http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=100&target=Nkessler&year=&month=2010.igem.org - User contributions [en]2024-03-29T15:30:42ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Bielefeld-Germany/Project/ApproachTeam:Bielefeld-Germany/Project/Approach2010-10-28T02:22:15Z<p>Nkessler: </p>
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= The Approach =<br />
<br />
First we looked for a sensor system which is able to detect substances and we searched for substances of interest. The system of choice had to be taken out of a different bacteria species than ''Escherichia coli'' in order to avoid any background. Therefore we checked the literature for a well reviewed sensor system, which is not part of the ''E. coli'' genome. Finally we decided to choose the phenolic sensing VirA/G signaling system of ''Agrobacterium tumefaciens''. It naturally detects acetosyringone, which is a secondary metabolite of plants that affects bacteria as an attractant. After repeated research to check for any other possible substances which could be detected by the system, we got a really long list of possibilities and picked 'capsaicin', which is responsible for the spiciness of edibles. <br />
<br />
<br />
The biological steps for creating a new VirA/G sensor system are:<br />
<br />
<br />
# extract the complete VirA/G signaling system out of ''A. tumefaciens''<br />
# create new BioBricks out of the existing environmental parts <br />
# get the new BioBricks working into ''E. coli''<br />
# choose a reporter gene as readout for the sensor system<br />
# modify the system for sensibility and specificity by error prone PCR, find and select the most promising mutants (directed mutagenesis)<br />
<br />
<br />
=== Preparing the system ===<br />
<br />
We tried to work with the already existing VirA receptor BioBrick from the registry (<partinfo>K238008</partinfo>). Unfortunately this VirA BioBrick did not work. The sequence data of the BioBrick did not fit to the published VirA sequence. So we had to extract the ''virA'' gene via PCR out of the ''A. tumefaciens'' TI-plasmid by ourselves in order to create a new BioBrick. <br />
<br />
VirA does not work without the help of the VirG protein. The existing ''virG'' gene in the iGEM registry contains illegal restriction sites. Moreover it needs the help of the RpoA Protein from ''A. tumefaciens'' to work as a transcription facor in ''E. coli''. We changed the sequence manually and had the gene synthesized by Mr. Gene.<br />
<br />
<br />
=== Starting point for BioBricks -> Go cloning===<br />
After discussing the possibility of creating only one big plasmid for mutated ''virA'' screenings, we unified that one big plasmid would minimize transformation efficiency and would be difficult to modify via error prone PCR. So we had to change the origin of a pSB1C3 plasmid in order to avoid incompatibility. Thus we cloned the R6K origin (<partinfo>J61001</partinfo>) into the <partinfo>pSB1C3</partinfo> plasmid and subsequently removed the original pMB1/ColE1 origin of replication. Therefore we digested the pSB1C3::R6K plasmid with Hin6I and seperated the resulting 6 fragments (1758 bp, 270 bp, 174 bp, 109 bp, 100 bp, 67 bp) by agarose gel electrophoresis. We extracted the largest fragment (1758 bp) and religated it. The resulting plasmid still contains 32 remaining basepairs of the pMB1 origin, but this fragment does not enable replication in pir<sup>-</sup> strains.<br />
<br />
[[Image:pSB1C3-R6K_300x300px.jpg]] [[Image:pSB1C3-R6K-ColE1del_300x300px.jpg]]<br />
<br />
<br />
=== The error prone PCR and screening ===<br />
<br />
After building a plasmid with R6K ori we were able to create the two constructs. The first one will be found inside the competent bacteria cells and contains the ''virG'' gene under the constitutive promoter <partinfo>J23110</partinfo>, a terminator (<partinfo>B0017</partinfo>), a ''vir'' promoter and a readout or selection gene (luciferase, mRFP and kanamycin resistance, respectively):<br />
<br />
[[Image:Screening_plasmid.jpg]]<br />
<br />
<br />
The second plasmid contains the (mutated) ''virA'' gene under the control of the constitutive promoter <partinfo>J23110</partinfo> and will be transformed and modified in one step via error prone PCR. <br />
<br />
<br />
[[Image:VirA plasmid.jpg]]<br />
<br />
<br />
The error prone PCR is a PCR under malfunction conditions for the polymerase. It is possible to regulate the frequency of the mutagenesis by editing unbalanced concentrations of nucleotides and co-factors to the PCR. So we are going to create a new BioBrick and transform it into our target in one step. Before we are able to do the error prone PCR we need to get the backbones done. <br />
<br />
<br />
=== Survival of the fittest ===<br />
<br />
We used high amounts of the antibiotic kanamycin in order to select the most specific system for our substances. By varying the amount of kanamycin we will be able to carefully select the best mutant out of our error-prone PCR tests. The mutated ''virA'' system will be induced after the error prone PCR by a mix of possible targets for the system (like capsaicin, dopamin, homovanillic acid etc.). It is important to avoid any acetosyringone, so we will be able to search for systems with new targets. <br />
<br />
<br />
=== Directed mutagenesis ===<br />
<br />
Alternatively to the mutagenesis strategy we developed two strategies for a directed mutagenesis: <br />
<br />
'''VirA_mut1''' <br />
<br />
Regarding to the results from Muscle, we used site directed mutagenesis (for Protocols see: ([http://openwetware.org/wiki/Site-directed_mutagenesis OpenWet Ware])) to change VirA protein sequence leucin 293 to tyrosine (L293Y = mut1).<br />
<br />
<br />
'''VirA_mut2'''<br />
<br />
After VirA_mut1 with its single mutation failed working in our screening system, we decided to mutate some further amino acids in the area of the reported binding region from the animal TRPV1 receptors.<br />
<br />
According to the literature amino acid residues from 288 to 293, regulate phenol structural specificity in the TRPV1 receptor. Substitutions in this region narrow the active phenols to those missing the para-acyl substituent ([http://www.springerlink.com/content/978-0-387-78884-5#section=169995&page=1 Lin ''et al.'', 2008]). Our VirA_mut2 construct contains the following substitutions:<br />
<center> A357H, R358P, R359T, L360L, D361K, Y361L </center><br />
<br />
<br />
[[Image:Bielefeld_VirA_mut2.jpg|300px|thumb|center|'''Alignment of crucial ligand binding regions from ''Agrobacterium tumefaciens'' (lane 1), chick (lane 2), ''homo sapiens'' (lane 3), ''rattus'' (lane 4) and ''mus muskulus'' (lane 5). The Box shows all six amino acids we mutagenized in our VirA_mut2 approach''']]<br />
<br />
=== The final construct===<br />
In order to get a visible light signal by the reporter gene luciferase we integrated genetic amplifiers into the final construct to enhance this signal. <br />
<br />
For a demonstration of this final system see the animation below: <br />
<br />
<br />
[[Image:Bielefeld_Vorgehen.gif]]</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/SubmittedTeam:Bielefeld-Germany/Results/Submitted2010-10-28T02:07:13Z<p>Nkessler: </p>
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= Submitted BioBricks =<br />
<br />
We have submitted the following [[Team:Bielefeld-Germany/Results/Characterization | working]] BioBricks. For a more detailed theoretical background to these BioBricks check our [[Team:Bielefeld-Germany/Project/Theory | theory pages]], too. <br />
<br />
<br />
===VirA receptor===<br />
[[Image:Vira_rezeptor.jpg|300px|thumb|right|'''Fig. 1: The VirA receptor of ''Agrobacterium tumefaciens''.''']]<br />
The VirA receptor is used by ''A. tumefaciens'' to detect acetosyringone and other phenolic substances which are secreted by plants after injury. In presence of these substances VirA phosphorylates itself and afterwards VirG, a response regulator which activates ''vir'' box containing promoters. These promoters control genes which are used for infecting the injured plant. <br />
Actually we tried to use the VirA receptor already existing in the partsregistry (<partinfo>K238008</partinfo>). But due to sequence problems (compare results in [[Team:Bielefeld-Germany/Results/Characterization | characterization]]) we had to isolate the ''virA'' gene from the Ti-plasmid of ''A. tumefaciens'' C58 ourselves and convert it into a BioBrick conform part. We removed an illegal ''PstI'' restriction site in the ''virA'' gene by site-directed mutagenesis. <br />
<br />
You can find this BioBrick here: <partinfo>K389001</partinfo><br />
<br />
You can find the K389001 BioBrick under the control of a constitutive promoter (<partinfo>J23110</partinfo>) here: <partinfo>K389010</partinfo>.<br />
<br />
===Mutated ''virG''===<br />
Phosphorylated VirG binds to ''vir'' box containing promoters and activates them. VirG is activated by the acetosyringone receptor VirA. <br />
This version of VirG activates ''virB'' promoters in ''Escherichia coli'' without the ''rpoA''-gene from ''Agrobacterium tumefaciens'' [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Subcloning_into_E._coli_and_receptor_function_in_new_host (compare to Theory)]. For this reason the point mutations G56V and I77V are brought into the molecule [http://www.springerlink.com/content/wmq06kua5qkma1au/ YC Jung ''et al.'', (2004)]. Because this BioBrick is synthesized, the codon usage is optimized for ''E. coli'' and illegal restriction sites were removed. When you use this ''virG'' gene in a VirA/G signaling system you do not need <partinfo>K238010</partinfo> anymore to get the system working in ''E. coli''. <br />
<br />
You can find this BioBrick here: <partinfo>K389002</partinfo><br />
<br />
===''virB''-promoter===<br />
''Vir''-promoters from ''A. tumefaciens'' are induced by phosphorylated VirG response regulators and control genes for infecting plants in their natural host. They are part of the VirA/G signal transduction system. <br />
We wanted to use the ''vir''-promoter from the partsregistry (<partinfo>K238011</partinfo>) but the same problems occurred as with the use of the VirA receptor BioBrick from the partsregistry. So we also had to create a new ''vir''-promoter BioBrick (again from Ti-plasmid of ''A. tumefaciens'' C58).<br />
<br />
You can find this BioBrick here: <partinfo>K389003</partinfo><br />
<br />
===Firefly luciferase===<br />
Bringing the firefly luciferase gene from Promega's pGL4.10[luc2] vector into a BioBrick compatible form as a sensitive reporter gene. To amplify the signal of the luciferase three different sensitivity tuners are assembled before the luciferase gene. The sensitivity tuners were created in 2007 by the iGEM team from Cambridge and amplify the readout signal. We also assembled the firefly luciferase behind three different strong constitutive promoters for gathering additional information about this reporter gene [https://2007.igem.org/Cambridge/Amplifier_project#Results Cambridge, (2007)]. <br />
<br />
You can find this BioBrick here: <partinfo>K389004</partinfo><br />
<br />
You can find this BioBrick with sensitivity tuner 1 here: <partinfo>K389401</partinfo><br />
<br />
You can find this BioBrick with sensitivity tuner 2 here: <partinfo>K389402</partinfo><br />
<br />
You can find this BioBrick with sensitivity tuner 3 here: <partinfo>K389403</partinfo><br />
<br />
You can find this BioBrick under the control of a weak constitutive promoter here: <partinfo>K389302</partinfo><br />
<br />
You can find this BioBrick under the control of a medium strong constitutive promoter here: <partinfo>K389307</partinfo><br />
<br />
You can find this BioBrick under the control of a strong constitutive promoter here: <partinfo>K389318</partinfo><br />
<br />
===Neomycin / kanamycin resistance===<br />
A neomycin / kanamycin resistance gene without promoter is isolated and brought into a BioBrick compatible form. We will use the BioBrick <partinfo>P1003</partinfo> as source for the kanamycin resistance gene. <br />
<br />
You can find this BioBrick here: <partinfo>K389005</partinfo><br />
<br />
<br />
===BioBrick for ''virA''-screenings===<br />
[[Image:Screening_plasmid.jpg|500px|thumb|right|'''Fig. 2: The BioBrick <partinfo>K389011</partinfo> for ''virA'' screenings.''']]<br />
This part contains our mutated ''virG'' BioBrick under the control of a constitutive promoter (<partinfo>J23110</partinfo>) and an antibiotic resistance (<partinfo>K389005</partinfo>) under the control of the ''virB'' promoter (<partinfo>K389003</partinfo>). The better the VirA receptor recognizes a substance, the stronger the antibiotic resistance will be expressed. <br />
<br />
This device is used to screen ''virA'' receptor mutants. The ''virA'' receptor gene is mutated in the <partinfo>BBa_K389010</partinfo> BioBrick. The better the receptor detects a substance, the more VirG is phosphorylated and the stronger the kanamycin resistance from this BioBrick is expressed. The screening system for a mutated ''virA'' gene contains this BioBrick in a plasmid with R6K ori and the BioBrick <partinfo>BBa_K389010</partinfo> in a plasmid with ColE1 ori. Both plasmids are transformed to and screened in ''E. coli'' EC100D because plasmids with R6K ori are only replicated in pir+ or pir116 ''E. coli'' strains. Once a receptor with high sensitivity for a screened substance is found, the plasmids are isolated and transformed to e.g. ''E. coli'' TOP10. Because the R6K ori does not work in this strain because it is not a pir+ / pir116 strain, it is easy to separate the mutated ''virA'' BioBrick from the screening plasmid.<br />
<br />
You can find this BioBrick here: <partinfo>K389011</partinfo><br />
<br />
<br />
===Reporter constructs===<br />
The reporter constructs are similar to the ''virA'' screening construct (compare fig. 2) but instead of the antibiotic resistance they carry a reporter gene. The amount of produced reporter shows the activity of the VirA receptor and the ''vir'' promoter, respectively. If the original ''vir'' promoter is too weak, we will use Cambridge's sensitivity tuners to increase the output signal of our biosensor ([https://2007.igem.org/Cambridge/Amplifier_project#Results Cambridge, 2007]). <br />
<br />
You can find this BioBrick with luciferase readout here: <partinfo>K389012</partinfo><br />
<br />
You can find this BioBrick with mRFP readout here: <partinfo>K389013</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase readout No. 1 here: <partinfo>K389411</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase readout No. 2 here: <partinfo>K389412</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase readout No. 3 here: <partinfo>K389413</partinfo><br />
<br />
<br />
===Characterization constructs===<br />
[[Image:Bielefeld_Vorgehen2.gif|400px|thumb|right|'''Fig. 3: Animation of the function of a complete characterization construct with sensitivity tuner (click to enlarge). In the system without sensitivity tuner the reporter gene is directly under the control of the ''vir'' promoter.''' ]]<br />
The characterization constructs are like the reporter constructs but a ''virA'' gene under the control of a constitutive promoter is assembled before them. These constructs are used to characterize the natural, unmutated VirA/G signaling system and the ''vir'' promoter, respectively. The natural system is induced with acetosyringone and the readout is measured to determine the behaviour of the VirA/G signaling system. <br />
<br />
You can find this BioBrick with luciferase readout here: <partinfo>K389015</partinfo><br />
<br />
You can find this BioBrick with mRFP readout here: <partinfo>K389016</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase readout No. 1 here: <partinfo>K389421</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase readout No. 2 here: <partinfo>K389422</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase readout No. 3 here: <partinfo>K389423</partinfo><br />
<br />
<br />
===VirA/G signaling system===<br />
A complete VirA/G signaling system without readout was submitted. The system contains a ''virA'' and our mutated ''virG'' gene under the control of a constitutive promoter and a ''vir'' promoter without reporter gene. <br />
<br />
You can find this BioBrick here: <partinfo>K389017</partinfo><br />
<br />
=References=<br />
*[https://2007.igem.org/Cambridge/Amplifier_project Cambridge iGEM Team wiki 2007, amplifier project]<br />
<br />
*YC Jung ''et al.'', (2004), ''Mutants of ''Agrobacterium tumefaciens virG'' Gene That Activate Transcription of ''vir'' Promoter in ''Escherichia coli'', Current Microbiol 49:334-340.</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/ResultsTeam:Bielefeld-Germany/Results2010-10-28T01:41:53Z<p>Nkessler: /* Results */</p>
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= Results =<br />
[[Image:Bielefeld_luc.jpg|300px|thumb|right| '''Figure 1: Visualization of induced (from left to right) <partinfo>K389421</partinfo>, <partinfo>K389422</partinfo> and <partinfo>K389423</partinfo> sensitivity tuner amplified vir-system.''']]<br />
<br />
On the next pages we are going to present you our BioBrick work.<br />
<br />
One approach of our project was to visualize a readout by a light emitting system. So a luciferase BioBrick was built. This BioBrick is a highly sensitive reporter gene which was successfully applied in different devices with a PoPS output. The characterization of this BioBrick can be found [[Team:Bielefeld-Germany/Results/Characterization/K389004 | here]]. <br />
<br />
The second step was to establish a complete VirA/G signaling system from ''Agrobacterium tumefaciens'' in ''Escherichia coli''. For this purpose three new BioBricks were created - ''virA'', ''virG'' and a ''vir'' promoter. The VirA receptor recognizes the phenolic substance acetosyringone and transmits this information to the VirG response regulator - a bacterial transcription factor which activates ''vir'' promoters. This natural VirA/G signaling system works in ''E. coli'' and was measured and characterized by using the reporter genes [[Team:Bielefeld-Germany/Results/Characterization/K389016 | mRFP]] and [[Team:Bielefeld-Germany/Results/Characterization/K389015 | luciferase]]. <br />
<br />
