of genomic services, forensics, agroscience, pharmaceutical, environmental, food and feed testing, we have established an outstanding team of experts and broad range of technologies. This unique constellation underlines our approach to offer best practise solutions and versatile concepts for our clients - academic institutions and large interdisciplinary operating companies of the world. </td> </tr> <tr class=subpage-sponsor> <td> <img src=https://static.igem.org/mediawiki/2013/1/18/Bonn_ThermoFisher.jpg class=bottom-sponsor width=250px id=sponsor-thermofisher> </td> <td> <h2> Thermo Fisher </h2> </br> Thermo Fisher Scientific Inc. (NYSE: TMO) is the world leader in serving science. Our mission is to enable our customers to make the world healthier, cleaner and safer. With revenues of $13 billion, we have approximately 39,000 employees and serve customers within pharmaceutical and biotech companies, hospitals and clinical diagnostic labs, universities, research institutions and government agencies, as well as in environmental and process control industries. We create value for our key stakeholders through three premier brands, Thermo Scientific, Fisher Scientific and Unity<sup> TM </sup> Lab Services, which offer a unique combination of innovative technologies, convenient purchasing options and a single solution for laboratory operations management. Our products and services help our customers solve complex analytical challenges, improve patient diagnostics and increase laboratory productivity. </td></tr></table> </div> </div> </div> </div> </div> "; | |
content.type="Team"; | content.type="Team"; | ||
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| + | case 136: | ||
| + | content.i = 136; | ||
| + | content.parents = [130]; | ||
| + | content.childs = []; | ||
| + | content.titleShort = "History"; | ||
| + | content.titleLong = "The History of the IGEM Team Bonn"; | ||
| + | content.summary = "The Team Bonn took part at IGEM 2012 for the first time. For 2013 many new members from different departments were attracted. After a crash course even freshmen were able to work in the laboratory." | ||
| + | content.text = "<h1>History</h1><p>Coming off a successful first competition entry and an amazing first jamboree in Amsterdam for our team, our team dove head-first into a off-season video production jaunt, winning first prize in a video competition organized by our state’s ministry of science, research and innovation. In our video, we addressed the contest’s theme of 'Biotechnology Today' by creating an animated short titled 'Synthetic Biology - A Simply Explained'. In addition to an invitation to the prize ceremony held at a cinema museum in nearby Düsseldorf, our winnings included 1500 Euros, which we put to good use as seed money for 2013’s iGEM competition!</p><p>In continuing our practice of recruiting first-year students for our team, we held a well-attended introductory presentation in november and managed to attract over 40 interested students and faculty from our University. After our initial warm-up iGEM team meetings, in which we introduced our new team members to the competition, presenting some successful projects and innovative human practice advances in the last year as well as brain-storming both wetlab and outreach ideas, we signed up 23 full members and developed both an ambitious wetlab project as well as a full schedule of human practice activities.</p><p>During our mid-winter break in February and March, our experienced and veteran team members took to Powerpoint and the teaching lab as we provided our new members with a two-week seminar- and practical-based crash course on molecular biology and its corresponding lab techniques, offering the very first biology lab experience for quite a few of our Freshmen. It is our philosophy that we try to provide early hands-on research experience as well as an eye-opener into the world of synthetic biology for interested students from any university department.</p><p>In March, our whole team, advisors and instructors in tow, crashed a departmental seminar talk at the nearby CAESAR institute held by Prof. Dr. Andreas Möglich, who has done extensive research on light-sensitive proteins. As a team that has been working with those very same light-sensitive LOV proteins for the last two years, it was a great opportunity to discuss the many pros and cons surrounding LOV proteins with an expert in the field! Based on Prof. Dr. Möglich’s recommendations, we tweaked our project outline, incorporating a novel light-sensitive protein switch in our proposed protein degradation system.</p><p>Following our introductory lab sessions focussing on mastering that arcane technique of pipetting Bradford Assays (to measure and improve pipetting accuracy, not to humilate the pipetter with low R2 values!), we started preliminary project-related work, gathering and cloning required genes from labs all over the world.</p><p>And thus it began!</p>"; | ||
| + | content.type = "Team"; | ||
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content.childs=[]; | content.childs=[]; | ||
content.titleShort = "Bonn"; | content.titleShort = "Bonn"; | ||
| - | content.titleLong = "Bonn"; | + | content.titleLong = "Our city Bonn"; |
| - | content.summary= " | + | content.summary= "Bonn is a beautiful city in North Rhine-Westphalia, Germany."; |
| - | content.text= " | + | content.text= "Bonn has a long and eventful history. The city was founded by the Romans in the year 12 before Christ and continued to grow ever since. Bonn also was the birthplace of the famous musician and composer Beethoven and even was the Capitol of West Germany for 41 years. Today Bonn has around 300 000 habitants and has become the cultural of the region. It is set on the river Rhine very close to Cologne. Everybody gets smitten with its charm and unique mixture of tradition and modern lifestyle. The city's historic center and the setting close to the river Rhine turn it into a wonderful place to live and study.<p style='text-align:center;'>the city's slogan:</br><i>"Stadt. City. Ville. Bonn"</i></p></br><p><a href=https://static.igem.org/mediawiki/2013/0/0b/Bonn_by_night.JPG><div class='content-image'align='center'><img src='https://static.igem.org/mediawiki/2013/0/0b/Bonn_by_night.JPG' width='600'></a>Bonn by night <sup>©Jan Hansen</sup></div></p><p><div class='content-image'align='center'><a href=https://static.igem.org/mediawiki/2013/a/a6/Bonn_poppelsdorfer_schloss.JPG><img src='https://static.igem.org/mediawiki/2013/a/a6/Bonn_poppelsdorfer_schloss.JPG' width='600'></a>The university <sup>©Jan Hansen</sup></div><p><a href=https://static.igem.org/mediawiki/2013/0/0b/Bonn_by_night.JPG><div class='content-image'align='center'><img src='https://static.igem.org/mediawiki/2013/7/75/Bonn_beethoven.JPG' width='600'></a>The famous Beethoven statue in the city center <sup><a href=http://www.bone-diagenesis.org/moziloCMS/gallery.php?gal=Bonn-nbsp~Impressions>[source]</a></sup></div></p><h2>References</h2><p><a name='1'>1.</a> <a href='http://www.bonn.de'>http://www.bonn.de</a></p><p><a name='2'>2.</a> <a href='http://de.wikipedia.org/wiki/Bonn'>http://de.wikipedia.org/wiki/Bonn</a></p>"; |
content.type="Team"; | content.type="Team"; | ||
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content.titleShort = "Methods"; | content.titleShort = "Methods"; | ||
| - | content.titleLong = " | + | content.titleLong = "All Protokolls and Methods used by iGEM team Bonn 2013"; |
content.summary= "The LOV-ipaA -vinculin system is a combined system for light inducible heterodimerisation. This powerful tool, which allows photocontroled complex formation was establish by Lungu et al. in 2012."; | content.summary= "The LOV-ipaA -vinculin system is a combined system for light inducible heterodimerisation. This powerful tool, which allows photocontroled complex formation was establish by Lungu et al. in 2012."; | ||
content.text= "<b><a href='#1'>1. 3A – Assembly</a></b></br></br><b><a href='#2'>2. Agarose preparation</a></b></br></br><b><a href='#3'>3.Agarose gel casting</a></b></br></br><b><a href='#4'>4.loading the agarose gel and starting electrophoresis</a></b></br></br><b><a href='#5'>5. Preparation of Antibiotic stocks</a></b></br></br><b><a href='#6'>6. Colony PCR</a></b></br></br><b><a href='#7'>7. Preparation of Glycerol Stocks (iGEM) </a></b></br></br><b><a href='#8'>8.Plasmid Preparation </a></b></br></br><b><a href='#8a'>8a. Midi-Prep (Promega)</a></b></br></br><b><a href='#8b'>8b. Mini-Prep (Promega)</a></b></br></br><b><a href='#9'>9.Preparation of LB agar plates</a></b></br></br><b><a href='#10'>10. PCR - Clean-Up (Macherey und Nagel)</a></b></br></br><b><a href='#11'>11. Preparation of chemocompetent DH5-alpha cells</a></b></br></br><b><a href='#12'>12. Re-transformation of BioBricks</a></b></br></br><b><a href='#13'>13. Strand directed Mutagenesis PCR</a></b></br></br><b><a href='#14'>14. Transformation using Ligation product (in DH5alpha or XL1Blue) </a></b></br></br></br></br><h2><a name='1'>1. 