In the third step we tried to modify the VirA receptor by directed mutagenesis using error-prone PCR, in order to detect substances other than acetosyringone (e.g. capsaicin). For that purpose we designed a [[Team:Bielefeld-Germany/Project/Theory#Screening_system | high-throughput screening system]] with a selective system based on the expression of a kanamycin resistance. In a short period of time we found many positive clones, but further analysis indicated that the VirA receptor was changed to constitutive activity. Thereby, due to the short time available, we did not manage to construct a receptor that was specific for other phenolic substance, e.g. capsaicin.<br />
<br />
<br />
We tried to create a light signal that is visible to the human eye as another part of our project. So genetic amplifiers ([http://parts.mit.edu/igem07/index.php/Cambridge/Amplifier_project#Results Cambridge, iGEM 2007, amplifier project]) were added to our luciferase BioBrick. This amplified luciferase readout was coupled with the VirA/G signaling system in order to visualize the induction behaviour. The amplifiers worked so far and gave a visible luciferase readout but the basal expression of the ''vir'' promoter was also amplified so the induced status of the VirA/G system was not discriminable properly from the uninduced status. <br />
<br />
<br />
<br />
<br />
<br />
We entered the following BioBricks to the partsregistry: <br />
<br />
<groupparts>iGEM010 Bielefeld-Germany</groupparts></div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/SubmittedTeam:Bielefeld-Germany/Results/Submitted2010-10-28T01:41:13Z<p>Nkessler: /* virB-promoter */</p>
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= Submitted BioBricks =<br />
<br />
We have submitted the following [[Team:Bielefeld-Germany/Results/Characterization | working]] BioBricks. For a more detailed theoretical background to these BioBricks check our [[Team:Bielefeld-Germany/Project/Theory | theory pages]], too. <br />
<br />
<br />
===VirA receptor===<br />
[[Image:Vira_rezeptor.jpg|300px|thumb|right|'''Fig. 1: The VirA receptor of ''Agrobacterium tumefaciens''.''']]<br />
The VirA receptor is used by ''A. tumefaciens'' to detect acetosyringone and other phenolic substances which are secreted by plants after injury. In presence of these substances VirA phosphorylates itself and afterwards VirG, a response regulator which activates ''vir'' box containing promoters. These promoters control genes which are used for infecting the injured plant. <br />
Actually we tried to use the VirA receptor already existing in the partsregistry (<partinfo>K238008</partinfo>). But due to sequence problems (compare results in [[Team:Bielefeld-Germany/Results/Characterization | characterization]]) we had to isolate the ''virA'' gene from the Ti-plasmid of ''A. tumefaciens'' C58 ourselves and convert it into a BioBrick conform part. We removed an illegal ''PstI'' restriction site in the ''virA'' gene by site-directed mutagenesis. <br />
<br />
You can find this BioBrick here: <partinfo>K389001</partinfo><br />
<br />
You can find the K389001 BioBrick under the control of a constitutive promoter (<partinfo>J23110</partinfo>) here: <partinfo>K389010</partinfo>.<br />
<br />
===Mutated ''virG''===<br />
Phosphorylated VirG binds to ''vir'' box containing promoters and activates them. VirG is activated by the acetosyringone receptor VirA. <br />
This version of VirG activates ''virB'' promoters in ''Escherichia coli'' without the ''rpoA''-gene from ''Agrobacterium tumefaciens'' [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Subcloning_into_E._coli_and_receptor_function_in_new_host (compare to Theory)]. For this reason the point mutations G56V and I77V are brought into the molecule [http://www.springerlink.com/content/wmq06kua5qkma1au/ YC Jung ''et al.'', (2004)]. Because this BioBrick is synthesized, the codon usage is optimized for ''E. coli'' and illegal restriction sites were removed. When you use this ''virG'' gene in a VirA/G signaling system you do not need <partinfo>K238010</partinfo> anymore to get the system working in ''E. coli''. <br />
<br />
You can find this BioBrick here: <partinfo>K389002</partinfo><br />
<br />
===''virB''-promoter===<br />
''Vir''-promoters from ''A. tumefaciens'' are induced by phosphorylated VirG response regulators and control genes for infecting plants in their natural host. They are part of the VirA/G signal transduction system. <br />
We wanted to use the ''vir''-promoter from the partsregistry (<partinfo>K238011</partinfo>) but the same problems occurred as with the use of the VirA receptor BioBrick from the partsregistry. So we also had to create a new ''vir''-promoter BioBrick (again from Ti-plasmid of ''A. tumefaciens'' C58).<br />
<br />
You can find this BioBrick here: <partinfo>K389003</partinfo><br />
<br />
===Firefly luciferase===<br />
Bringing the firefly luciferase gene from Promega's pGL4.10[luc2] vector into a BioBrick compatible form as a sensitive reporter gene. To amplify the signal of the luciferase three different sensitivity tuners are assembled before the luciferase gene. The sensitivity tuners were created in 2007 by the iGEM team from Cambridge and amplify the read-out signal. We also assembled the firefly luciferase behind three different strong constitutive promoters for gathering additional information about this reporter gene [http://parts.mit.edu/igem07/index.php/Cambridge/Amplifier_project#Results Cambridge, (2007)]. <br />
<br />
You can find this BioBrick here: <partinfo>K389004</partinfo><br />
<br />
You can find this BioBrick with sensitivity tuner 1 here: <partinfo>K389401</partinfo><br />
<br />
You can find this BioBrick with sensitivity tuner 2 here: <partinfo>K389402</partinfo><br />
<br />
You can find this BioBrick with sensitivity tuner 3 here: <partinfo>K389403</partinfo><br />
<br />
You can find this BioBrick under the control of a weak constitutive promoter here: <partinfo>K389302</partinfo><br />
<br />
You can find this BioBrick under the control of a medium strong constitutive promoter here: <partinfo>K389307</partinfo><br />
<br />
You can find this BioBrick under the control of a strong constitutive promoter here: <partinfo>K389318</partinfo><br />
<br />
===Neomycin / kanamycin resistance===<br />
A neomycin / kanamycin resistance gene without promoter is isolated and brought into a BioBrick compatible form. We will use the BioBrick <partinfo>P1003</partinfo> as source for the kanamycin resistance gene. <br />
<br />
You can find this BioBrick here: <partinfo>K389005</partinfo><br />
<br />
<br />
===BioBrick for ''virA''-screenings===<br />
[[Image:Screening_plasmid.jpg|500px|thumb|right|'''Fig. 2: The BioBrick <partinfo>K389011</partinfo> for ''virA'' screenings.''']]<br />
This part contains our mutated ''virG'' BioBrick under the control of a constitutive promoter (<partinfo>J23110</partinfo>) and an antibiotic resistance (<partinfo>K389005</partinfo>) under the control of the ''virB'' promoter (<partinfo>K389003</partinfo>). The better the VirA receptor recognizes a substance the stronger will the antibiotic resistance be expressed. <br />
<br />
This device is used to screen ''virA'' receptor mutants. The ''virA'' receptor gene is mutated in the <partinfo>BBa_K389010</partinfo> BioBrick. The better the receptor detects a substance, the more VirG is phosphorylated and the stronger the kanamycin resistance from this BioBrick is expressed. The screening system for a mutated ''virA'' gene contains this BioBrick in a plasmid with R6K ori and the BioBrick <partinfo>BBa_K389010</partinfo> in a plasmid with ColE1 ori. Both plasmids are transformed to and screened in ''E. coli'' EC100D because plasmids with R6K ori are only replicated in pir+ or pir116 ''E. coli'' strains. Once a receptor with high sensitivity for a screened substance is found, the plasmids are isolated and transformed to ''e.g. E. coli'' TOP10. Because the R6K ori does not work in this strain because it is not a pir+ / pir116 strain, it is easy to separate the mutated ''virA'' BioBrick from the screening plasmid.<br />
<br />
You can find this BioBrick here: <partinfo>K389011</partinfo><br />
<br />
<br />
===Reporter constructs===<br />
The reporter constructs are similar to the ''virA'' screening construct (compare fig. 2) but instead of the antibiotic resistance they carry a reporter gene. The amount of produced reporter shows the activity of the VirA receptor and the ''vir'' promoter, respectively. If the original ''vir'' promoter is too weak, we will use Cambridge's sensitivity tuners to increase the output signal of our biosensor ([http://parts.mit.edu/igem07/index.php/Cambridge/Amplifier_project#Results Cambridge, 2007]). <br />
<br />
You can find this BioBrick with luciferase read-out here: <partinfo>K389012</partinfo><br />
<br />
You can find this BioBrick with mRFP read-out here: <partinfo>K389013</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 1 here: <partinfo>K389411</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 2 here: <partinfo>K389412</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 3 here: <partinfo>K389413</partinfo><br />
<br />
<br />
===Characterization constructs===<br />
[[Image:Bielefeld_Vorgehen2.gif|400px|thumb|right|'''Fig. 3: Animation of the function of a complete characterization construct with sensitivity tuner (click to enlarge). In the system without sensitivity tuner the reporter gene is directly under the control of the ''vir'' promoter.''' ]]<br />
The characterization constructs are like the reporter constructs but a ''virA'' gene under the control of a constitutive promoter is assembled before them. These constructs are used to characterize the natural, unmutated VirA/G signaling system and the ''vir'' promoter, respectively. The natural system is induced with acetosyringone and the read-out is measured to determine the behaviour of the VirA/G signaling system. <br />
<br />
You can find this BioBrick with luciferase read-out here: <partinfo>K389015</partinfo><br />
<br />
You can find this BioBrick with mRFP read-out here: <partinfo>K389016</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 1 here: <partinfo>K389421</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 2 here: <partinfo>K389422</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 3 here: <partinfo>K389423</partinfo><br />
<br />
<br />
===VirA/G signaling system===<br />
A complete VirA/G signaling system without read-out was submitted. The system contains a ''virA'' and our mutated ''virG'' gene under the control of a constitutive promoter and a ''vir'' promoter without reporter gene. <br />
<br />
You can find this BioBrick here: <partinfo>K389017</partinfo><br />
<br />
=References=<br />
*[http://parts.mit.edu/igem07/index.php/Cambridge/Amplifier_project Cambridge iGEM Team wiki 2007, amplifier project]<br />
<br />
*YC Jung ''et al.'', (2004), ''Mutants of ''Agrobacterium tumefaciens virG'' Gene That Activate Transcription of ''vir'' Promoter in ''Escherichia coli'', Current Microbiol 49:334-340.</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/SubmittedTeam:Bielefeld-Germany/Results/Submitted2010-10-28T01:25:04Z<p>Nkessler: /* Mutated virG */</p>
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= Submitted BioBricks =<br />
<br />
We have submitted the following [[Team:Bielefeld-Germany/Results/Characterization | working]] BioBricks. For a more detailed theoretical background to these BioBricks check our [[Team:Bielefeld-Germany/Project/Theory | theory pages]], too. <br />
<br />
<br />
===VirA receptor===<br />
[[Image:Vira_rezeptor.jpg|300px|thumb|right|'''Fig. 1: The VirA receptor of ''Agrobacterium tumefaciens''.''']]<br />
The VirA receptor is used by ''A. tumefaciens'' to detect acetosyringone and other phenolic substances which are secreted by plants after injury. In presence of these substances VirA phosphorylates itself and afterwards VirG, a response regulator which activates ''vir'' box containing promoters. These promoters control genes which are used for infecting the injured plant. <br />
Actually we tried to use the VirA receptor already existing in the partsregistry (<partinfo>K238008</partinfo>). But due to sequence problems (compare results in [[Team:Bielefeld-Germany/Results/Characterization | characterization]]) we had to isolate the ''virA'' gene from the Ti-plasmid of ''A. tumefaciens'' C58 ourselves and convert it into a BioBrick conform part. We removed an illegal ''PstI'' restriction site in the ''virA'' gene by site-directed mutagenesis. <br />
<br />
You can find this BioBrick here: <partinfo>K389001</partinfo><br />
<br />
You can find the K389001 BioBrick under the control of a constitutive promoter (<partinfo>J23110</partinfo>) here: <partinfo>K389010</partinfo>.<br />
<br />
===Mutated ''virG''===<br />
Phosphorylated VirG binds to ''vir'' box containing promoters and activates them. VirG is activated by the acetosyringone receptor VirA. <br />
This version of VirG activates ''virB'' promoters in ''Escherichia coli'' without the ''rpoA''-gene from ''Agrobacterium tumefaciens'' [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Subcloning_into_E._coli_and_receptor_function_in_new_host (compare to Theory)]. For this reason the point mutations G56V and I77V are brought into the molecule [http://www.springerlink.com/content/wmq06kua5qkma1au/ YC Jung ''et al.'', (2004)]. Because this BioBrick is synthesized, the codon usage is optimized for ''E. coli'' and illegal restriction sites were removed. When you use this ''virG'' gene in a VirA/G signaling system you do not need <partinfo>K238010</partinfo> anymore to get the system working in ''E. coli''. <br />
<br />
You can find this BioBrick here: <partinfo>K389002</partinfo><br />
<br />
===''virB''-promoter===<br />
''Vir''-promoters from ''A. tumefaciens'' are induced by phosphorylated VirG response regulators and control genes for infecting plants in their natural host. They are part of the VirA/G signal transduction system. <br />
We wanted to use the ''vir''-promoter from the partsregistry (<partinfo>K238011</partinfo>) but the same problems occurred like with the use of the VirA receptor BioBrick from the partsregistry. So we also have to create a new ''vir''-promoter BioBrick (again from Ti-plasmid of ''A. tumefaciens'' C58).<br />
<br />
You can find this BioBrick here: <partinfo>K389003</partinfo><br />
<br />
===Firefly luciferase===<br />
Bringing the firefly luciferase gene from Promega's pGL4.10[luc2] vector into a BioBrick compatible form as a sensitive reporter gene. To amplify the signal of the luciferase three different sensitivity tuners are assembled before the luciferase gene. The sensitivity tuners were created in 2007 by the iGEM team from Cambridge and amplify the read-out signal. We also assembled the firefly luciferase behind three different strong constitutive promoters for gathering additional information about this reporter gene [http://parts.mit.edu/igem07/index.php/Cambridge/Amplifier_project#Results Cambridge, (2007)]. <br />
<br />
You can find this BioBrick here: <partinfo>K389004</partinfo><br />
<br />
You can find this BioBrick with sensitivity tuner 1 here: <partinfo>K389401</partinfo><br />
<br />
You can find this BioBrick with sensitivity tuner 2 here: <partinfo>K389402</partinfo><br />
<br />
You can find this BioBrick with sensitivity tuner 3 here: <partinfo>K389403</partinfo><br />
<br />
You can find this BioBrick under the control of a weak constitutive promoter here: <partinfo>K389302</partinfo><br />
<br />
You can find this BioBrick under the control of a medium strong constitutive promoter here: <partinfo>K389307</partinfo><br />
<br />
You can find this BioBrick under the control of a strong constitutive promoter here: <partinfo>K389318</partinfo><br />
<br />
===Neomycin / kanamycin resistance===<br />
A neomycin / kanamycin resistance gene without promoter is isolated and brought into a BioBrick compatible form. We will use the BioBrick <partinfo>P1003</partinfo> as source for the kanamycin resistance gene. <br />
<br />
You can find this BioBrick here: <partinfo>K389005</partinfo><br />
<br />
<br />
===BioBrick for ''virA''-screenings===<br />
[[Image:Screening_plasmid.jpg|500px|thumb|right|'''Fig. 2: The BioBrick <partinfo>K389011</partinfo> for ''virA'' screenings.''']]<br />
This part contains our mutated ''virG'' BioBrick under the control of a constitutive promoter (<partinfo>J23110</partinfo>) and an antibiotic resistance (<partinfo>K389005</partinfo>) under the control of the ''virB'' promoter (<partinfo>K389003</partinfo>). The better the VirA receptor recognizes a substance the stronger will the antibiotic resistance be expressed. <br />
<br />
This device is used to screen ''virA'' receptor mutants. The ''virA'' receptor gene is mutated in the <partinfo>BBa_K389010</partinfo> BioBrick. The better the receptor detects a substance, the more VirG is phosphorylated and the stronger the kanamycin resistance from this BioBrick is expressed. The screening system for a mutated ''virA'' gene contains this BioBrick in a plasmid with R6K ori and the BioBrick <partinfo>BBa_K389010</partinfo> in a plasmid with ColE1 ori. Both plasmids are transformed to and screened in ''E. coli'' EC100D because plasmids with R6K ori are only replicated in pir+ or pir116 ''E. coli'' strains. Once a receptor with high sensitivity for a screened substance is found, the plasmids are isolated and transformed to ''e.g. E. coli'' TOP10. Because the R6K ori does not work in this strain because it is not a pir+ / pir116 strain, it is easy to separate the mutated ''virA'' BioBrick from the screening plasmid.<br />
<br />
You can find this BioBrick here: <partinfo>K389011</partinfo><br />
<br />
<br />
===Reporter constructs===<br />
The reporter constructs are similar to the ''virA'' screening construct (compare fig. 2) but instead of the antibiotic resistance they carry a reporter gene. The amount of produced reporter shows the activity of the VirA receptor and the ''vir'' promoter, respectively. If the original ''vir'' promoter is too weak, we will use Cambridge's sensitivity tuners to increase the output signal of our biosensor ([http://parts.mit.edu/igem07/index.php/Cambridge/Amplifier_project#Results Cambridge, 2007]). <br />
<br />
You can find this BioBrick with luciferase read-out here: <partinfo>K389012</partinfo><br />
<br />
You can find this BioBrick with mRFP read-out here: <partinfo>K389013</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 1 here: <partinfo>K389411</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 2 here: <partinfo>K389412</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 3 here: <partinfo>K389413</partinfo><br />
<br />
<br />
===Characterization constructs===<br />