3A – Assembly</a></h2><hr><i>NOTE: Enzymes and buffers were provided by Promega </i></br></br><b>Restriction (50 µl Reaction)</b></br>- 25 µl Mastermix Restriction-Enzyme Buffer (2x, with BSA)</br>- add 1 µl from every restriction enzyme to 500ng backbone equimolar DNA</br>- fill up to 50 µl dest. water</br>- incubate for 1.5h-3h at 37ºC </br>- inactivate for 20min at 70ºC (no clean-up, if directly used for ligation) </br></br><b>Ligation (20 µl Reaction)</b></br>- 2,0 µl equimolare restriction samples (inserts)</br>- 20ng/1.5 µl backbone</br>- fill up to 17.7 µl with dest. water</br>- incubate 5min at 37ºC</br>- add 2 µl Ligation buffer (10x) and 0.3 µl T4 DNA Ligase </br>- incubate for 3h RT or 15ºC over night </br>- inactivate for 10min at 70ºC</br></br></br><h2><a name='2'>2.Agarose preparation</a></h2><hr><b>Materials:</b></br>- Agarose </br>- 500ml bottle</br>- TBE (1x)</br></br><b>Procedure:</b></br>- dissolve 1g agarose in 100ml TBE (1x), resulting in 1% agarose</br>- heat for 2 minutes in a microwave at maximal power</br>- mix</br>- cook until it boils (1 min)</br>- mix carefully</br><i>2 clues for a successful boiling: 1. no cords, 2. boiling retardation</i></br>- store in 65ºC incubator</br></br></br><h2><a name='3'>3.Agarose gel casting</a></h2><hr>- assemble the gel chamber (chamber + 2 fences + 1 - 2 gel combs ) under the ethidiumbromid hood</br>- prepare 50ml falcon tube (label it!)</br>- fill 40ml warm agarose in the falcon tube</br>- add 4 µl ethidiumbromid (1:10.000) under the ethidiumbromid hood</br>- mix by inverting 2-3 times</br>- fill agarose in the gel chamber</br>- wait until the gel becomes solid (about 20minutes)</br></br></br><h2><a name='4'>4.loading the agarose gel and starting electrophoresis</a></h2><hr>- add LoadingDye (1:6) to your sample</br>- remove comb and fence from the gel</br>- place agarose gel in the electrophoresis chamber</br>- pipette samples carefully in the pockets (small pockets: up to 20 µl, big pockets: up to 50 µl)</br>- Place lid on the electrophoresis chamber and connect the electrodes to it.</br>- set parameters (high resolution: 120V, 20-30minutes, low resolution: 130V, 15min)</br>- evaluate gel under UV-light</br></br></br><h2><a name='5'>5. Preparation of Antibiotic stocks</a></h2><hr><b>Ampicillin:</b></br>- dissolve 100 mg ampicillin in 1 ml dest. water</br>- store at 20 ºC</br></br><b>Chloramphenicol:</b></br>- dissolve 18 mg chloramphenicol in 1 ml ethanol</br>- store at 20 ºC</br></br></br><h2><a name='6'>6. Colony PCR</a></h2><hr>- inoculate 10 µl dest. water with colony.</br>- use 1 µl of this water for one reaction:</br><img src='https://static.igem.org/mediawiki/2013/9/91/Bonn_MS_Methods1.png' width='550px'></br></br></br><h2><a name='7'>7.Preparation of Glycerol Stocks (iGEM)</a></h2><hr>- autoclave glycerol (60%)</br>- add 0,5 ml Glycerol to 1,5 ml cell culture in a cryo tube</br>- mix</br>- shock freeze in liquid nitrogen</br>- store at -80 ºC</br></br></br><h2><a name='8'>8. Plasmid Preparation</a></h2><hr><h2><a name='8a'>8a. Midi-Prep (Promega)</a></h2><hr>- centrifuge 50 ml of liquid cell culture for 10min at 5000g </br>- decant supernatant</br>- resuspend with 3 ml resuspension solution </br>- add 3 ml cell lysis solution and incubate for maximal 3 min at room temperature </br>- add 5 ml neutralization solution </br>- centrifuge for 20 min at 20 ºC, 5000g </br>- vacuum pump lysat through cleaning column into binding column </br>- abolish cleaning column</br>- vacuum pump with 10 ml endotoxin removal wash solution</br>- vacuum pump with 20 ml column wash solution</br>- dry membrane by vacuum</br>- add 600 µl nuclease free water on membrane</br>- centrifuge for 5 min at 1750 g into a fresh tube</br></br></br><h2><a name='8b'>8b. Mini-Prep (Promega)</a></h2><hr>- fill 1,5 ml overnight-culture in a new tube</br>- centrifuge for 30 seconds at maximal speed </br>- decant supernatant</br>- Repeat previous steps 2-5 times (depending on growth density)</br>- resuspend with 600 µl dest water </br>- add 100 µl cell lysis buffer </br>- after 1min (maximum 2min) add 350 µl of neutralization buffer </br>- centrifuge 3min at maximal speed</br>- place mini column in a collection tube and transfer supernatant into PureYield^TM Mini column</br>- centrifuge for 15 sec at maximal speed</br>- add 200 µl Endotoxin Removal Wash</br>- centrifuge for 15 sec at maximal speed</br>- add 400 µl column Wash solution</br>- centrifuge for 30 sec at maximal speed</br>- place mini column in a new tube</br>- add 30 µl elution buffer to the mini column, incubation at RT for 1 min</br>- centrifuge