[[Image:Bielefeld_Vorgehen2.gif|400px|thumb|right|'''Fig. 3: Animation of the function of a complete characterization construct with sensitivity tuner (click to enlarge). In the system without sensitivity tuner the reporter gene is directly under the control of the ''vir'' promoter.''' ]]<br />
The characterization constructs are like the reporter constructs but a ''virA'' gene under the control of a constitutive promoter is assembled before them. These constructs are used to characterize the natural, unmutated VirA/G signaling system and the ''vir'' promoter, respectively. The natural system is induced with acetosyringone and the read-out is measured to determine the behaviour of the VirA/G signaling system. <br />
<br />
You can find this BioBrick with luciferase read-out here: <partinfo>K389015</partinfo><br />
<br />
You can find this BioBrick with mRFP read-out here: <partinfo>K389016</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 1 here: <partinfo>K389421</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 2 here: <partinfo>K389422</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 3 here: <partinfo>K389423</partinfo><br />
<br />
<br />
===VirA/G signaling system===<br />
A complete VirA/G signaling system without read-out was submitted. The system contains a ''virA'' and our mutated ''virG'' gene under the control of a constitutive promoter and a ''vir'' promoter without reporter gene. <br />
<br />
You can find this BioBrick here: <partinfo>K389017</partinfo><br />
<br />
=References=<br />
*[http://parts.mit.edu/igem07/index.php/Cambridge/Amplifier_project Cambridge iGEM Team wiki 2007, amplifier project]<br />
<br />
*YC Jung ''et al.'', (2004), ''Mutants of ''Agrobacterium tumefaciens virG'' Gene That Activate Transcription of ''vir'' Promoter in ''Escherichia coli'', Current Microbiol 49:334-340.</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389011Team:Bielefeld-Germany/Results/Characterization/K3890112010-10-28T00:59:52Z<p>Nkessler: /* Characterization of K389011 */</p>
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<br />
=Characterization of <partinfo>K389011</partinfo>=<br />
The induction of ''virA'' with acetosyringone was tested with different response devices. One of the read out systems was the <partinfo>BB_K389011</partinfo> with a kanamycin resistance cassette under control of the ''virG'' promoter. To measure the expression of the resistance gene, hundreds of colonies including <partinfo>BB_K389011</partinfo> and <partinfo>BB_K389010</partinfo> (''virA'') were transferred to agar plates with rising concentrations of kanamycin. The colonies which were able to grow on each plate were divided by the total number of colonies on a plate without kanamycin to calculate the survival rate &phi;. The ability of the bacterial population to withstand the antibiotic was determined without any inducing substance and in the presence of acetosyringone.<br />
The results of this experiment are summarized in the figure below. The two curves (induced and non-induced population) indicate a common tendency. Under rising concentrations of kanamycin (x-axis) the survival rate of each population (y-axis) first decreases slowly but drops dramatically at a certain concentration leading to the death of all bacteria. This minimal inhibitory concentration (MIC), which completely inhibits visible growth of a population, is higher in the curve of induced bacteria.<br />
<br />
<br />
[[Image:Bielefeld_LD50_Graph2.jpg|600px|thumb|center|Ratio of surviving colonies of ''E. coli'' EC100D carrying unmutated <partinfo>K389010</partinfo> and <partinfo>K389011</partinfo> plated on PA agar plates with chloramphenicol, ampicillin and different concentrations of kanamycin. Comparison between cells that were induced with acetosyringone with cells that were not induced.]]<br />
<br />
<br />
The interpretation of this results leads to two different conclusions.<br />
First, approximately 70 % of the uninduced bacteria can withstand common working concentrations of kanamycin (25 – 50 µg mL<sup>-1</sup>). This result shows that the whole receptor system seems to be not very tight regulated, causing a relatively high basal transcription. The observed leakiness might either be caused by the presence of transcription factors in ''E. coli'' other than VirG that can initiate the transcription at the ''virB'' promoter or by a significant activity of the VirA kinase domain without acetosyrigone.<br />
The second result of the determination of MIC of kanamycin is that the induction of VirA can be detected by the growth of colonies on agar plates with 100 µg mL<sup>-1</sup>. As indicated in the figure none of the uninduced bacteria could grow at this concentration, while more than 70 % of the induced population showed a sufficient expression of the resistance cassette.<br />
According to these results, we chose to use a kanamycin selection system during our directed mutagenesis. The idea was to select bacteria with mutated ''virA'' variants that can be activated by substances other than acetosyringone (e.g. capsaicin) by their growth on agar plates with 100 µg mL<sup>-1</sup> kanamycin.</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389015Team:Bielefeld-Germany/Results/Characterization/K3890152010-10-28T00:51:25Z<p>Nkessler: /* References */</p>
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<br />
=Characterization of <partinfo>K389015</partinfo>=<br />
<br />
On this page the experiments and results that lead to the <partinfo>K389015</partinfo> characterization data presented on our [[Team:Bielefeld-Germany/Results/Characterization | characterization page]] are shown in detail. <br />
<br />
<br />
== Growth functions and Luciferase expression for <partinfo>K389015</partinfo>==<br />
<br />
To characterize this part we performed several cultivations with different concentrations of [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] as inducer and measured the luminescence emitted by the luciferasereaction with Luciferin ([[#Protocols|Protocol]]). We used ''Escherichia coli'' DB3.1 carrying the pSB1C3::K389015 plasmid. Even without inducer the bacteria carrying the plasmid showed decelerated growth. In addition acetosyringone affected the growth rates (we used a stocksolution of 20 mM acetosyringone solved in 10 % (v/v) DMSO). Growth curves, averaged specific growth rates and doubling times are shown below. It can be observed, that ''E. coli'' carrying the pSB1C3::K389015 plasmid growths nearly linear. <br />
<br />
<br />
[[Image:K389015growth.jpg|600px|thumb|center|'''Fig. 1: Growth curves for ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389015</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.''']]<br />
<br />
<br />
The specific growth rates µ and doubling times t<sub>d</sub> are calculated with the OD<sub>600</sub> and following formulas:<br />
<br />
<br />
[[Image:Bielefeld_Specific_growth_rate.jpg|175px|center]] <div align="right">(1)</div><br />
<br />
<br />
[[Image:Bielefeld_Doubling_time.jpg|175px|center]] <div align="right">(2)</div><br />
<br />
<br />
<center><br />
Table 1: Averaged specific growth rates and doubling times for cultivations of ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389015</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| ''E. coli'' DB3.1<br />
!style="border-style: solid; border-width: 1px"| µ / h<sup>-1</sup><br />
!style="border-style: solid; border-width: 1px"| t<sub>d</sub> / h<br />
|-<br />
|style="border-style: solid; border-width: 1px"| without plasmid<br />
|style="border-style: solid; border-width: 1px"| 0.35<br />
|style="border-style: solid; border-width: 1px"| 1.98<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389015<br />
|style="border-style: solid; border-width: 1px"| 0.31<br />
|style="border-style: solid; border-width: 1px"| 2.24<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389015 with 400 µM acetosyringone<br />
|style="border-style: solid; border-width: 1px"| 0.26<br />
|style="border-style: solid; border-width: 1px"| 2.67<br />
|}<br />
<br />
</center><br />
<br />
<br />
Exemplary induction curves with the luminescence normalized to OD<sub>600</sub> are shown in Fig. 2. We observed a basal transcription, but the induction with acetosyringone is undoubtedly. The detailed [[#Data_Analysis | data analysis]] and [[#Transfer_function | transfer function]] is described below. <br />
<br />
<br />
[[Image:K389015induction.jpg|600px|thumb|center|'''Fig. 2: Induction curves for ''E. coli'' DB3.1 carrying <partinfo>K389015</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol. The relative luminescence units (RLU) are normalized to OD<sub>600</sub> and plotted against the cultivation time in h''']]<br />
<br />
==Transfer function==<br />
The data for the transfer function was measured and analyzed as [[Team:Bielefeld-Germany/Results/Characterization/K389015#Data analysis | described below]]. Nelson ''et al.'' suggests using a dose response function and fitting it with a logistical equation for the data analysis of receptor systems ([http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html Nelson ''et al.'', 2002]). The data was fitted with a function of the form<br />
<br />
<br />
[[Image:Bielefeld_Doseresponse_fit.jpg|175px|center]] <div align="right">(3)</div><br />
<br />
<br />
with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x<sub>0</sub>). Figure 3 shows the measured [[Team:Bielefeld-Germany/Results/Characterization/K389015#Data analysis | ratio ɸ<sub>I</sub>]] between induced (i) and uninduced (u) relative luminescence units (RLU) per OD<sub>600</sub> plotted against the logarithm of the concentration of the inductor [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] in µM. The fit has an R<sup>2</sup> = 0.98. <br />
<br />
<br />
[[Image:Bielefeld_Fit_Luc_final.jpg|600px|thumb|center|'''Fig. 3: Transfer function for the part <partinfo>K389015</partinfo> (R<sup>2</sup> = 0.98).''']]<br />
<br />
<br />
The important data from the transfer function is summarized in table 1: <br />
<br />
<center><br />
Table 2: Data from the transfer function for the part <partinfo>K389015</partinfo>.<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Parameter<br />
!style="border-style: solid; border-width: 1px"| Value<br />
|-<br />
|style="border-style: solid; border-width: 1px"| Hill coefficient<br />
|style="border-style: solid; border-width: 1px"| 1.092<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [http://partsregistry.org/Switch_Point Switch point]<br />
|style="border-style: solid; border-width: 1px"| 31.6 µM<br />
|-<br />
|style="border-style: solid; border-width: 1px"| Top asymptote<br />
|style="border-style: solid; border-width: 1px"| 2.16<br />
|}<br />
<br />
</center><br />
<br />
<br />
The fully induced VirA/G signaling system with luciferase readout has a 2.2 fold increased expression compared to the uninduced system. The Hill coefficient is > 1, so a positive cooperation can be observed ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 D Chu ''et al.'', 2009]). The [http://partsregistry.org/Switch_Point switch point] of the system is at about 32 µM, so this is the concentration at which the device output is 50% of the maximum output.<br />
<br />
==Response time==<br />
<br />
The system needs at least one hour to show a measurable reaction to an induction with acetosyringone. In the following illustration the reaction of the system to induction with 200 µM acetosyringone in the exponential growth phase is shown. For a good separation of the induced system from the uninduced system at least two hours are needed. <br />
<br />
[[Image:Bielefeld_Induktionsverhalten_luciferase.jpg|600px|center|thumb|'''Fig. 4: Cultivation of <partinfo>K389015</partinfo> in ''Escherichia coli'' DB3.1. Induction with 200 µM acetosyringone during the exponential growth phase.''']]<br />
<br />
==Data Analysis==<br />
Because the luciferase accumulation is very different in different cultivations, the uninduced negative control was used as internal standard. To show the behaviour of the VirA/G signaling system when induced, the ratio ɸ<sub>I</sub> between induced (i) and uninduced (u) relative luminescence units (RLU) per OD<sub>600</sub> is calculated: <br />
<br />
<br />
[[Image:Bielefeld_Quotient_RLU.jpg|200px|center]] <div align="right">(4)</div><br />
<br />
<br />
[[#Response time | As seen above]], at least one hour is needed to separate the induced luminescence signal from the uninduced, so ɸ<sub>I</sub> > 1. Within a cultivation ɸ<sub>I</sub> is rising during the first hours and is decreasing after it reached a maximum at OD<sub>600</sub> ~ 1. This is shown in figure 3: <br />
<br />
<br />
[[Image:Bielefeld_Luc_fit_ratio_to_OD.jpg|650px|thumb|center|'''Fig. 5A: Typical development of ɸ<sub>I</sub> plotted to OD<sub>600</sub> of a cultivation with <partinfo>K389015</partinfo> in ''E. coli'' DB3.1 with polynomial fit (5th order, R<sup>2</sup> = 0.75, induced with 200 µM acetosyringone). Fig. 5B: Another typical development of ɸ<sub>I</sub> plotted to OD<sub>600</sub> of a cultivation with <partinfo>K389015</partinfo> in ''E. coli'' DB3.1 with polynomial fit (5th order, R<sup>2</sup> = 0.87, induced with 400 µM acetosyringone).''']]<br />
<br />
<br />
To measure the ratio of increasing promoter activity by inducing the system ɸ<sub>I</sub> samples for analyzation should be taken at OD<sub>600</sub> = 1 +/- 0.5. The highest ɸ<sub>I</sub> in this range of the cultivation is taken for the calculation of the [[#Transfer function | transfer function]].<br />
<br />
==Plasmid conformation analysis==<br />
A plasmid conformation analysis for the BioBrick <partinfo>K389015</partinfo> in <partinfo>pSB1C3</partinfo> was performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] by Capillary Gel Electrophoresis (CGE). The chromatogram is shown in fig. 6 and the results in tab. 3. The data shows a high percentage of covalently closed circular (ccc) plasmid DNA. This is the biological active shape of plasmids so a high percentage of ccc plasmid DNA indicates a high quality of plasmid DNA ([http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory]). <br />
<br />
<br />
[[Image:Bielefeld_CGE_K389015.jpg|600px|thumb|center|'''Fig. 6: Chromatogram of the CGE of the BioBrick <partinfo>K389015</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).''']]<br />
<br />
<br />
<center><br />
Table 3: Data from the CGE of the BioBrick <partinfo>K389015</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Conformation<br />
!style="border-style: solid; border-width: 1px"| Ratio / %<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ccc monomer<br />
|style="border-style: solid; border-width: 1px"| 91<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ccc dimer<br />
|style="border-style: solid; border-width: 1px"| 3.7<br />
|-<br />
|style="border-style: solid; border-width: 1px"| oc<br />
|style="border-style: solid; border-width: 1px"| 5.3<br />
|}<br />
<br />
</center><br />
<br />
<br />
=References=<br />
*Chu D, Zabet NR, Mitavskiy B (2009) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 Models of transcription factor binding: Sensitivity of activation functions to model assumptions], ''J Theor Biol'' 257(3):419-429. <br />
<br />
*Greg Nelson, Jayaram Chandrashekar, Mark A. Hoon, Luxin Feng, Grace Zhao, Nicholas J. P. Ryba & Charles S. Zuker (2002) ''[http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html An amino-acid taste receptor ]'', Nature 416: 199-202. <br />
<br />
*[http://www.promega.com/tbs/tb281/tb281.pdf Promega Luciferase Assay System]<br />
<br />
*[http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory Homepage]</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389015Team:Bielefeld-Germany/Results/Characterization/K3890152010-10-28T00:45:32Z<p>Nkessler: /* Transfer function */</p>
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=Characterization of <partinfo>K389015</partinfo>=<br />
<br />
On this page the experiments and results that lead to the <partinfo>K389015</partinfo> characterization data presented on our [[Team:Bielefeld-Germany/Results/Characterization | characterization page]] are shown in detail. <br />
<br />
<br />
== Growth functions and Luciferase expression for <partinfo>K389015</partinfo>==<br />
<br />
To characterize this part we performed several cultivations with different concentrations of [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] as inducer and measured the luminescence emitted by the luciferasereaction with Luciferin ([[#Protocols|Protocol]]). We used ''Escherichia coli'' DB3.1 carrying the pSB1C3::K389015 plasmid. Even without inducer the bacteria carrying the plasmid showed decelerated growth. In addition acetosyringone affected the growth rates (we used a stocksolution of 20 mM acetosyringone solved in 10 % (v/v) DMSO). Growth curves, averaged specific growth rates and doubling times are shown below. It can be observed, that ''E. coli'' carrying the pSB1C3::K389015 plasmid growths nearly linear. <br />
<br />
<br />
[[Image:K389015growth.jpg|600px|thumb|center|'''Fig. 1: Growth curves for ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389015</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.''']]<br />
<br />
<br />
The specific growth rates µ and doubling times t<sub>d</sub> are calculated with the OD<sub>600</sub> and following formulas:<br />
<br />
<br />
[[Image:Bielefeld_Specific_growth_rate.jpg|175px|center]] <div align="right">(1)</div><br />
<br />
<br />
[[Image:Bielefeld_Doubling_time.jpg|175px|center]] <div align="right">(2)</div><br />
<br />
<br />
<center><br />
Table 1: Averaged specific growth rates and doubling times for cultivations of ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389015</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| ''E. coli'' DB3.1<br />
!style="border-style: solid; border-width: 1px"| µ / h<sup>-1</sup><br />
!style="border-style: solid; border-width: 1px"| t<sub>d</sub> / h<br />
|-<br />
|style="border-style: solid; border-width: 1px"| without plasmid<br />
|style="border-style: solid; border-width: 1px"| 0.35<br />
|style="border-style: solid; border-width: 1px"| 1.98<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389015<br />
|style="border-style: solid; border-width: 1px"| 0.31<br />
|style="border-style: solid; border-width: 1px"| 2.24<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389015 with 400 µM acetosyringone<br />
|style="border-style: solid; border-width: 1px"| 0.26<br />
|style="border-style: solid; border-width: 1px"| 2.67<br />
|}<br />
<br />
</center><br />
<br />
<br />