for 15 seconds at maximal speed </br>- store DNA at -20 ºC</br></br></br><h2><a name='9'>9. Preparation of LB agar plates</a></h2><hr>1l LB agar will result in approximately 30 plates</br></br>- dissolve 15g agar and 20g LB in 1l dest. Water</br>- autoclave</br>- cool down to 60-70ºC </br>- add antibiotics (1:1000) under the laminar airflow cabinet</br>- mix</br>- cast plates (approximately 20ml / plate)</br>- dry for 2h by room temperature</br>- store at 4ºC</br></br></br><h2><a name='10'>10. PCR - Clean-Up (Macherey und Nagel) </a></h2><hr><b>Gel Extraction: </b></br>1. add double amount NTI to gel</br>2. Incubate 3-7minutes at 50ºC and at 1000rpm (until gel is dissolved)</br>- continue with regular Clean-up Protocol (from step 3.) </br></br><b>Cleanup: </b></br>1. fill up sample with dest. water to 50 µl, if necessary</br>2. add double amount NTI to the sample</br>3. place column in a collection and add transfer solution to the column</br>4. centrifuge 30 seconds at 11.000g and discard flow through </br>5. add 700 µl NT3 </br>6. centrifuge 30 seconds at 11.000g and discard flow through </br>7. repeat step 5) and 6)</br>8. centrifuge 1minute at 11.000g</br>9. place column in a new tube and dry column at 70ºC for 5min</br></br>small parts (<1000bp): </br>9a. place column in a new tube and add 30 µl Elution buffer </br>9b. incubate 1min at room temperature</br>9c. Centrifuge 1minute at 11.000g </br></br>Bigger Parts: (>1000bp) </br>9A. place column in a new tube and add 20 µl Elution buffer </br>9B. incubate at 70ºC for 5minutes </br>9C. centrifuge at 50g for 1minute</br>9D. centrifuge at 11.000g for 1minute</br>9E. repeat step 9A. to 9D.</br></br></br><h2><a name='11'>11. Preparation of chemocompetent DH5-alpha cells</a></h2><hr>- Start with 200 ml Overnight culture with OD<sub>600</sub> of 0,6-0,8</br>- centrifuge at 4 ºC, 4500 g, 10 minutes </br>- decant supernatant</br>- resuspend with 40 ml inoune transformation buffer </br>- centrifuge at 4 ºC, 4500 g, 10 minutes </br>- decant supernatant</br>- resuspend in 20 ml inoune transformation buffer </br>- add 1,5 ml DMSO</br>- incubate 10 minutes on ice</br>- transfer 100 – 200 µl into precooled tubes</br>- shock freeze in liquid nitrogen</br></br></br><h2><a name='12'> 12. Re-transformation of Bio Bricks</a></h2><hr>- add 10 µl sterile dest water to DNA on plate </br>- incubate for 10 min at RT</br>- take 2 µl, leave rest on plate </br>- store plates at -20 ºC </br>- add the 2 µl DNA solution to 5 µl competent DH5-alpha</br>- incubate for 30 min on ice </br>- heat shock for 45 s at 42 ºC </br>- incubate 3 min on ice </br>- add 250 µl LB medium at 37 ºC </br>- incubate for 45 min at 37 ºC, 800 rpm </br>- plate 300 µl on Agar-plate with appropriate antibiotic </br>- dry 15min at RT</br>- incubate at 37ºC over night</br></br></br><h2><a name='13'> 13. Strand directed Mutagenesis PCR</a></h2><hr>Prepare master mix and add template as follows: </br><img src='https://static.igem.org/mediawiki/2013/b/b6/Bonn_MS_Methods2.png' width='550px'></br>- start PCR-Program: </br>1. initial denaturation 94ºC for 120 seconds </br>2. Denaturating 94ºC for 30 seconds</br>3. Annealing 94ºC for 30 seconds</br>4. Elongation 68 for 720 seconds</br>5. Repeat step 2) to 4) 12x </br></br></br><h2><a name='14'>14. Transformation using Ligation product (in DH5alpha or XL1Blue) </a></h2><hr>- thaw bacteria on ice </br>- add 2-4 µl Ligation mixture to 50 µl bacteria</br>- incubate 30minutes on ice </br>- heat shock 30-45 seconds (XL1Blue preferably 35 seconds) at 42ºC</br>- incubate 6min on ice</br>- add 250 µl LB medium at 37ºC</br>- incubate for 45minutes at 37ºC, 800rpm </br>- plate 300 µl on appropriate antibiotic</br>- dry 15 minutes at RT</br>- incubate at 37ºC over night</br>"; | content.text= "<b><a href='#1'>1. 3A – Assembly</a></b></br></br><b><a href='#2'>2. Agarose preparation</a></b></br></br><b><a href='#3'>3.Agarose gel casting</a></b></br></br><b><a href='#4'>4.loading the agarose gel and starting electrophoresis</a></b></br></br><b><a href='#5'>5. Preparation of Antibiotic stocks</a></b></br></br><b><a href='#6'>6. Colony PCR</a></b></br></br><b><a href='#7'>7. Preparation of Glycerol Stocks (iGEM) </a></b></br></br><b><a href='#8'>8.Plasmid Preparation </a></b></br></br><b><a href='#8a'>8a. Midi-Prep (Promega)</a></b></br></br><b><a href='#8b'>8b. Mini-Prep (Promega)</a></b></br></br><b><a href='#9'>9.Preparation of LB agar plates</a></b></br></br><b><a href='#10'>10. PCR - Clean-Up (Macherey und Nagel)</a></b></br></br><b><a href='#11'>11. Preparation of chemocompetent DH5-alpha cells</a></b></br></br><b><a href='#12'>12. Re-transformation of BioBricks</a></b></br></br><b><a href='#13'>13. Strand directed Mutagenesis PCR</a></b></br></br><b><a href='#14'>14. Transformation using Ligation product (in DH5alpha or XL1Blue) </a></b></br></br></br></br><h2><a name='1'>1. 3A – Assembly</a></h2><hr><i>NOTE: Enzymes and buffers were provided by Promega </i></br></br><b>Restriction (50 µl Reaction)</b></br>- 25 µl Mastermix Restriction-Enzyme Buffer (2x, with BSA)</br>- add 1 µl from every restriction enzyme to 500ng backbone equimolar DNA</br>- fill up to 50 µl dest. water</br>- incubate for 1.5h-3h at 37ºC </br>- inactivate for 20min at 70ºC (no clean-up, if directly used for ligation) </br></br><b>Ligation (20 µl Reaction)</b></br>- 2,0 µl equimolare restriction samples (inserts)</br>- 20ng/1.5 µl backbone</br>- fill up to 17.7 µl with dest. water</br>- incubate 5min at 37ºC</br>- add 2 µl Ligation buffer (10x) and 0.3 µl T4 DNA Ligase </br>- incubate for 3h RT or 15ºC over night </br>- inactivate for 10min at 70ºC</br></br></br><h2><a name='2'>2.Agarose preparation</a></h2><hr><b>Materials:</b></br>- Agarose </br>- 500ml bottle</br>- TBE (1x)</br></br><b>Procedure:</b></br>- dissolve 1g agarose in 100ml TBE (1x), resulting in 1% agarose</br>- heat for 2 minutes in a microwave at maximal power</br>- mix</br>- cook until it boils (1 min)</br>- mix carefully</br><i>2 clues for a successful boiling: 1. no cords, 2. boiling retardation</i></br>- store in 65ºC incubator</br></br></br><h2><a name='3'>3.Agarose gel casting</a></h2><hr>- assemble the gel chamber (chamber + 2 fences + 1 - 2 gel combs ) under the ethidiumbromid hood</br>- prepare 50ml falcon tube (label it!)</br>- fill 40ml warm agarose in the falcon tube</br>- add 4 µl ethidiumbromid (1:10.000) under the ethidiumbromid hood</br>- mix by inverting 2-3 times</br>- fill agarose in the gel chamber</br>- wait until the gel becomes solid (about 20minutes)</br></br></br><h2><a name='4'>4.loading the agarose gel and starting electrophoresis</a></h2><hr>- add LoadingDye (1:6) to your sample</br>- remove comb and fence from the gel</br>- place agarose gel in the electrophoresis chamber</br>- pipette samples carefully in the pockets (small pockets: up to 20 µl, big pockets: up to 50 µl)</br>- Place lid on the electrophoresis chamber and connect the electrodes to it.</br>- set parameters (high resolution: 120V, 20-30minutes, low resolution: 130V, 15min)</br>- evaluate gel under UV-light</br></br></br><h2><a name='5'>5. Preparation of Antibiotic stocks</a></h2><hr><b>Ampicillin:</b></br>- dissolve 100 mg ampicillin in 1 ml dest. water</br>- store at 20 ºC</br></br><b>Chloramphenicol:</b></br>- dissolve 18 mg chloramphenicol in 1 ml ethanol</br>- store at 20 ºC</br></br></br><h2><a name='6'>6. Colony PCR</a></h2><hr>- inoculate 10 µl dest. water with colony.</br>- use 1 µl of this water for one reaction:</br><img src='https://static.igem.org/mediawiki/2013/9/91/Bonn_MS_Methods1.png' width='550px'></br></br></br><h2><a name='7'>7.Preparation of Glycerol Stocks (iGEM)</a></h2><hr>- autoclave glycerol (60%)</br>- add 0,5 ml Glycerol to 1,5 ml cell culture in a cryo tube</br>- mix</br>- shock freeze in liquid nitrogen</br>- store at -80 ºC</br></br></br><h2><a name='8'>8. Plasmid Preparation</a></h2><hr><h2><a name='8a'>8a. Midi-Prep (Promega)</a></h2><hr>- centrifuge 50 ml of liquid cell culture for 10min at 5000g </br>- decant supernatant</br>- resuspend with 3 ml resuspension solution </br>- add 3 ml cell lysis solution and incubate for maximal 3 min at room temperature </br>- add 5 ml neutralization solution </br>- centrifuge for 20 min at 20 ºC, 5000g </br>- vacuum pump lysat through cleaning column into binding column </br>- abolish cleaning column</br>- vacuum pump with 10 ml endotoxin removal wash solution</br>- vacuum pump with 20 ml column wash solution</br>- dry membrane by vacuum</br>- add 600 µl nuclease free water on membrane</br>- centrifuge for 5 min at 1750 g into a fresh tube</br></br></br><h2><a name='8b'>8b. Mini-Prep (Promega)</a></h2><hr>- fill 1,5 ml overnight-culture in a new tube</br>- centrifuge for 30 seconds at maximal speed </br>- decant supernatant</br>- Repeat previous steps 2-5 times (depending on growth density)</br>- resuspend with 600 µl dest water </br>- add 100 µl cell lysis buffer </br>- after 1min (maximum 2min) add 350 µl of neutralization buffer </br>- centrifuge 3min at maximal speed</br>- place mini column in a collection tube and transfer