Exemplary induction curves with the luminescence normalized to OD<sub>600</sub> are shown in Fig. 2. We observed a basal transcription, but the induction with acetosyringone is undoubtedly. The detailed [[#Data_Analysis | data analysis]] and [[#Transfer_function | transfer function]] is described below. <br />
<br />
<br />
[[Image:K389015induction.jpg|600px|thumb|center|'''Fig. 2: Induction curves for ''E. coli'' DB3.1 carrying <partinfo>K389015</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol. The relative luminescence units (RLU) are normalized to OD<sub>600</sub> and plotted against the cultivation time in h''']]<br />
<br />
==Transfer function==<br />
The data for the transfer function was measured and analyzed as [[Team:Bielefeld-Germany/Results/Characterization/K389015#Data analysis | described below]]. Nelson ''et al.'' suggests using a dose response function and fitting it with a logistical equation for the data analysis of receptor systems ([http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html Nelson ''et al.'', 2002]). The data was fitted with a function of the form<br />
<br />
<br />
[[Image:Bielefeld_Doseresponse_fit.jpg|175px|center]] <div align="right">(3)</div><br />
<br />
<br />
with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x<sub>0</sub>). Figure 3 shows the measured [[Team:Bielefeld-Germany/Results/Characterization/K389015#Data analysis | ratio ɸ<sub>I</sub>]] between induced (i) and uninduced (u) relative luminescence units (RLU) per OD<sub>600</sub> plotted against the logarithm of the concentration of the inductor [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] in µM. The fit has an R<sup>2</sup> = 0.98. <br />
<br />
<br />
[[Image:Bielefeld_Fit_Luc_final.jpg|600px|thumb|center|'''Fig. 3: Transfer function for the part <partinfo>K389015</partinfo> (R<sup>2</sup> = 0.98).''']]<br />
<br />
<br />
The important data from the transfer function is summarized in table 1: <br />
<br />
<center><br />
Table 2: Data from the transfer function for the part <partinfo>K389015</partinfo>.<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Parameter<br />
!style="border-style: solid; border-width: 1px"| Value<br />
|-<br />
|style="border-style: solid; border-width: 1px"| Hill coefficient<br />
|style="border-style: solid; border-width: 1px"| 1.092<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [http://partsregistry.org/Switch_Point Switch point]<br />
|style="border-style: solid; border-width: 1px"| 31.6 µM<br />
|-<br />
|style="border-style: solid; border-width: 1px"| Top asymptote<br />
|style="border-style: solid; border-width: 1px"| 2.16<br />
|}<br />
<br />
</center><br />
<br />
<br />
The fully induced VirA/G signaling system with luciferase readout has a 2.2 fold increased expression compared to the uninduced system. The Hill coefficient is > 1, so a positive cooperation can be observed ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 D Chu ''et al.'', 2009]). The [http://partsregistry.org/Switch_Point switch point] of the system is at about 32 µM, so this is the concentration at which the device output is 50% of the maximum output.<br />
<br />
==Response time==<br />
<br />
The system needs at least one hour to show a measurable reaction to an induction with acetosyringone. In the following illustration the reaction of the system to induction with 200 µM acetosyringone in the exponential growth phase is shown. For a good separation of the induced system from the uninduced system at least two hours are needed. <br />
<br />
[[Image:Bielefeld_Induktionsverhalten_luciferase.jpg|600px|center|thumb|'''Fig. 4: Cultivation of <partinfo>K389015</partinfo> in ''Escherichia coli'' DB3.1. Induction with 200 µM acetosyringone during the exponential growth phase.''']]<br />
<br />
==Data Analysis==<br />
Because the luciferase accumulation is very different in different cultivations, the uninduced negative control was used as internal standard. To show the behaviour of the VirA/G signaling system when induced, the ratio ɸ<sub>I</sub> between induced (i) and uninduced (u) relative luminescence units (RLU) per OD<sub>600</sub> is calculated: <br />
<br />
<br />
[[Image:Bielefeld_Quotient_RLU.jpg|200px|center]] <div align="right">(4)</div><br />
<br />
<br />
[[#Response time | As seen above]], at least one hour is needed to separate the induced luminescence signal from the uninduced, so ɸ<sub>I</sub> > 1. Within a cultivation ɸ<sub>I</sub> is rising during the first hours and is decreasing after it reached a maximum at OD<sub>600</sub> ~ 1. This is shown in figure 3: <br />
<br />
<br />
[[Image:Bielefeld_Luc_fit_ratio_to_OD.jpg|650px|thumb|center|'''Fig. 5A: Typical development of ɸ<sub>I</sub> plotted to OD<sub>600</sub> of a cultivation with <partinfo>K389015</partinfo> in ''E. coli'' DB3.1 with polynomial fit (5th order, R<sup>2</sup> = 0.75, induced with 200 µM acetosyringone). Fig. 5B: Another typical development of ɸ<sub>I</sub> plotted to OD<sub>600</sub> of a cultivation with <partinfo>K389015</partinfo> in ''E. coli'' DB3.1 with polynomial fit (5th order, R<sup>2</sup> = 0.87, induced with 400 µM acetosyringone).''']]<br />
<br />
<br />
To measure the ratio of increasing promoter activity by inducing the system ɸ<sub>I</sub> samples for analyzation should be taken at OD<sub>600</sub> = 1 +/- 0.5. The highest ɸ<sub>I</sub> in this range of the cultivation is taken for the calculation of the [[#Transfer function | transfer function]].<br />
<br />
==Plasmid conformation analysis==<br />
A plasmid conformation analysis for the BioBrick <partinfo>K389015</partinfo> in <partinfo>pSB1C3</partinfo> was performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] by Capillary Gel Electrophoresis (CGE). The chromatogram is shown in fig. 6 and the results in tab. 3. The data shows a high percentage of covalently closed circular (ccc) plasmid DNA. This is the biological active shape of plasmids so a high percentage of ccc plasmid DNA indicates a high quality of plasmid DNA ([http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory]). <br />
<br />
<br />
[[Image:Bielefeld_CGE_K389015.jpg|600px|thumb|center|'''Fig. 6: Chromatogram of the CGE of the BioBrick <partinfo>K389015</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).''']]<br />
<br />
<br />
<center><br />
Table 3: Data from the CGE of the BioBrick <partinfo>K389015</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Conformation<br />
!style="border-style: solid; border-width: 1px"| Ratio / %<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ccc monomer<br />
|style="border-style: solid; border-width: 1px"| 91<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ccc dimer<br />
|style="border-style: solid; border-width: 1px"| 3.7<br />
|-<br />
|style="border-style: solid; border-width: 1px"| oc<br />
|style="border-style: solid; border-width: 1px"| 5.3<br />
|}<br />
<br />
</center><br />
<br />
<br />
=References=<br />
Chu D, Zabet NR, Mitavskiy B (2009) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 Models of transcription factor binding: Sensitivity of activation functions to model assumptions], ''J Theor Biol'' 257(3):419-429. <br />
<br />
Greg Nelson, Jayaram Chandrashekar, Mark A. Hoon, Luxin Feng, Grace Zhao, Nicholas J. P. Ryba & Charles S. Zuker (2002) ''[http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html An amino-acid taste receptor ]'', Nature 416: 199-202. <br />
<br />
[http://www.promega.com/tbs/tb281/tb281.pdf Promega Luciferase Assay System]<br />
<br />
[http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory Homepage]</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389015Team:Bielefeld-Germany/Results/Characterization/K3890152010-10-28T00:41:24Z<p>Nkessler: /* Growth functions and Luciferase expression for K389015 */</p>
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<br />
=Characterization of <partinfo>K389015</partinfo>=<br />
<br />
On this page the experiments and results that lead to the <partinfo>K389015</partinfo> characterization data presented on our [[Team:Bielefeld-Germany/Results/Characterization | characterization page]] are shown in detail. <br />
<br />
<br />
== Growth functions and Luciferase expression for <partinfo>K389015</partinfo>==<br />
<br />
To characterize this part we performed several cultivations with different concentrations of [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] as inducer and measured the luminescence emitted by the luciferasereaction with Luciferin ([[#Protocols|Protocol]]). We used ''Escherichia coli'' DB3.1 carrying the pSB1C3::K389015 plasmid. Even without inducer the bacteria carrying the plasmid showed decelerated growth. In addition acetosyringone affected the growth rates (we used a stocksolution of 20 mM acetosyringone solved in 10 % (v/v) DMSO). Growth curves, averaged specific growth rates and doubling times are shown below. It can be observed, that ''E. coli'' carrying the pSB1C3::K389015 plasmid growths nearly linear. <br />
<br />
<br />
[[Image:K389015growth.jpg|600px|thumb|center|'''Fig. 1: Growth curves for ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389015</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.''']]<br />
<br />
<br />
The specific growth rates µ and doubling times t<sub>d</sub> are calculated with the OD<sub>600</sub> and following formulas:<br />
<br />
<br />
[[Image:Bielefeld_Specific_growth_rate.jpg|175px|center]] <div align="right">(1)</div><br />
<br />
<br />
[[Image:Bielefeld_Doubling_time.jpg|175px|center]] <div align="right">(2)</div><br />
<br />
<br />
<center><br />
Table 1: Averaged specific growth rates and doubling times for cultivations of ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389015</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| ''E. coli'' DB3.1<br />
!style="border-style: solid; border-width: 1px"| µ / h<sup>-1</sup><br />
!style="border-style: solid; border-width: 1px"| t<sub>d</sub> / h<br />
|-<br />
|style="border-style: solid; border-width: 1px"| without plasmid<br />
|style="border-style: solid; border-width: 1px"| 0.35<br />
|style="border-style: solid; border-width: 1px"| 1.98<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389015<br />
|style="border-style: solid; border-width: 1px"| 0.31<br />
|style="border-style: solid; border-width: 1px"| 2.24<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389015 with 400 µM acetosyringone<br />
|style="border-style: solid; border-width: 1px"| 0.26<br />
|style="border-style: solid; border-width: 1px"| 2.67<br />
|}<br />
<br />
</center><br />
<br />
<br />
Exemplary induction curves with the luminescence normalized to OD<sub>600</sub> are shown in Fig. 2. We observed a basal transcription, but the induction with acetosyringone is undoubtedly. The detailed [[#Data_Analysis | data analysis]] and [[#Transfer_function | transfer function]] is described below. <br />
<br />
<br />
[[Image:K389015induction.jpg|600px|thumb|center|'''Fig. 2: Induction curves for ''E. coli'' DB3.1 carrying <partinfo>K389015</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol. The relative luminescence units (RLU) are normalized to OD<sub>600</sub> and plotted against the cultivation time in h''']]<br />
<br />
==Transfer function==<br />
The data for the transfer function was measured and analyzed as [[Team:Bielefeld-Germany/Results/Characterization/K389015#Data analysis | described below]]. Nelson ''et al'' suggests using a dose response function and fitting it with a logistical equation for the data analysis of receptor systems ([http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html Nelson ''et al.'', 2002]). The data was fitted with a function of the form<br />
<br />
<br />
[[Image:Bielefeld_Doseresponse_fit.jpg|175px|center]] <div align="right">(3)</div><br />
<br />
<br />
with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x<sub>0</sub>). Figure 3 shows the measured [[Team:Bielefeld-Germany/Results/Characterization/K389015#Data analysis | ratio ɸ<sub>I</sub>]] between induced (i) and uninduced (u) relative luminescence units (RLU) per OD<sub>600</sub> plotted against the logarithm of the concentration of the inductor [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] in µM. The fit has an R<sup>2</sup> = 0.98. <br />
<br />
<br />
[[Image:Bielefeld_Fit_Luc_final.jpg|600px|thumb|center|'''Fig. 3: Transfer function for the part <partinfo>K389015</partinfo> (R<sup>2</sup> = 0.98).''']]<br />
<br />
<br />
The important data from the transfer function is summarized in table 1: <br />
<br />
<center><br />
Table 2: Data from the transfer function for the part <partinfo>K389015</partinfo>.<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Parameter<br />
!style="border-style: solid; border-width: 1px"| Value<br />
|-<br />
|style="border-style: solid; border-width: 1px"| Hill coefficient<br />
|style="border-style: solid; border-width: 1px"| 1.092<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [http://partsregistry.org/Switch_Point Switch point]<br />
|style="border-style: solid; border-width: 1px"| 31.6 µM<br />
|-<br />
|style="border-style: solid; border-width: 1px"| Top asymptote<br />
|style="border-style: solid; border-width: 1px"| 2.16<br />
|}<br />
<br />
</center><br />
<br />
<br />
The fully induced VirA/G signaling system with luciferase read-out has a 2.2 fold increased expression compared to the uninduced system. The Hill coefficient is > 1, so a positive cooperation can be observed ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 D Chu ''et al.'', 2009]). The [http://partsregistry.org/Switch_Point switch point] of the system is at about 32 µM, so this is the concentration at which the device output is 50% of the maximum output.<br />
<br />
==Response time==<br />
<br />
The system needs at least one hour to show a measurable reaction to an induction with acetosyringone. In the following illustration the reaction of the system to induction with 200 µM acetosyringone in the exponential growth phase is shown. For a good separation of the induced system from the uninduced system at least two hours are needed. <br />
<br />
[[Image:Bielefeld_Induktionsverhalten_luciferase.jpg|600px|center|thumb|'''Fig. 4: Cultivation of <partinfo>K389015</partinfo> in ''Escherichia coli'' DB3.1. Induction with 200 µM acetosyringone during the exponential growth phase.''']]<br />
<br />
==Data Analysis==<br />
Because the luciferase accumulation is very different in different cultivations, the uninduced negative control was used as internal standard. To show the behaviour of the VirA/G signaling system when induced, the ratio ɸ<sub>I</sub> between induced (i) and uninduced (u) relative luminescence units (RLU) per OD<sub>600</sub> is calculated: <br />
<br />
<br />
[[Image:Bielefeld_Quotient_RLU.jpg|200px|center]] <div align="right">(4)</div><br />
<br />
<br />
[[#Response time | As seen above]], at least one hour is needed to separate the induced luminescence signal from the uninduced, so ɸ<sub>I</sub> > 1. Within a cultivation ɸ<sub>I</sub> is rising during the first hours and is decreasing after it reached a maximum at OD<sub>600</sub> ~ 1. This is shown in figure 3: <br />
<br />
<br />
[[Image:Bielefeld_Luc_fit_ratio_to_OD.jpg|650px|thumb|center|'''Fig. 5A: Typical development of ɸ<sub>I</sub> plotted to OD<sub>600</sub> of a cultivation with <partinfo>K389015</partinfo> in ''E. coli'' DB3.1 with polynomial fit (5th order, R<sup>2</sup> = 0.75, induced with 200 µM acetosyringone). Fig. 5B: Another typical development of ɸ<sub>I</sub> plotted to OD<sub>600</sub> of a cultivation with <partinfo>K389015</partinfo> in ''E. coli'' DB3.1 with polynomial fit (5th order, R<sup>2</sup> = 0.87, induced with 400 µM acetosyringone).''']]<br />
<br />
<br />
To measure the ratio of increasing promoter activity by inducing the system ɸ<sub>I</sub> samples for analyzation should be taken at OD<sub>600</sub> = 1 +/- 0.5. The highest ɸ<sub>I</sub> in this range of the cultivation is taken for the calculation of the [[#Transfer function | transfer function]].<br />
<br />
==Plasmid conformation analysis==<br />
A plasmid conformation analysis for the BioBrick <partinfo>K389015</partinfo> in <partinfo>pSB1C3</partinfo> was performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] by Capillary Gel Electrophoresis (CGE). The chromatogram is shown in fig. 6 and the results in tab. 3. The data shows a high percentage of covalently closed circular (ccc) plasmid DNA. This is the biological active shape of plasmids so a high percentage of ccc plasmid DNA indicates a high quality of plasmid DNA ([http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory]). <br />
<br />
<br />
[[Image:Bielefeld_CGE_K389015.jpg|600px|thumb|center|'''Fig. 6: Chromatogram of the CGE of the BioBrick <partinfo>K389015</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).''']]<br />
<br />
<br />
<center><br />
Table 3: Data from the CGE of the BioBrick <partinfo>K389015</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Conformation<br />
!style="border-style: solid; border-width: 1px"| Ratio / %<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ccc monomer<br />
|style="border-style: solid; border-width: 1px"| 91<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ccc dimer<br />
|style="border-style: solid; border-width: 1px"| 3.7<br />
|-<br />
|style="border-style: solid; border-width: 1px"| oc<br />
|style="border-style: solid; border-width: 1px"| 5.3<br />
|}<br />
<br />
</center><br />
<br />
<br />
=References=<br />
Chu D, Zabet NR, Mitavskiy B (2009) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 Models of transcription factor binding: Sensitivity of activation functions to model assumptions], ''J Theor Biol'' 257(3):419-429. <br />
<br />
Greg Nelson, Jayaram Chandrashekar, Mark A. Hoon, Luxin Feng, Grace Zhao, Nicholas J. P. Ryba & Charles S. Zuker (2002) ''[http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html An amino-acid taste receptor ]'', Nature 416: 199-202. <br />
<br />
[http://www.promega.com/tbs/tb281/tb281.pdf Promega Luciferase Assay System]<br />
<br />
[http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory Homepage]</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389016Team:Bielefeld-Germany/Results/Characterization/K3890162010-10-28T00:38:26Z<p>Nkessler: /* References */</p>
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<li><a href="/Team:Bielefeld-Germany/Results">Results</a></li><br />
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<br />
=Characterization of <partinfo>K389016</partinfo>=<br />
<br />