supernatant into PureYield^TM Mini column</br>- centrifuge for 15 sec at maximal speed</br>- add 200 µl Endotoxin Removal Wash</br>- centrifuge for 15 sec at maximal speed</br>- add 400 µl column Wash solution</br>- centrifuge for 30 sec at maximal speed</br>- place mini column in a new tube</br>- add 30 µl elution buffer to the mini column, incubation at RT for 1 min</br>- centrifuge for 15 seconds at maximal speed </br>- store DNA at -20 ºC</br></br></br><h2><a name='9'>9. Preparation of LB agar plates</a></h2><hr>1l LB agar will result in approximately 30 plates</br></br>- dissolve 15g agar and 20g LB in 1l dest. Water</br>- autoclave</br>- cool down to 60-70ºC </br>- add antibiotics (1:1000) under the laminar airflow cabinet</br>- mix</br>- cast plates (approximately 20ml / plate)</br>- dry for 2h by room temperature</br>- store at 4ºC</br></br></br><h2><a name='10'>10. PCR - Clean-Up (Macherey und Nagel) </a></h2><hr><b>Gel Extraction: </b></br>1. add double amount NTI to gel</br>2. Incubate 3-7minutes at 50ºC and at 1000rpm (until gel is dissolved)</br>- continue with regular Clean-up Protocol (from step 3.) </br></br><b>Cleanup: </b></br>1. fill up sample with dest. water to 50 µl, if necessary</br>2. add double amount NTI to the sample</br>3. place column in a collection and add transfer solution to the column</br>4. centrifuge 30 seconds at 11.000g and discard flow through </br>5. add 700 µl NT3 </br>6. centrifuge 30 seconds at 11.000g and discard flow through </br>7. repeat step 5) and 6)</br>8. centrifuge 1minute at 11.000g</br>9. place column in a new tube and dry column at 70ºC for 5min</br></br>small parts (<1000bp): </br>9a. place column in a new tube and add 30 µl Elution buffer </br>9b. incubate 1min at room temperature</br>9c. Centrifuge 1minute at 11.000g </br></br>Bigger Parts: (>1000bp) </br>9A. place column in a new tube and add 20 µl Elution buffer </br>9B. incubate at 70ºC for 5minutes </br>9C. centrifuge at 50g for 1minute</br>9D. centrifuge at 11.000g for 1minute</br>9E. repeat step 9A. to 9D.</br></br></br><h2><a name='11'>11. Preparation of chemocompetent DH5-alpha cells</a></h2><hr>- Start with 200 ml Overnight culture with OD<sub>600</sub> of 0,6-0,8</br>- centrifuge at 4 ºC, 4500 g, 10 minutes </br>- decant supernatant</br>- resuspend with 40 ml inoune transformation buffer </br>- centrifuge at 4 ºC, 4500 g, 10 minutes </br>- decant supernatant</br>- resuspend in 20 ml inoune transformation buffer </br>- add 1,5 ml DMSO</br>- incubate 10 minutes on ice</br>- transfer 100 – 200 µl into precooled tubes</br>- shock freeze in liquid nitrogen</br></br></br><h2><a name='12'> 12. Re-transformation of Bio Bricks</a></h2><hr>- add 10 µl sterile dest water to DNA on plate </br>- incubate for 10 min at RT</br>- take 2 µl, leave rest on plate </br>- store plates at -20 ºC </br>- add the 2 µl DNA solution to 5 µl competent DH5-alpha</br>- incubate for 30 min on ice </br>- heat shock for 45 s at 42 ºC </br>- incubate 3 min on ice </br>- add 250 µl LB medium at 37 ºC </br>- incubate for 45 min at 37 ºC, 800 rpm </br>- plate 300 µl on Agar-plate with appropriate antibiotic </br>- dry 15min at RT</br>- incubate at 37ºC over night</br></br></br><h2><a name='13'> 13. Strand directed Mutagenesis PCR</a></h2><hr>Prepare master mix and add template as follows: </br><img src='https://static.igem.org/mediawiki/2013/b/b6/Bonn_MS_Methods2.png' width='550px'></br>- start PCR-Program: </br>1. initial denaturation 94ºC for 120 seconds </br>2. Denaturating 94ºC for 30 seconds</br>3. Annealing 94ºC for 30 seconds</br>4. Elongation 68 for 720 seconds</br>5. Repeat step 2) to 4) 12x </br></br></br><h2><a name='14'>14. Transformation using Ligation product (in DH5alpha or XL1Blue) </a></h2><hr>- thaw bacteria on ice </br>- add 2-4 µl Ligation mixture to 50 µl bacteria</br>- incubate 30minutes on ice </br>- heat shock 30-45 seconds (XL1Blue preferably 35 seconds) at 42ºC</br>- incubate 6min on ice</br>- add 250 µl LB medium at 37ºC</br>- incubate for 45minutes at 37ºC, 800rpm </br>- plate 300 µl on appropriate antibiotic</br>- dry 15 minutes at RT</br>- incubate at 37ºC over night</br>"; | ||
| Line 975: | Line 904: | ||
content.type="Project"; | content.type="Project"; | ||
break; */ | break; */ | ||
| + | |||
| + | /* | ||
| + | case 112: | ||
| + | content.i = 112; | ||
| + | content.parents = [100]; | ||
| + | content.childs = [109]; | ||
| + | content.titleShort = "Comic"; | ||
| + | content.titleLong = "Comic – The adventures of Obi Wan E.Coli"; | ||