On this page the experiments and results that lead to the <partinfo>K389016</partinfo> characterization data presented on our [[Team:Bielefeld-Germany/Results/Characterization | characterization page]] are shown in detail. <br />
<br />
== Growth functions and mRFP expression for <partinfo>K389016</partinfo>==<br />
<br />
To characterize this part we performed several cultivations with different concentrations of acetosyringone as inducer and measured the fluorescence emitted by mRFP ([[Team:Bielefeld-Germany/Project/Protocols#Measuring of mRFP | Protocol]]). We used ''Escherichia Coli'' DB3.1 carrying the pSB1C3::K389016 plasmid. Even without inducer the bacteria carrying the plasmid showed decelerated growth. In addition acetosyringone affected the growth rates (we used a stock solution of 20 mM acetosyringone solved in 10 % (v/v) DMSO). Growth curves, averaged specific growth rates and doubling times are shown below. It can be observed, that ''E. coli'' carrying the pSB1C3::K389016 plasmid growths nearly linear. <br />
<br />
<br />
[[Image:K389016growth.jpg|600px|thumb|center|'''Fig. 1: Growth curves for ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.''']]<br />
<br />
<br />
The specific growth rates µ and doubling times t<sub>d</sub> are calculated with the OD<sub>600</sub> and following formulas:<br />
<br />
[[Image:Bielefeld_Specific_growth_rate.jpg|175px|center]] <div align="right">(1)</div><br />
<br />
<br />
[[Image:Bielefeld_Doubling_time.jpg|175px|center]] <div align="right">(2)</div><br />
<br />
<br />
<center>Table 1: Averaged specific growth rates and doubling times for cultivations of ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| ''E. coli'' DB3.1<br />
!style="border-style: solid; border-width: 1px"| µ / h<sup>-1</sup><br />
!style="border-style: solid; border-width: 1px"| t<sub>d</sub> / h<br />
|-<br />
|style="border-style: solid; border-width: 1px"| without plasmid<br />
|style="border-style: solid; border-width: 1px"| 0.35<br />
|style="border-style: solid; border-width: 1px"| 1.98<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389016<br />
|style="border-style: solid; border-width: 1px"| 0.27<br />
|style="border-style: solid; border-width: 1px"| 2.57<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389016 with 150 µM acetosyringone<br />
|style="border-style: solid; border-width: 1px"| 0.25<br />
|style="border-style: solid; border-width: 1px"| 2.77<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389016 with 1000 µM acetosyringone<br />
|style="border-style: solid; border-width: 1px"| 0.23<br />
|style="border-style: solid; border-width: 1px"| 3.01<br />
|}<br />
<br />
</center><br />
<br />
<br />
Exemplary induction curves with the fluorescence normalized to OD<sub>600</sub> are shown in Fig.2. We observed a basal transcription, but the induction with acetosyringone is undoubtedly. The detailed [[#Data analysis for BBa_K389016 | data analysis]] and [[#Transfer function of BBa_K389016 | transfer function]] is described below. <br />
<br />
<br />
[[Image:K389016induction.jpg|600px|thumb|center|'''Fig. 2: Induction Curves for ''E. coli'' DB3.1 carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol. The relative fluorescence units are normalized to OD<sub>600</sub> and plotted against the ime in h''']]<br />
<br />
==Transfer function of <partinfo>K389016</partinfo>==<br />
The data for the transfer function was measured and analyzed as [[Team:Bielefeld-Germany/Results/Characterization/K389016#Data analysis for BBa_K389016 | described below]]. [http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html Nelson ''et al.'' (2002)] suggest using a dose response function and fitting it with a logistical equation for the data analysis of receptor systems. The data was fitted with a function of the form<br />
<br />
<br />
[[Image:Bielefeld_Doseresponse_fit.jpg|175px|center]] <div align="right">(3)</div><br />
<br />
<br />
with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x<sub>0</sub>). Figure 3 shows the measured normalized specific production rates q<sub>P,n</sub> (eq. 8) plotted against the logarithm of the concentration of the inductor [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] in µM. The fit has an R<sup>2</sup> = 0.99. <br />
<br />
<br />
[[Image:Bielefeld_Final_RFP_fit.jpg|600px|thumb|center|'''Fig. 3: Transfer function for the part <partinfo>K389016</partinfo> (R<sup>2</sup> = 0.99).''']]<br />
<br />
<br />
The important data from the transfer function is summarized in table 2: <br />
<br />
<br />
<center>Table 2: Data from the transfer function for the part <partinfo>K389016</partinfo>.<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Parameter<br />
!style="border-style: solid; border-width: 1px"| Value<br />
|-<br />
|style="border-style: solid; border-width: 1px"| Hill coefficient<br />
|style="border-style: solid; border-width: 1px"| 1.673<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [http://partsregistry.org/Switch_Point Switch point]<br />
|style="border-style: solid; border-width: 1px"| 26.5 µM<br />
|-<br />
|style="border-style: solid; border-width: 1px"| Top asymptote<br />
|style="border-style: solid; border-width: 1px"| 2.62<br />
|}<br />
<br />
</center><br />
<br />
<br />
So the fully induced VirA/G signaling system has a 2.6 fold increased expression compared to the uninduced system. The Hill coefficient is > 1, so a positive cooperation can be observed ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 D Chu ''et al.'', 2009]). The [http://partsregistry.org/Switch_Point switch point] of the system is at about 25 µM, so this is the concentration at which the device output is 50% of the maximum output.<br />
<br />
==Data analysis for <partinfo>K389016</partinfo>==<br />
The data analysis is made in three steps. First step is the processing of the fluorescence raw data gained by the fluorescence plate reader for every sample: <br />
<br />
<br />
[[Image:Bielefeld_RFU_corrected.jpg|250px|center]] <div align="right">(4)</div><br />
<br />
<br />
In the second step the RFU<sub>corrected</sub> of every sample is plotted against the cultivation time it was drawn. The data is fitted by an exponential fit of the following style: <br />
<br />
<br />
[[Image:Bielefeld_Expfit.jpg|100px|center]] <div align="right">(5)</div><br />
<br />
<br />
The accumulation of mRFP in the cells is always exponential. A typical fitted product accumulation curve is shown below: <br />
<br />
<br />
[[Image:Bielefeld_ExpFit_auf_RFU.jpg|500px|thumb|center|'''Fig. 4: Exponential fit on the measured RFU plotted against cultivation time of a cultivation of <partinfo>K389016</partinfo> in ''Escherichia coli'' DB3.1 in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol and 150 µM acetosyringone. ''']]<br />
<br />
<br />
The product accumulation in a cultivation can be described as: <br />
<br />
<br />
[[Image:Bielefeld_Produktbildung.jpg|100px|center]] <div align="right">(6)</div><br />
<br />
<br />
with the amount of product P, the cell count X and the specific production rate q<sub>P</sub>. <br />
<br />
RFU is commensurate to the concentration of mRFP (P) and the OD<sub>600</sub> is commensurate to the cell count (X) ([http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Canton and Labno, 2004]): <br />
<br />
<br />
[[Image:Bielefeld_RFUpropP.jpg|100px|center]] <div align="right">(7)</div><br />
<br />
[[Image:Bielefeld_ODpropX.jpg|80px|center]] <div align="right">(8)</div><br />
<br />
<br />
With these assumptions it is possible to calculate the specific production rate of mRFP q<sub>P</sub> in the third step: the specific production rate for every sample of a cultivation is calculated by the derivation of the exponential fit line which describes the accumulation of product in the culture (dRFU/dt) and the measured OD<sub>600</sub> data: <br />
<br />
<br />
[[Image:Bielefeld_specific_production_rate.jpg|150px|center]] <div align="right">(9)</div><br />
<br />
<br />
The specific production rates q<sub>P</sub> of all samples of all cultivations made with a specific inductor concentration c are averaged and normalized against the specific production rate of the uninduced system q<sub>P,0</sub>: <br />
<br />
<br />
[[Image:Bielefeld_QPN.jpg|100px|center]] <div align="right">(10)</div><br />
<br />
<br />
This normalized specific production rate we calculated is commensurate to relative promotor units (RPU) which is commensurate to PoPS (polymerase per seconds) ([http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Canton and Labno, 2004]; [http://partsregistry.org/Part:BBa_J23101:Experience Pasotti ''et al.'', 2009]): <br />
<br />
<br />
[[Image:Bielefeld_QPNRPUPoPS.jpg|150px|center]] <div align="right">(11)</div><br />
<br />
==Plasmid conformation analysis==<br />
A plasmid conformation analysis for the BioBrick <partinfo>K389016</partinfo> in <partinfo>pSB1C3</partinfo> was performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] by Capillary Gel Electrophoresis (CGE). The chromatogram is shown in fig. 5 and the results in tab. 3. The data shows a high percentage of covalently closed circular (ccc) plasmid DNA. This is the biological active shape of plasmids so a high percentage of ccc plasmid DNA indicates a high quality of plasmid DNA ([http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory]). <br />
<br />
<br />
[[Image:Bielefeld_CGE_K389016_2.jpg|600px|thumb|center|'''Fig. 5: Chromatogram of the CGE of the BioBrick <partinfo>K389016</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).''']]<br />
<br />
<br />
<br />
<center> Table 3: Data from the CGE of the BioBrick <partinfo>K389016</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Conformation<br />
!style="border-style: solid; border-width: 1px"| Ratio / %<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ccc monomer<br />
|style="border-style: solid; border-width: 1px"| 91.2<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ccc dimer<br />
|style="border-style: solid; border-width: 1px"| 3.2<br />
|-<br />
|style="border-style: solid; border-width: 1px"| oc<br />
|style="border-style: solid; border-width: 1px"| 5.6<br />
|}<br />
<br />
</center><br />
<br />
==Different possible inducers==<br />
A list of tested possible inducers for a VirA/G signaling system is shown in tab. 4. These inducers where tested as a mix. The specific production rate of mRFP q<sub>P</sub> measured as described [[Team:Bielefeld-Germany/Results/Characterization/K389016#Data analysis for BBa_K389016 | above]] for the mix did not significantly differ from the synthesis rate of the uninduced system (t-Test, p < 0.005). So none of the possible inducers listet in tab. 4 induce the VirA/G signaling system significantly in the measured concentration range. In tab. 4 the chemical structures of the testet possible inducers are shown, too. Acetosyringone is also in tab. 4 although it was not testet in the inducer mix to show the chemical similarity of the tested possible inducers to the natural inducer of the VirA/G signaling system. <br />
<br />
[[Team:Bielefeld-Germany/Project/Outlook | For more information about the possible inducers click here. ]]<br />
<br />
<br />
<br />
<center> Table 4: Tested possible inducers for a VirA/G signaling system and acetosyringone with concentrations and chemical structure that were tested. <br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Inducer<br />
!style="border-style: solid; border-width: 1px"| Concentration / µM<br />
!style="border-style: solid; border-width: 1px"| Chemical structure<br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Capsaicin'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"|<html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1548942" width="200px" /></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Dopamine'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=681" width="200"></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Homovanillic acid'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1738" width="200"></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''3-Methoxytyramine'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=11957621" width="200"></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Acetosyringone'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=17198" width="200px" /></html><br />
|}<br />
<br />
</center><br />
<br />
=References=<br />
*Canton B and Labno A (2004) [http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Data processing of Part BBa_F2620]. <br />
<br />
*Chu D, Zabet NR, Mitavskiy B (2009) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 Models of transcription factor binding: Sensitivity of activation functions to model assumptions], ''J Theor Biol'' 257(3):419-429. <br />
<br />
*Greg Nelson, Jayaram Chandrashekar, Mark A. Hoon, Luxin Feng, Grace Zhao, Nicholas J. P. Ryba & Charles S. Zuker (2002) ''[http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html An amino-acid taste receptor ]'', Nature 416: 199-202. <br />
<br />
*Pasotti L, Zucca S, Del Fabbro E (2009) Characterization experiment on BBa_J23100, BBa_J23101, BBa_J23118, http://partsregistry.org/Part:BBa_J23101:Experience.<br />
<br />
*[http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory]</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389016Team:Bielefeld-Germany/Results/Characterization/K3890162010-10-28T00:35:28Z<p>Nkessler: /* Different possible inducers */</p>
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=Characterization of <partinfo>K389016</partinfo>=<br />
<br />
On this page the experiments and results that lead to the <partinfo>K389016</partinfo> characterization data presented on our [[Team:Bielefeld-Germany/Results/Characterization | characterization page]] are shown in detail. <br />
<br />
== Growth functions and mRFP expression for <partinfo>K389016</partinfo>==<br />
<br />
To characterize this part we performed several cultivations with different concentrations of acetosyringone as inducer and measured the fluorescence emitted by mRFP ([[Team:Bielefeld-Germany/Project/Protocols#Measuring of mRFP | Protocol]]). We used ''Escherichia Coli'' DB3.1 carrying the pSB1C3::K389016 plasmid. Even without inducer the bacteria carrying the plasmid showed decelerated growth. In addition acetosyringone affected the growth rates (we used a stock solution of 20 mM acetosyringone solved in 10 % (v/v) DMSO). Growth curves, averaged specific growth rates and doubling times are shown below. It can be observed, that ''E. coli'' carrying the pSB1C3::K389016 plasmid growths nearly linear. <br />
<br />
<br />
[[Image:K389016growth.jpg|600px|thumb|center|'''Fig. 1: Growth curves for ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.''']]<br />
<br />
<br />
The specific growth rates µ and doubling times t<sub>d</sub> are calculated with the OD<sub>600</sub> and following formulas:<br />
<br />
[[Image:Bielefeld_Specific_growth_rate.jpg|175px|center]] <div align="right">(1)</div><br />
<br />
<br />
[[Image:Bielefeld_Doubling_time.jpg|175px|center]] <div align="right">(2)</div><br />
<br />
<br />
<center>Table 1: Averaged specific growth rates and doubling times for cultivations of ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| ''E. coli'' DB3.1<br />
!style="border-style: solid; border-width: 1px"| µ / h<sup>-1</sup><br />
!style="border-style: solid; border-width: 1px"| t<sub>d</sub> / h<br />
|-<br />
|style="border-style: solid; border-width: 1px"| without plasmid<br />
|style="border-style: solid; border-width: 1px"| 0.35<br />
|style="border-style: solid; border-width: 1px"| 1.98<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389016<br />
|style="border-style: solid; border-width: 1px"| 0.27<br />
|style="border-style: solid; border-width: 1px"| 2.57<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389016 with 150 µM acetosyringone<br />
|style="border-style: solid; border-width: 1px"| 0.25<br />
|style="border-style: solid; border-width: 1px"| 2.77<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389016 with 1000 µM acetosyringone<br />
|style="border-style: solid; border-width: 1px"| 0.23<br />
|style="border-style: solid; border-width: 1px"| 3.01<br />
|}<br />
<br />
</center><br />
<br />
<br />
Exemplary induction curves with the fluorescence normalized to OD<sub>600</sub> are shown in Fig.2. We observed a basal transcription, but the induction with acetosyringone is undoubtedly. The detailed [[#Data analysis for BBa_K389016 | data analysis]] and [[#Transfer function of BBa_K389016 | transfer function]] is described below. <br />
<br />
<br />
[[Image:K389016induction.jpg|600px|thumb|center|'''Fig. 2: Induction Curves for ''E. coli'' DB3.1 carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol. The relative fluorescence units are normalized to OD<sub>600</sub> and plotted against the ime in h''']]<br />
<br />
==Transfer function of <partinfo>K389016</partinfo>==<br />
The data for the transfer function was measured and analyzed as [[Team:Bielefeld-Germany/Results/Characterization/K389016#Data analysis for BBa_K389016 | described below]]. [http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html Nelson ''et al.'' (2002)] suggest using a dose response function and fitting it with a logistical equation for the data analysis of receptor systems. The data was fitted with a function of the form<br />
<br />
<br />
[[Image:Bielefeld_Doseresponse_fit.jpg|175px|center]] <div align="right">(3)</div><br />
<br />
<br />
with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x<sub>0</sub>). Figure 3 shows the measured normalized specific production rates q<sub>P,n</sub> (eq. 8) plotted against the logarithm of the concentration of the inductor [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] in µM. The fit has an R<sup>2</sup> = 0.99. <br />
<br />
<br />
[[Image:Bielefeld_Final_RFP_fit.jpg|600px|thumb|center|'''Fig. 3: Transfer function for the part <partinfo>K389016</partinfo> (R<sup>2</sup> = 0.99).''']]<br />
<br />
<br />
The important data from the transfer function is summarized in table 2: <br />
<br />
<br />
<center>Table 2: Data from the transfer function for the part <partinfo>K389016</partinfo>.<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Parameter<br />
!style="border-style: solid; border-width: 1px"| Value<br />
|-<br />
|style="border-style: solid; border-width: 1px"| Hill coefficient<br />
|style="border-style: solid; border-width: 1px"| 1.673<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [http://partsregistry.org/Switch_Point Switch point]<br />
|style="border-style: solid; border-width: 1px"| 26.5 µM<br />
|-<br />
|style="border-style: solid; border-width: 1px"| Top asymptote<br />
|style="border-style: solid; border-width: 1px"| 2.62<br />
|}<br />
<br />
</center><br />
<br />
<br />
So the fully induced VirA/G signaling system has a 2.6 fold increased expression compared to the uninduced system. The Hill coefficient is > 1, so a positive cooperation can be observed ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 D Chu ''et al.'', 2009]). The [http://partsregistry.org/Switch_Point switch point] of the system is at about 25 µM, so this is the concentration at which the device output is 50% of the maximum output.<br />
<br />
==Data analysis for <partinfo>K389016</partinfo>==<br />
The data analysis is made in three steps. First step is the processing of the fluorescence raw data gained by the fluorescence plate reader for every sample: <br />
<br />
<br />
[[Image:Bielefeld_RFU_corrected.jpg|250px|center]] <div align="right">(4)</div><br />
<br />
<br />
In the second step the RFU<sub>corrected</sub> of every sample is plotted against the cultivation time it was drawn. The data is fitted by an exponential fit of the following style: <br />
<br />
<br />
[[Image:Bielefeld_Expfit.jpg|100px|center]] <div align="right">(5)</div><br />
<br />
<br />
The accumulation of mRFP in the cells is always exponential. A typical fitted product accumulation curve is shown below: <br />
<br />
<br />
[[Image:Bielefeld_ExpFit_auf_RFU.jpg|500px|thumb|center|'''Fig. 4: Exponential fit on the measured RFU plotted against cultivation time of a cultivation of <partinfo>K389016</partinfo> in ''Escherichia coli'' DB3.1 in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol and 150 µM acetosyringone. ''']]<br />
<br />
<br />
The product accumulation in a cultivation can be described as: <br />
<br />
<br />
[[Image:Bielefeld_Produktbildung.jpg|100px|center]] <div align="right">(6)</div><br />
<br />
<br />
with the amount of product P, the cell count X and the specific production rate q<sub>P</sub>. <br />
<br />
RFU is commensurate to the concentration of mRFP (P) and the OD<sub>600</sub> is commensurate to the cell count (X) ([http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Canton and Labno, 2004]): <br />
<br />
<br />
[[Image:Bielefeld_RFUpropP.jpg|100px|center]] <div align="right">(7)</div><br />
<br />
[[Image:Bielefeld_ODpropX.jpg|80px|center]] <div align="right">(8)</div><br />
<br />
<br />
With these assumptions it is possible to calculate the specific production rate of mRFP q<sub>P</sub> in the third step: the specific production rate for every sample of a cultivation is calculated by the derivation of the exponential fit line which describes the accumulation of product in the culture (dRFU/dt) and the measured OD<sub>600</sub> data: <br />
<br />
<br />
[[Image:Bielefeld_specific_production_rate.jpg|150px|center]] <div align="right">(9)</div><br />
<br />
<br />
The specific production rates q<sub>P</sub> of all samples of all cultivations made with a specific inductor concentration c are averaged and normalized against the specific production rate of the uninduced system q<sub>P,0</sub>: <br />
<br />
<br />
[[Image:Bielefeld_QPN.jpg|100px|center]] <div align="right">(10)</div><br />
<br />
<br />
This normalized specific production rate we calculated is commensurate to relative promotor units (RPU) which is commensurate to PoPS (polymerase per seconds) ([http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Canton and Labno, 2004]; [http://partsregistry.org/Part:BBa_J23101:Experience Pasotti ''et al.'', 2009]): <br />
<br />
<br />
[[Image:Bielefeld_QPNRPUPoPS.jpg|150px|center]] <div align="right">(11)</div><br />
<br />
==Plasmid conformation analysis==<br />
A plasmid conformation analysis for the BioBrick <partinfo>K389016</partinfo> in <partinfo>pSB1C3</partinfo> was performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] by Capillary Gel Electrophoresis (CGE). The chromatogram is shown in fig. 5 and the results in tab. 3. The data shows a high percentage of covalently closed circular (ccc) plasmid DNA. This is the biological active shape of plasmids so a high percentage of ccc plasmid DNA indicates a high quality of plasmid DNA ([http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory]). <br />
<br />
<br />
[[Image:Bielefeld_CGE_K389016_2.jpg|600px|thumb|center|'''Fig. 5: Chromatogram of the CGE of the BioBrick <partinfo>K389016</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).''']]<br />
<br />
<br />
<br />
<center> Table 3: Data from the CGE of the BioBrick <partinfo>K389016</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Conformation<br />
!style="border-style: solid; border-width: 1px"| Ratio / %<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ccc monomer<br />
|style="border-style: solid; border-width: 1px"| 91.2<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ccc dimer<br />
|style="border-style: solid; border-width: 1px"| 3.2<br />
|-<br />
|style="border-style: solid; border-width: 1px"| oc<br />
|style="border-style: solid; border-width: 1px"| 5.6<br />
|}<br />
<br />
</center><br />
<br />
==Different possible inducers==<br />
A list of tested possible inducers for a VirA/G signaling system is shown in tab. 4. These inducers where tested as a mix. The specific production rate of mRFP q<sub>P</sub> measured as described [[Team:Bielefeld-Germany/Results/Characterization/K389016#Data analysis for BBa_K389016 | above]] for the mix did not significantly differ from the synthesis rate of the uninduced system (t-Test, p < 0.005). So none of the possible inducers listet in tab. 4 induce the VirA/G signaling system significantly in the measured concentration range. In tab. 4 the chemical structures of the testet possible inducers are shown, too. Acetosyringone is also in tab. 4 although it was not testet in the inducer mix to show the chemical similarity of the tested possible inducers to the natural inducer of the VirA/G signaling system. <br />
<br />
[[Team:Bielefeld-Germany/Project/Outlook | For more information about the possible inducers click here. ]]<br />
<br />
<br />
<br />
<center> Table 4: Tested possible inducers for a VirA/G signaling system and acetosyringone with concentrations and chemical structure that were tested. <br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Inducer<br />
!style="border-style: solid; border-width: 1px"| Concentration / µM<br />
!style="border-style: solid; border-width: 1px"| Chemical structure<br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Capsaicin'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"|<html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1548942" width="200px" /></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Dopamine'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=681" width="200"></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Homovanillic acid'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1738" width="200"></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''3-Methoxytyramine'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=11957621" width="200"></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Acetosyringone'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=17198" width="200px" /></html><br />
|}<br />
<br />
</center><br />
<br />
=References=<br />
Canton B and Labno A (2004) [http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Data processing of Part BBa_F2620]. <br />
<br />
Chu D, Zabet NR, Mitavskiy B (2009) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 Models of transcription factor binding: Sensitivity of activation functions to model assumptions], ''J Theor Biol'' 257(3):419-429. <br />
<br />
Greg Nelson, Jayaram Chandrashekar, Mark A. Hoon, Luxin Feng, Grace Zhao, Nicholas J. P. Ryba & Charles S. Zuker (2002) ''[http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html An amino-acid taste receptor ]'', Nature 416: 199-202. <br />
<br />
Pasotti L, Zucca S, Del Fabbro E (2009) Characterization experiment on BBa_J23100, BBa_J23101, BBa_J23118, http://partsregistry.org/Part:BBa_J23101:Experience.<br />
<br />
[http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory]</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389016Team:Bielefeld-Germany/Results/Characterization/K3890162010-10-28T00:18:32Z<p>Nkessler: /* Data analysis for K389016 */</p>
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=Characterization of <partinfo>K389016</partinfo>=<br />
<br />
On this page the experiments and results that lead to the <partinfo>K389016</partinfo> characterization data presented on our [[Team:Bielefeld-Germany/Results/Characterization | characterization page]] are shown in detail. <br />
<br />
== Growth functions and mRFP expression for <partinfo>K389016</partinfo>==<br />
<br />
To characterize this part we performed several cultivations with different concentrations of acetosyringone as inducer and measured the fluorescence emitted by mRFP ([[Team:Bielefeld-Germany/Project/Protocols#Measuring of mRFP | Protocol]]). We used ''Escherichia Coli'' DB3.1 carrying the pSB1C3::K389016 plasmid. Even without inducer the bacteria carrying the plasmid showed decelerated growth. In addition acetosyringone affected the growth rates (we used a stock solution of 20 mM acetosyringone solved in 10 % (v/v) DMSO). Growth curves, averaged specific growth rates and doubling times are shown below. It can be observed, that ''E. coli'' carrying the pSB1C3::K389016 plasmid growths nearly linear. <br />
<br />
<br />
[[Image:K389016growth.jpg|600px|thumb|center|'''Fig. 1: Growth curves for ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.''']]<br />
<br />
<br />
The specific growth rates µ and doubling times t<sub>d</sub> are calculated with the OD<sub>600</sub> and following formulas:<br />
<br />
[[Image:Bielefeld_Specific_growth_rate.jpg|175px|center]] <div align="right">(1)</div><br />
<br />
<br />
[[Image:Bielefeld_Doubling_time.jpg|175px|center]] <div align="right">(2)</div><br />
<br />
<br />
<center>Table 1: Averaged specific growth rates and doubling times for cultivations of ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| ''E. coli'' DB3.1<br />
!style="border-style: solid; border-width: 1px"| µ / h<sup>-1</sup><br />
!style="border-style: solid; border-width: 1px"| t<sub>d</sub> / h<br />
|-<br />
|style="border-style: solid; border-width: 1px"| without plasmid<br />
|style="border-style: solid; border-width: 1px"| 0.35<br />
|style="border-style: solid; border-width: 1px"| 1.98<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389016<br />
|style="border-style: solid; border-width: 1px"| 0.27<br />
|style="border-style: solid; border-width: 1px"| 2.57<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389016 with 150 µM acetosyringone<br />
|style="border-style: solid; border-width: 1px"| 0.25<br />
|style="border-style: solid; border-width: 1px"| 2.77<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389016 with 1000 µM acetosyringone<br />
|style="border-style: solid; border-width: 1px"| 0.23<br />
|style="border-style: solid; border-width: 1px"| 3.01<br />
|}<br />
<br />
</center><br />
<br />
<br />
Exemplary induction curves with the fluorescence normalized to OD<sub>600</sub> are shown in Fig.2. We observed a basal transcription, but the induction with acetosyringone is undoubtedly. The detailed [[#Data analysis for BBa_K389016 | data analysis]] and [[#Transfer function of BBa_K389016 | transfer function]] is described below. <br />
<br />
<br />
[[Image:K389016induction.jpg|600px|thumb|center|'''Fig. 2: Induction Curves for ''E. coli'' DB3.1 carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol. The relative fluorescence units are normalized to OD<sub>600</sub> and plotted against the ime in h''']]<br />
<br />
==Transfer function of <partinfo>K389016</partinfo>==<br />
The data for the transfer function was measured and analyzed as [[Team:Bielefeld-Germany/Results/Characterization/K389016#Data analysis for BBa_K389016 | described below]]. [http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html Nelson ''et al.'' (2002)] suggest using a dose response function and fitting it with a logistical equation for the data analysis of receptor systems. The data was fitted with a function of the form<br />
<br />
<br />
[[Image:Bielefeld_Doseresponse_fit.jpg|175px|center]] <div align="right">(3)</div><br />
<br />
<br />
with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x<sub>0</sub>). Figure 3 shows the measured normalized specific production rates q<sub>P,n</sub> (eq. 8) plotted against the logarithm of the concentration of the inductor [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] in µM. The fit has an R<sup>2</sup> = 0.99. <br />
<br />
<br />
[[Image:Bielefeld_Final_RFP_fit.jpg|600px|thumb|center|'''Fig. 3: Transfer function for the part <partinfo>K389016</partinfo> (R<sup>2</sup> = 0.99).''']]<br />
<br />
<br />
The important data from the transfer function is summarized in table 2: <br />
<br />
<br />
<center>Table 2: Data from the transfer function for the part <partinfo>K389016</partinfo>.<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Parameter<br />
!style="border-style: solid; border-width: 1px"| Value<br />
|-<br />
|style="border-style: solid; border-width: 1px"| Hill coefficient<br />
|style="border-style: solid; border-width: 1px"| 1.673<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [http://partsregistry.org/Switch_Point Switch point]<br />
|style="border-style: solid; border-width: 1px"| 26.5 µM<br />
|-<br />
|style="border-style: solid; border-width: 1px"| Top asymptote<br />
|style="border-style: solid; border-width: 1px"| 2.62<br />
|}<br />
<br />
</center><br />
<br />
<br />
So the fully induced VirA/G signaling system has a 2.6 fold increased expression compared to the uninduced system. The Hill coefficient is > 1, so a positive cooperation can be observed ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 D Chu ''et al.'', 2009]). The [http://partsregistry.org/Switch_Point switch point] of the system is at about 25 µM, so this is the concentration at which the device output is 50% of the maximum output.<br />
<br />
==Data analysis for <partinfo>K389016</partinfo>==<br />
The data analysis is made in three steps. First step is the processing of the fluorescence raw data gained by the fluorescence plate reader for every sample: <br />
<br />
<br />
[[Image:Bielefeld_RFU_corrected.jpg|250px|center]] <div align="right">(4)</div><br />
<br />
<br />
In the second step the RFU<sub>corrected</sub> of every sample is plotted against the cultivation time it was drawn. The data is fitted by an exponential fit of the following style: <br />
<br />
<br />
[[Image:Bielefeld_Expfit.jpg|100px|center]] <div align="right">(5)</div><br />
<br />
<br />
The accumulation of mRFP in the cells is always exponential. A typical fitted product accumulation curve is shown below: <br />
<br />
<br />
[[Image:Bielefeld_ExpFit_auf_RFU.jpg|500px|thumb|center|'''Fig. 4: Exponential fit on the measured RFU plotted against cultivation time of a cultivation of <partinfo>K389016</partinfo> in ''Escherichia coli'' DB3.1 in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol and 150 µM acetosyringone. ''']]<br />
<br />
<br />
The product accumulation in a cultivation can be described as: <br />
<br />
<br />
[[Image:Bielefeld_Produktbildung.jpg|100px|center]] <div align="right">(6)</div><br />
<br />
<br />
with the amount of product P, the cell count X and the specific production rate q<sub>P</sub>. <br />
<br />
RFU is commensurate to the concentration of mRFP (P) and the OD<sub>600</sub> is commensurate to the cell count (X) ([http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Canton and Labno, 2004]): <br />
<br />
<br />
[[Image:Bielefeld_RFUpropP.jpg|100px|center]] <div align="right">(7)</div><br />
<br />
[[Image:Bielefeld_ODpropX.jpg|80px|center]] <div align="right">(8)</div><br />
<br />
<br />
With these assumptions it is possible to calculate the specific production rate of mRFP q<sub>P</sub> in the third step: the specific production rate for every sample of a cultivation is calculated by the derivation of the exponential fit line which describes the accumulation of product in the culture (dRFU/dt) and the measured OD<sub>600</sub> data: <br />
<br />
<br />
[[Image:Bielefeld_specific_production_rate.jpg|150px|center]] <div align="right">(9)</div><br />
<br />
<br />
The specific production rates q<sub>P</sub> of all samples of all cultivations made with a specific inductor concentration c are averaged and normalized against the specific production rate of the uninduced system q<sub>P,0</sub>: <br />
<br />
<br />
[[Image:Bielefeld_QPN.jpg|100px|center]] <div align="right">(10)</div><br />
<br />
<br />
This normalized specific production rate we calculated is commensurate to relative promotor units (RPU) which is commensurate to PoPS (polymerase per seconds) ([http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Canton and Labno, 2004]; [http://partsregistry.org/Part:BBa_J23101:Experience Pasotti ''et al.'', 2009]): <br />
<br />
<br />
[[Image:Bielefeld_QPNRPUPoPS.jpg|150px|center]] <div align="right">(11)</div><br />
<br />
==Plasmid conformation analysis==<br />
A plasmid conformation analysis for the BioBrick <partinfo>K389016</partinfo> in <partinfo>pSB1C3</partinfo> was performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] by Capillary Gel Electrophoresis (CGE). The chromatogram is shown in fig. 5 and the results in tab. 3. The data shows a high percentage of covalently closed circular (ccc) plasmid DNA. This is the biological active shape of plasmids so a high percentage of ccc plasmid DNA indicates a high quality of plasmid DNA ([http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory]). <br />
<br />
<br />
[[Image:Bielefeld_CGE_K389016_2.jpg|600px|thumb|center|'''Fig. 5: Chromatogram of the CGE of the BioBrick <partinfo>K389016</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).''']]<br />
<br />
<br />
<br />
<center> Table 3: Data from the CGE of the BioBrick <partinfo>K389016</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Conformation<br />
!style="border-style: solid; border-width: 1px"| Ratio / %<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ccc monomer<br />
|style="border-style: solid; border-width: 1px"| 91.2<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ccc dimer<br />
|style="border-style: solid; border-width: 1px"| 3.2<br />
|-<br />