| + | content.summary = "Read about the adventures of Obi Wan E.Coli"; | ||
| + | content.text = "<p style=display:block;text-align:center;><img src=https://static.igem.org/mediawiki/2013/c/c1/Bonn_Comic_episode1_page1.jpg width=650px></p><p style=display:block;text-align:center;><img src=https://static.igem.org/mediawiki/2013/a/ad/Bonn_Comic_episode1_page2.jpg width=650px></p><p style=display:block;text-align:center;><img src=https://static.igem.org/mediawiki/2013/1/19/Bonn_Comic_episode1_page3.jpg width=650px></p><p style=display:block;text-align:center;><img src=https://static.igem.org/mediawiki/2013/8/80/Bonn_Comic_episode1_page4.jpg width=650px></p><p style=display:block;text-align:center;><img src=https://static.igem.org/mediawiki/2013/6/6c/Comic_EN_2.0_%281%29.jpg width=650px></p><p style=display:block;text-align:center;><img src=https://static.igem.org/mediawiki/2013/8/84/Comic_EN_2.0_%282%29.jpg width=650px></p><p style=display:block;text-align:center;><img src=https://static.igem.org/mediawiki/2013/9/97/Comic_EN_2.0_%283%29.jpg width=650px></p><p style=display:block;text-align:center;><img src=https://static.igem.org/mediawiki/2013/1/19/Comic_EN_2.0_%284%29.jpg width=650px></p><p style=display:block;text-align:center;><img src=https://static.igem.org/mediawiki/2013/8/8d/Bonn_Comic_EN_3.0_%281%29.jpg width=650px></p><p style=display:block;text-align:center;><img src=https://static.igem.org/mediawiki/2013/5/58/Bonn_Comic_EN_3.0_%282%29.jpg width=650px></p><p style=display:block;text-align:center;><img src=https://static.igem.org/mediawiki/2013/8/8c/Bonn_Comic_EN_3.0_%283%29.jpg width=650px></p><p style=display:block;text-align:center;><img src=https://static.igem.org/mediawiki/2013/b/b4/Bonn_Comic_EN_3.0_%284%29.jpg width=650px>"; | ||
| + | content.type = "Human Practice"; | ||
| + | break; | ||
| + | */ | ||
| + | |||
| + | case 200: | ||
| + | content.i = 200; | ||
| + | content.parents=[37]; | ||
| + | content.childs=[]; | ||
| + | content.titleShort = "Safety"; | ||
| + | content.titleLong = "Safety"; | ||
| + | content.summary= "Since safety is an important concern for any iGEM team, we gave deep thought to it. In this article our reflections about safety measures are presented."; | ||
| + | content.text= " Safety is an important issue for all of the IGEM teams. Taking care of the health and safety of our teammates is of the utmost importance for us. In our opinion, only a safe lab is a good environment for research and work. On our safety page we would like to give you a brief overview over the precautions we take to ensure a safe work environment in the lab. </br></br> <h2> A </h2> Do the biological materials used in your lab work pose any of the following risks: </br> </br> 1. Risks to the safety and health of team members or others working in the lab? </br> In our lab, we work with E. coli (strain: DH5alpha;XL1Blue;w3110 sspB-; w3110 sspB- LacI-DAS+4;Top10; DH10 Beta). All these E.Coli strains are classified as risk group 1, as they can cause irritation of the skin, the eyes, and the respiratory tract; additionally, they can affect the kidneys. Although our E.coli strains are nonpathogenic, we wear protective clothes and examination gloves to ensure safety. </br> We also use ethidum bromide in our lab, which is toxic and can cause cancer. To minimize the risk of accidental contact with this dangerous substance, we have fume hoods especially designated for working with ethidium bromide. Furthermore, the use of nitrile gloves as well as lab coats is mandatory when we are working with potentially harmful substances and any waste contaminated with ethidium bromide is kept separate from other waste. </br> In addition, every team member had to take a course about biosafety and regulations in Germany (as required in §8-12 of the GenTSV, the German act of genetic engineering). </br></br> 2. Risks to the safety and health of the general public, if released by design or by accident? </br> There is little risk to the general public, since our bacteria strains are nonpathogenic and in general do not cause harm to humans. And yet, the contact of an immunocompromised person with our bacteria strains could lead to health issues. </br></br> 3. Risk to the environment, if released by design or by accident? </br> Since our bacteria and genetic parts are non pathogenic and the light induced protein degradation is not a threat to the environment, there is minimal risk to the environment if our (modified) bacteria were released . In addition, our lab is equipped with a special air conditioning system which