|style="border-style: solid; border-width: 1px"| oc<br />
|style="border-style: solid; border-width: 1px"| 5.6<br />
|}<br />
<br />
</center><br />
<br />
==Different possible inducers==<br />
A list of testet possible inducers for a VirA/G signaling system is shown in tab. 4. These inducers where testet as a mix. The specific production rate of mRFP q<sub>P</sub> measured as described [[Team:Bielefeld-Germany/Results/Characterization/K389016#Data analysis for BBa_K389016 | above]] for the mix did not significantly differ from the synthesis rate of the uninduced system (t-Test, p < 0.005). So none of the possible inducers listet in tab. 4 induce the VirA/G signaling system significantly in the measured concentration range. In tab. 4 the chemical structures of the testet possible inducers are shown, too. Acetosyringone is also in tab. 4 although it was not testet in the inducer mix to show the chemical similarity of the testet possible inducers to the natural inducer of the VirA/G signaling system. <br />
<br />
[[Team:Bielefeld-Germany/Project/Outlook | For more information about the possible inducers click here. ]]<br />
<br />
<br />
<br />
<center> Table 4: Testet possible inducers for a VirA/G signaling system and acetosyringone with concentrations and chemical structure that were tested. <br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Inducer<br />
!style="border-style: solid; border-width: 1px"| Concentration / µM<br />
!style="border-style: solid; border-width: 1px"| Chemical structure<br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Capsaicin'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"|<html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1548942" width="200px" /></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Dopamine'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=681" width="200"></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Homovanillic acid'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1738" width="200"></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''3-Methoxytyramine'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=11957621" width="200"></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Acetosyringone'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=17198" width="200px" /></html><br />
|}<br />
<br />
</center><br />
<br />
=References=<br />
Canton B and Labno A (2004) [http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Data processing of Part BBa_F2620]. <br />
<br />
Chu D, Zabet NR, Mitavskiy B (2009) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 Models of transcription factor binding: Sensitivity of activation functions to model assumptions], ''J Theor Biol'' 257(3):419-429. <br />
<br />
Greg Nelson, Jayaram Chandrashekar, Mark A. Hoon, Luxin Feng, Grace Zhao, Nicholas J. P. Ryba & Charles S. Zuker (2002) ''[http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html An amino-acid taste receptor ]'', Nature 416: 199-202. <br />
<br />
Pasotti L, Zucca S, Del Fabbro E (2009) Characterization experiment on BBa_J23100, BBa_J23101, BBa_J23118, http://partsregistry.org/Part:BBa_J23101:Experience.<br />
<br />
[http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory]</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389016Team:Bielefeld-Germany/Results/Characterization/K3890162010-10-28T00:13:30Z<p>Nkessler: /* Transfer function of K389016 */</p>
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<li><a href="/Team:Bielefeld-Germany/Results">Results</a></li><br />
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<li><a href="/Team:Bielefeld-Germany/Results/Used">Used</a></li><br />
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<br />
=Characterization of <partinfo>K389016</partinfo>=<br />
<br />
On this page the experiments and results that lead to the <partinfo>K389016</partinfo> characterization data presented on our [[Team:Bielefeld-Germany/Results/Characterization | characterization page]] are shown in detail. <br />
<br />
== Growth functions and mRFP expression for <partinfo>K389016</partinfo>==<br />
<br />
To characterize this part we performed several cultivations with different concentrations of acetosyringone as inducer and measured the fluorescence emitted by mRFP ([[Team:Bielefeld-Germany/Project/Protocols#Measuring of mRFP | Protocol]]). We used ''Escherichia Coli'' DB3.1 carrying the pSB1C3::K389016 plasmid. Even without inducer the bacteria carrying the plasmid showed decelerated growth. In addition acetosyringone affected the growth rates (we used a stock solution of 20 mM acetosyringone solved in 10 % (v/v) DMSO). Growth curves, averaged specific growth rates and doubling times are shown below. It can be observed, that ''E. coli'' carrying the pSB1C3::K389016 plasmid growths nearly linear. <br />
<br />
<br />
[[Image:K389016growth.jpg|600px|thumb|center|'''Fig. 1: Growth curves for ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.''']]<br />
<br />
<br />
The specific growth rates µ and doubling times t<sub>d</sub> are calculated with the OD<sub>600</sub> and following formulas:<br />
<br />
[[Image:Bielefeld_Specific_growth_rate.jpg|175px|center]] <div align="right">(1)</div><br />
<br />
<br />
[[Image:Bielefeld_Doubling_time.jpg|175px|center]] <div align="right">(2)</div><br />
<br />
<br />
<center>Table 1: Averaged specific growth rates and doubling times for cultivations of ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| ''E. coli'' DB3.1<br />
!style="border-style: solid; border-width: 1px"| µ / h<sup>-1</sup><br />
!style="border-style: solid; border-width: 1px"| t<sub>d</sub> / h<br />
|-<br />
|style="border-style: solid; border-width: 1px"| without plasmid<br />
|style="border-style: solid; border-width: 1px"| 0.35<br />
|style="border-style: solid; border-width: 1px"| 1.98<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389016<br />
|style="border-style: solid; border-width: 1px"| 0.27<br />
|style="border-style: solid; border-width: 1px"| 2.57<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389016 with 150 µM acetosyringone<br />
|style="border-style: solid; border-width: 1px"| 0.25<br />
|style="border-style: solid; border-width: 1px"| 2.77<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389016 with 1000 µM acetosyringone<br />
|style="border-style: solid; border-width: 1px"| 0.23<br />
|style="border-style: solid; border-width: 1px"| 3.01<br />
|}<br />
<br />
</center><br />
<br />
<br />
Exemplary induction curves with the fluorescence normalized to OD<sub>600</sub> are shown in Fig.2. We observed a basal transcription, but the induction with acetosyringone is undoubtedly. The detailed [[#Data analysis for BBa_K389016 | data analysis]] and [[#Transfer function of BBa_K389016 | transfer function]] is described below. <br />
<br />
<br />
[[Image:K389016induction.jpg|600px|thumb|center|'''Fig. 2: Induction Curves for ''E. coli'' DB3.1 carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol. The relative fluorescence units are normalized to OD<sub>600</sub> and plotted against the ime in h''']]<br />
<br />
==Transfer function of <partinfo>K389016</partinfo>==<br />
The data for the transfer function was measured and analyzed as [[Team:Bielefeld-Germany/Results/Characterization/K389016#Data analysis for BBa_K389016 | described below]]. [http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html Nelson ''et al.'' (2002)] suggest using a dose response function and fitting it with a logistical equation for the data analysis of receptor systems. The data was fitted with a function of the form<br />
<br />
<br />
[[Image:Bielefeld_Doseresponse_fit.jpg|175px|center]] <div align="right">(3)</div><br />
<br />
<br />
with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x<sub>0</sub>). Figure 3 shows the measured normalized specific production rates q<sub>P,n</sub> (eq. 8) plotted against the logarithm of the concentration of the inductor [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] in µM. The fit has an R<sup>2</sup> = 0.99. <br />
<br />
<br />
[[Image:Bielefeld_Final_RFP_fit.jpg|600px|thumb|center|'''Fig. 3: Transfer function for the part <partinfo>K389016</partinfo> (R<sup>2</sup> = 0.99).''']]<br />
<br />
<br />
The important data from the transfer function is summarized in table 2: <br />
<br />
<br />
<center>Table 2: Data from the transfer function for the part <partinfo>K389016</partinfo>.<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Parameter<br />
!style="border-style: solid; border-width: 1px"| Value<br />
|-<br />
|style="border-style: solid; border-width: 1px"| Hill coefficient<br />
|style="border-style: solid; border-width: 1px"| 1.673<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [http://partsregistry.org/Switch_Point Switch point]<br />
|style="border-style: solid; border-width: 1px"| 26.5 µM<br />
|-<br />
|style="border-style: solid; border-width: 1px"| Top asymptote<br />
|style="border-style: solid; border-width: 1px"| 2.62<br />
|}<br />
<br />
</center><br />
<br />
<br />
So the fully induced VirA/G signaling system has a 2.6 fold increased expression compared to the uninduced system. The Hill coefficient is > 1, so a positive cooperation can be observed ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 D Chu ''et al.'', 2009]). The [http://partsregistry.org/Switch_Point switch point] of the system is at about 25 µM, so this is the concentration at which the device output is 50% of the maximum output.<br />
<br />
==Data analysis for <partinfo>K389016</partinfo>==<br />
The data analysis is made in three steps. First step is the processing of the fluorescence raw data gained by the fluorescence plate reader for every sample: <br />
<br />
<br />
[[Image:Bielefeld_RFU_corrected.jpg|250px|center]] <div align="right">(4)</div><br />
<br />
<br />
In the second step the RFU<sub>corrected</sub> of every sample is plotted against the cultivation time it was drawn. The data is fitted by an exponential fit of the following style: <br />
<br />
<br />
[[Image:Bielefeld_Expfit.jpg|100px|center]] <div align="right">(5)</div><br />
<br />
<br />
The accumulation of mRFP in the cells is always exponential. A typical fitted product accumulation curve is shown below: <br />
<br />
<br />
[[Image:Bielefeld_ExpFit_auf_RFU.jpg|500px|thumb|center|'''Fig. 4: Exponential fit on the measured RFU plotted against cultivation time of a cultivation of <partinfo>K389016</partinfo> in ''Escherichia coli'' DB3.1 in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol and 150 µM acetosyringone. ''']]<br />
<br />
<br />
The product accumulation in a cultivation can be described as: <br />
<br />
<br />
[[Image:Bielefeld_Produktbildung.jpg|100px|center]] <div align="right">(6)</div><br />
<br />
<br />
with the amount of product P, the cell count X and the specific production rate q<sub>P</sub>. <br />
<br />
RFU is commensurate to the concentration of mRFP (P) and the OD<sub>600</sub> is commensurate to the cell count (X) ([[http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis | Canton and Labno, 2004]]): <br />
<br />
<br />
[[Image:Bielefeld_RFUpropP.jpg|100px|center]] <div align="right">(7)</div><br />
<br />
[[Image:Bielefeld_ODpropX.jpg|80px|center]] <div align="right">(8)</div><br />
<br />
<br />
With these assumptions it is possible to calculate the specific production rate of mRFP q<sub>P</sub> in the third step: the specific production rate for every sample of a cultivation is calculated by the derivation of the exponential fit line which describes the accumulation of product in the culture (dRFU/dt) and the measured OD<sub>600</sub> data: <br />
<br />
<br />
[[Image:Bielefeld_specific_production_rate.jpg|150px|center]] <div align="right">(9)</div><br />
<br />
<br />
The specific production rates q<sub>P</sub> of all samples of all cultivations made with a specific inductor concentration c are averaged and normalized against the specific production rate of the uninduced system q<sub>P,0</sub>: <br />
<br />
<br />
[[Image:Bielefeld_QPN.jpg|100px|center]] <div align="right">(10)</div><br />
<br />
<br />
This normalized specific production rate we calculated is commensurate to relative promotor units (RPU) which is commensurate to PoPS (polymerase per seconds) ([http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Canton and Labno, 2004]; [http://partsregistry.org/Part:BBa_J23101:Experience Pasotti ''et al.'', 2009]): <br />
<br />
<br />
[[Image:Bielefeld_QPNRPUPoPS.jpg|150px|center]] <div align="right">(11)</div><br />
<br />
==Plasmid conformation analysis==<br />
A plasmid conformation analysis for the BioBrick <partinfo>K389016</partinfo> in <partinfo>pSB1C3</partinfo> was performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] by Capillary Gel Electrophoresis (CGE). The chromatogram is shown in fig. 5 and the results in tab. 3. The data shows a high percentage of covalently closed circular (ccc) plasmid DNA. This is the biological active shape of plasmids so a high percentage of ccc plasmid DNA indicates a high quality of plasmid DNA ([http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory]). <br />
<br />
<br />
[[Image:Bielefeld_CGE_K389016_2.jpg|600px|thumb|center|'''Fig. 5: Chromatogram of the CGE of the BioBrick <partinfo>K389016</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).''']]<br />
<br />
<br />
<br />
<center> Table 3: Data from the CGE of the BioBrick <partinfo>K389016</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Conformation<br />
!style="border-style: solid; border-width: 1px"| Ratio / %<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ccc monomer<br />
|style="border-style: solid; border-width: 1px"| 91.2<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ccc dimer<br />
|style="border-style: solid; border-width: 1px"| 3.2<br />
|-<br />
|style="border-style: solid; border-width: 1px"| oc<br />
|style="border-style: solid; border-width: 1px"| 5.6<br />
|}<br />
<br />
</center><br />
<br />
==Different possible inducers==<br />
A list of testet possible inducers for a VirA/G signaling system is shown in tab. 4. These inducers where testet as a mix. The specific production rate of mRFP q<sub>P</sub> measured as described [[Team:Bielefeld-Germany/Results/Characterization/K389016#Data analysis for BBa_K389016 | above]] for the mix did not significantly differ from the synthesis rate of the uninduced system (t-Test, p < 0.005). So none of the possible inducers listet in tab. 4 induce the VirA/G signaling system significantly in the measured concentration range. In tab. 4 the chemical structures of the testet possible inducers are shown, too. Acetosyringone is also in tab. 4 although it was not testet in the inducer mix to show the chemical similarity of the testet possible inducers to the natural inducer of the VirA/G signaling system. <br />
<br />
[[Team:Bielefeld-Germany/Project/Outlook | For more information about the possible inducers click here. ]]<br />
<br />
<br />
<br />
<center> Table 4: Testet possible inducers for a VirA/G signaling system and acetosyringone with concentrations and chemical structure that were tested. <br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Inducer<br />
!style="border-style: solid; border-width: 1px"| Concentration / µM<br />
!style="border-style: solid; border-width: 1px"| Chemical structure<br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Capsaicin'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"|<html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1548942" width="200px" /></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Dopamine'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=681" width="200"></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Homovanillic acid'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1738" width="200"></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''3-Methoxytyramine'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=11957621" width="200"></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Acetosyringone'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=17198" width="200px" /></html><br />
|}<br />
<br />
</center><br />
<br />
=References=<br />
Canton B and Labno A (2004) [http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Data processing of Part BBa_F2620]. <br />
<br />
Chu D, Zabet NR, Mitavskiy B (2009) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 Models of transcription factor binding: Sensitivity of activation functions to model assumptions], ''J Theor Biol'' 257(3):419-429. <br />
<br />
Greg Nelson, Jayaram Chandrashekar, Mark A. Hoon, Luxin Feng, Grace Zhao, Nicholas J. P. Ryba & Charles S. Zuker (2002) ''[http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html An amino-acid taste receptor ]'', Nature 416: 199-202. <br />
<br />
Pasotti L, Zucca S, Del Fabbro E (2009) Characterization experiment on BBa_J23100, BBa_J23101, BBa_J23118, http://partsregistry.org/Part:BBa_J23101:Experience.<br />
<br />
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<div id="checkParameter" style="margin-right:250px;">{{{1}}}</div></div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389016Team:Bielefeld-Germany/Results/Characterization/K3890162010-10-27T23:46:15Z<p>Nkessler: /* Growth functions and mRFP expression for K389016 */</p>
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<br />
=Characterization of <partinfo>K389016</partinfo>=<br />
<br />
On this page the experiments and results that lead to the <partinfo>K389016</partinfo> characterization data presented on our [[Team:Bielefeld-Germany/Results/Characterization | characterization page]] are shown in detail. <br />
<br />
== Growth functions and mRFP expression for <partinfo>K389016</partinfo>==<br />
<br />
To characterize this part we performed several cultivations with different concentrations of acetosyringone as inducer and measured the fluorescence emitted by mRFP ([[Team:Bielefeld-Germany/Project/Protocols#Measuring of mRFP | Protocol]]). We used ''Escherichia Coli'' DB3.1 carrying the pSB1C3::K389016 plasmid. Even without inducer the bacteria carrying the plasmid showed decelerated growth. In addition acetosyringone affected the growth rates (we used a stock solution of 20 mM acetosyringone solved in 10 % (v/v) DMSO). Growth curves, averaged specific growth rates and doubling times are shown below. It can be observed, that ''E. coli'' carrying the pSB1C3::K389016 plasmid growths nearly linear. <br />
<br />
<br />
[[Image:K389016growth.jpg|600px|thumb|center|'''Fig. 1: Growth curves for ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.''']]<br />