maintains a slight low pressure environment in the lab and thus prevents the air from inside of the lab from getting out when the doors are opened. The risk of (genetically altered) bacteria being released by accident is therefore reduced. </br> </br> 4.Risks to security through malicious misuse by individuals, groups, or countries? </br> The risk of malicious misuse can never be completely eliminated. Although there are cameras around the campus as well as other security installations, an element of risk remains. If our project was stolen and our bacteria were released, the risks for the public would be minimal, since our bacteria strains are nonpathogenic and our project mainly deals with the light-inducible degradation of proteins, which is hardly dangerous. </br></br> 5. If your project moved from a small-scale lab study to become widely used as a commercial/industrial product, what new risks might arise? </br> Since our light-inducible degradation system is mostly thought to be applicable in restricted areas (labs, bioreactors) the risks are low. Of course, any industrial application of our system should mot be designed without additional fail-safes to ensure multiple layers of safety. </br> One possible “risk” of our project is inherent to any other biological system; namely, that mutations in our device's gene sequence lead to unforeseen consequences. We assume that it is most likely that a mutation in our device will lead to the device's failure, i.e. leading to an accumulation of the protein that is supposed to be degraded. Whether this poses merely efficiency problems or more serious biosafety risks depends on the specific application and should be considered by persons responsible for implementing that instance of our technology. </br></br> 6. Does your project include any design features to address safety risks? (For example: kill switches, auxotrophic chassis, etc.) Note that including such features is not mandatory to participate in iGEM, but many groups choose to include them. </br> We developed a kill switch that is induced by blue light. This switch is capable of killing bacteria that are accidentally removed from a restricted area - for example a red-light hood – and which are then exposed to daylight in a short period of time. </br></br> 7. What safety training have you received (or plan to receive in the future)? Provide a brief description, and a link to your institution’s safety training requirements, if available. </br> Every team member had to take a course about safe work in the lab. We also have advisors who teach us how to work properly and safely and inform us about safety measures, such as wearing protective clothing. We also plan to implement a disinfection checkpoint that our team members will have to pass when leaving our entering the lab to decrease the chance of contamination. </br></br> 8. Under what biosafety provisions will / do you work? </br> Please provide a link to your institution biosafety guidelines. </br> The institution's biosafety guidelines are the national safety guidelines. <sup><a href=#2001>200.1</a></sup> </br> </br></br> <h2>B</h2> Does your institution have an Institutional Biosafety Committee, or an equivalent group? If yes, have you discussed your project with them? Describe any concerns they raised with your project, and any changes you made to your project plan based on their review. </br> Our university does not have a committee for biosafety. We are using the guidelines from the central biosafety committee from our federal government. We also work together with our supervisor, discussing our project and to address any safety issues that might arise. </br></br></br> <h2>C</h2> Does your country have national biosafety regulations or guidelines? If so, please provide a link to these regulations or guidelines if possible. <sup><a href=#2001>200.1</a></sup> <sup><a href=#2002>200.2</a></sup> </br></br></br> <h2>D</h2> According to the WHO Biosafety Manual, what is the BioSafety Level rating of your lab? </br> Our Biosafety Level is 1. <h2><b>References</b></h2></br> <a name=2001>200.1</a> <a href= http://www.gesetze-im-internet.de/gentsv/index.html#BJNR023400990BJNE001503320> http://www.gesetze-im-internet.de/gentsv/index.html#BJNR023400990BJNE001503320 </a> </br> <a name=2002>200.2</a> <a href= http://www.gesetze-im-internet.de/gentsv/anhang_iii_34.html> http://www.gesetze-im-internet.de/gentsv/anhang_iii_34.html </a>"; | ||
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Latest revision as of 13:27, 1 December 2013