<br />
<br />
The specific growth rates µ and doubling times t<sub>d</sub> are calculated with the OD<sub>600</sub> and following formulas:<br />
<br />
[[Image:Bielefeld_Specific_growth_rate.jpg|175px|center]] <div align="right">(1)</div><br />
<br />
<br />
[[Image:Bielefeld_Doubling_time.jpg|175px|center]] <div align="right">(2)</div><br />
<br />
<br />
<center>Table 1: Averaged specific growth rates and doubling times for cultivations of ''E. coli'' DB3.1 without plasmid and carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol.<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| ''E. coli'' DB3.1<br />
!style="border-style: solid; border-width: 1px"| µ / h<sup>-1</sup><br />
!style="border-style: solid; border-width: 1px"| t<sub>d</sub> / h<br />
|-<br />
|style="border-style: solid; border-width: 1px"| without plasmid<br />
|style="border-style: solid; border-width: 1px"| 0.35<br />
|style="border-style: solid; border-width: 1px"| 1.98<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389016<br />
|style="border-style: solid; border-width: 1px"| 0.27<br />
|style="border-style: solid; border-width: 1px"| 2.57<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389016 with 150 µM acetosyringone<br />
|style="border-style: solid; border-width: 1px"| 0.25<br />
|style="border-style: solid; border-width: 1px"| 2.77<br />
|-<br />
|style="border-style: solid; border-width: 1px"| carrying K389016 with 1000 µM acetosyringone<br />
|style="border-style: solid; border-width: 1px"| 0.23<br />
|style="border-style: solid; border-width: 1px"| 3.01<br />
|}<br />
<br />
</center><br />
<br />
<br />
Exemplary induction curves with the fluorescence normalized to OD<sub>600</sub> are shown in Fig.2. We observed a basal transcription, but the induction with acetosyringone is undoubtedly. The detailed [[#Data analysis for BBa_K389016 | data analysis]] and [[#Transfer function of BBa_K389016 | transfer function]] is described below. <br />
<br />
<br />
[[Image:K389016induction.jpg|600px|thumb|center|'''Fig. 2: Induction Curves for ''E. coli'' DB3.1 carrying <partinfo>K389016</partinfo> with different acetosyringone concentrations in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol. The relative fluorescence units are normalized to OD<sub>600</sub> and plotted against the ime in h''']]<br />
<br />
==Transfer function of <partinfo>K389016</partinfo>==<br />
The data for the transfer function was measured and analyzed as [[Team:Bielefeld-Germany/Results/Characterization/K389016#Data analysis for BBa_K389016 | described below]]. Nelson ''et al.'' (2002) suggest using a dose response function and fitting it with a logistical equation for the data analysis of receptor systems ([http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html Nelson ''et al.'', 2002]). The data was fitted with a function of the form<br />
<br />
<br />
[[Image:Bielefeld_Doseresponse_fit.jpg|175px|center]] <div align="right">(3)</div><br />
<br />
<br />
with the Hill coefficient p, the bottom asymptote A1, the top asymptote A2 and the switch point log(x<sub>0</sub>). Figure 3 shows the measured normalized specific production rates q<sub>P,n</sub> (eq. 8) plotted against the logarithm of the concentration of the inductor [http://www.chemblink.com/products/2478-38-8.htm acetosyringone] in µM. The fit has an R<sup>2</sup> = 0.99. <br />
<br />
<br />
[[Image:Bielefeld_Final_RFP_fit.jpg|600px|thumb|center|'''Fig. 3: Transfer function for the part <partinfo>K389016</partinfo> (R<sup>2</sup> = 0.99).''']]<br />
<br />
<br />
The important data from the transfer function is summarized in table 2: <br />
<br />
<br />
<center>Table 2: Data from the transfer function for the part <partinfo>K389016</partinfo>.<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Parameter<br />
!style="border-style: solid; border-width: 1px"| Value<br />
|-<br />
|style="border-style: solid; border-width: 1px"| Hill coefficient<br />
|style="border-style: solid; border-width: 1px"| 1.673<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [http://partsregistry.org/Switch_Point Switch point]<br />
|style="border-style: solid; border-width: 1px"| 26.5 µM<br />
|-<br />
|style="border-style: solid; border-width: 1px"| Top asymptote<br />
|style="border-style: solid; border-width: 1px"| 2.62<br />
|}<br />
<br />
</center><br />
<br />
<br />
So the fully induced VirA/G signaling system has a 2.6 fold increased expression compared to the uninduced system. The Hill coefficient is > 1, so a positive cooperation can be observed ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 D Chu ''et al.'', 2009]). The [http://partsregistry.org/Switch_Point switch point] of the system is at about 25 µM, so this is the concentration at which the device output is 50% of the maximum output.<br />
<br />
==Data analysis for <partinfo>K389016</partinfo>==<br />
The data analysis is made in three steps. First step is the processing of the fluorescence raw data gained by the fluorescence plate reader for every sample: <br />
<br />
<br />
[[Image:Bielefeld_RFU_corrected.jpg|250px|center]] <div align="right">(4)</div><br />
<br />
<br />
In the second step the RFU<sub>corrected</sub> of every sample is plotted against the cultivation time it was drawn. The data is fitted by an exponential fit of the following style: <br />
<br />
<br />
[[Image:Bielefeld_Expfit.jpg|100px|center]] <div align="right">(5)</div><br />
<br />
<br />
The accumulation of mRFP in the cells is always exponential. A typical fitted product accumulation curve is shown below: <br />
<br />
<br />
[[Image:Bielefeld_ExpFit_auf_RFU.jpg|500px|thumb|center|'''Fig. 4: Exponential fit on the measured RFU plotted against cultivation time of a cultivation of <partinfo>K389016</partinfo> in ''Escherichia coli'' DB3.1 in LB medium with 10 mg ml<sup>-1</sup> chloramphenicol and 150 µM acetosyringone. ''']]<br />
<br />
<br />
The product accumulation in a cultivation can be described as: <br />
<br />
<br />
[[Image:Bielefeld_Produktbildung.jpg|100px|center]] <div align="right">(6)</div><br />
<br />
<br />
with the amount of product P, the cell count X and the specific production rate q<sub>P</sub>. <br />
<br />
RFU is commensurate to the concentration of mRFP (P) and the OD<sub>600</sub> is commensurate to the cell count (X) ([[http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis | Canton and Labno, 2004]]): <br />
<br />
<br />
[[Image:Bielefeld_RFUpropP.jpg|100px|center]] <div align="right">(7)</div><br />
<br />
[[Image:Bielefeld_ODpropX.jpg|80px|center]] <div align="right">(8)</div><br />
<br />
<br />
With these assumptions it is possible to calculate the specific production rate of mRFP q<sub>P</sub> in the third step: the specific production rate for every sample of a cultivation is calculated by the derivation of the exponential fit line which describes the accumulation of product in the culture (dRFU/dt) and the measured OD<sub>600</sub> data: <br />
<br />
<br />
[[Image:Bielefeld_specific_production_rate.jpg|150px|center]] <div align="right">(9)</div><br />
<br />
<br />
The specific production rates q<sub>P</sub> of all samples of all cultivations made with a specific inductor concentration c are averaged and normalized against the specific production rate of the uninduced system q<sub>P,0</sub>: <br />
<br />
<br />
[[Image:Bielefeld_QPN.jpg|100px|center]] <div align="right">(10)</div><br />
<br />
<br />
This normalized specific production rate we calculated is commensurate to relative promotor units (RPU) which is commensurate to PoPS (polymerase per seconds) ([http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Canton and Labno, 2004]; [http://partsregistry.org/Part:BBa_J23101:Experience Pasotti ''et al.'', 2009]): <br />
<br />
<br />
[[Image:Bielefeld_QPNRPUPoPS.jpg|150px|center]] <div align="right">(11)</div><br />
<br />
==Plasmid conformation analysis==<br />
A plasmid conformation analysis for the BioBrick <partinfo>K389016</partinfo> in <partinfo>pSB1C3</partinfo> was performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] by Capillary Gel Electrophoresis (CGE). The chromatogram is shown in fig. 5 and the results in tab. 3. The data shows a high percentage of covalently closed circular (ccc) plasmid DNA. This is the biological active shape of plasmids so a high percentage of ccc plasmid DNA indicates a high quality of plasmid DNA ([http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory]). <br />
<br />
<br />
[[Image:Bielefeld_CGE_K389016_2.jpg|600px|thumb|center|'''Fig. 5: Chromatogram of the CGE of the BioBrick <partinfo>K389016</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).''']]<br />
<br />
<br />
<br />
<center> Table 3: Data from the CGE of the BioBrick <partinfo>K389016</partinfo> in <partinfo>pSB1C3</partinfo> performed by the [http://web.plasmidfactory.com/de/ PlasmidFactory] (Bielefeld).<br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Conformation<br />
!style="border-style: solid; border-width: 1px"| Ratio / %<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ccc monomer<br />
|style="border-style: solid; border-width: 1px"| 91.2<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ccc dimer<br />
|style="border-style: solid; border-width: 1px"| 3.2<br />
|-<br />
|style="border-style: solid; border-width: 1px"| oc<br />
|style="border-style: solid; border-width: 1px"| 5.6<br />
|}<br />
<br />
</center><br />
<br />
==Different possible inducers==<br />
A list of testet possible inducers for a VirA/G signaling system is shown in tab. 4. These inducers where testet as a mix. The specific production rate of mRFP q<sub>P</sub> measured as described [[Team:Bielefeld-Germany/Results/Characterization/K389016#Data analysis for BBa_K389016 | above]] for the mix did not significantly differ from the synthesis rate of the uninduced system (t-Test, p < 0.005). So none of the possible inducers listet in tab. 4 induce the VirA/G signaling system significantly in the measured concentration range. In tab. 4 the chemical structures of the testet possible inducers are shown, too. Acetosyringone is also in tab. 4 although it was not testet in the inducer mix to show the chemical similarity of the testet possible inducers to the natural inducer of the VirA/G signaling system. <br />
<br />
[[Team:Bielefeld-Germany/Project/Outlook | For more information about the possible inducers click here. ]]<br />
<br />
<br />
<br />
<center> Table 4: Testet possible inducers for a VirA/G signaling system and acetosyringone with concentrations and chemical structure that were tested. <br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Inducer<br />
!style="border-style: solid; border-width: 1px"| Concentration / µM<br />
!style="border-style: solid; border-width: 1px"| Chemical structure<br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Capsaicin'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"|<html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1548942" width="200px" /></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Dopamine'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=681" width="200"></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Homovanillic acid'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1738" width="200"></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''3-Methoxytyramine'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=11957621" width="200"></html><br />
|-<br />
|style="border-style: solid; border-width: 1px"| '''Acetosyringone'''<br />
|style="border-style: solid; border-width: 1px"| 200<br />
|style="border-style: solid; border-width: 1px"| <html><img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=17198" width="200px" /></html><br />
|}<br />
<br />
</center><br />
<br />
=References=<br />
Canton B and Labno A (2004) [http://partsregistry.org/Part:BBa_F2620:Experience/Endy/Data_analysis Data processing of Part BBa_F2620]. <br />
<br />
Chu D, Zabet NR, Mitavskiy B (2009) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WMD-4V42JG5-1&_user=10&_rdoc=1&_fmt=&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=b6431553217aca1129c5b441f4b78425 Models of transcription factor binding: Sensitivity of activation functions to model assumptions], ''J Theor Biol'' 257(3):419-429. <br />
<br />
Greg Nelson, Jayaram Chandrashekar, Mark A. Hoon, Luxin Feng, Grace Zhao, Nicholas J. P. Ryba & Charles S. Zuker (2002) ''[http://www.nature.com/nature/journal/v416/n6877/abs/nature726.html An amino-acid taste receptor ]'', Nature 416: 199-202. <br />
<br />
Pasotti L, Zucca S, Del Fabbro E (2009) Characterization experiment on BBa_J23100, BBa_J23101, BBa_J23118, http://partsregistry.org/Part:BBa_J23101:Experience.<br />
<br />
[http://web.plasmidfactory.com/en/service_CGE.html PlasmidFactory]</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/CharacterizationTeam:Bielefeld-Germany/Results/Characterization2010-10-27T22:54:54Z<p>Nkessler: /* K389421, K389422, K389423: Sensitivity Tuner amlified Vir-test system */</p>
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<br />
<br />
=<partinfo>K238008</partinfo>: ''virA''=<br />
We wanted to use this part in our project, but could only obtain unexpected/faulty restriction patterns. Finally we chose to sequence the part, hoping to find the cause for the maintained restriction patterns. Unfortunately we could not approve the sequence of <partinfo>BBa_K238008</partinfo> deposited in the parts registry so that we chose to design our own VirA BioBrick. I strongly recommend using our VirA since it has been approved by multiple means, e.g. restriction patterns and sequencing (<partinfo>K389001</partinfo>).<br />
<br />
=<partinfo>BBa_K238011</partinfo>: ''vir''-promoter=<br />
We made a restriction analysis and sequenced parts of this BioBrick.<br />
<br />
<br />
=<partinfo>P1010</partinfo>: ''ccdB''-gene=<br />
The ''ccdB'' gene targets the gyrase of ''Escherichia coli'' and is lethal for all ''E. coli'' strains without the gyrase mutation gyrA462 ([http://openwetware.org/wiki/CcdB Openwetware]). The ''ccdB'' BioBrick is used for the 3A-assembly as a positive selection marker. <br />
We transformed this BioBrick into ''E. coli'' JM109, DH5α, TOP10, XL1-Blue, EC100D and DB3.1. ''E. coli'' JM109, XL1-Blue and DH5α seem to be ''ccdB'' resistant because there were as much colonies after P1010 transformation as observed with DB3.1. The P1010 works as expected in ''E. coli'' TOP10, EC100D (no colonies after transformation) and DB3.1 (many colonies after transformation).<br />
<br />
<br />
<center>Table 1: Results of the transformation of the cell-death gene ''ccdB'', BioBrick <partinfo>P1010</partinfo>, into different strains of ''E. coli''. <br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| ''E. coli'' strain<br />
!style="border-style: solid; border-width: 1px"| Resistant to ''ccdB''?<br />
!style="border-style: solid; border-width: 1px"| Expected result?<br />
!style="border-style: solid; border-width: 1px"| Gyrase genotype <br> ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T39-47PNXC3-F3&_user=2459438&_coverDate=01%2F28%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=dfcdeab4c210c1f4ec70de318d013c15&searchtype=a Metcalf ''et al.'', 1994]; [http://openwetware.org/wiki/E._coli_genotypes Openwetware])<br />
|-<br />
|style="border-style: solid; border-width: 1px"| DB3.1<br />
|style="border-style: solid; border-width: 1px"| yes <br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| gyrA462<br />
|-<br />
|style="border-style: solid; border-width: 1px"| DH5α<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| gyrA96<br />
|-<br />
|style="border-style: solid; border-width: 1px"| EC100D<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| WT<br />
|-<br />
|style="border-style: solid; border-width: 1px"| JM109<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| gyrA96<br />
|-<br />
|style="border-style: solid; border-width: 1px"| TOP10<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| WT<br />
|-<br />
|style="border-style: solid; border-width: 1px"| XL1-Blue<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| gyrA96<br />
|-<br />
|}<br />
</center><br />
<br />
<br />
It seems that not only the gyrase mutation gyrA462 is causing a ''ccdB'' resistance. Also the gyrase mutation gyrA96 gives ''E. coli'' a ''ccdB'' resistance. This should be kept in mind when assembling BioBricks with the 3A assembly.<br />
<br />
=<partinfo>K389004</partinfo>: Luciferase from pGL4.10[luc2]=<br />
[[Team:Bielefeld-Germany/Results/Characterization/K389004#mRFP vs. luciferase as reporter gene | For a comparison between mRFP and luciferase as reporter genes click here. ]]<br />
<br />
Some important parameters determined by the characterization experiments are shown in tab. 2. For more information concerning these experiments click on the corresponding link in tab. 2 or click here: <br />
<br />
<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389004">Detailed information...</a></div></html><br />
<br />
<br />
<center>Table 2: Parameters for <partinfo>K389004</partinfo>. <br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Experiment<br />
!style="border-style: solid; border-width: 1px"| Result<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Accumulation of luciferase | Behaviour during cultivation]]<br />
|style="border-style: solid; border-width: 1px"| <br />
* production is growth dependent<br />
* degradation in stationary growth phase<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Kinetic of luciferin conversion | Kinetic of luciferin conversion]]<br />
|style="border-style: solid; border-width: 1px"| max. output between 20 - 40 s<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Sensitivity | Limit of detection (LOD)]]<br />
|style="border-style: solid; border-width: 1px"| 162 RLU ~ 0.3 % of <partinfo>J23103</partinfo> output<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Sensitivity | Limit of quantification (LOQ)]]<br />
|style="border-style: solid; border-width: 1px"| 306 RLU ~ 0.7 % of <partinfo>J23103</partinfo> output<br />
|}<br />
</center><br />
<br />
=<partinfo>K389011</partinfo>: VirA screening device=<br />
<br />
<br />
<br />
<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389011">Detailed information...</a></div></html><br />
<br />
<br />
<br />
[[Image:Bielefeld_LD50_Graph2.jpg|600px|thumb|center|Ratio of surviving colonies of ''E. coli'' EC100D carrying unmutated <partinfo>K389010</partinfo> and <partinfo>K389011</partinfo> plated on PA agar plates with chloramphenicol, ampicillin and different concentrations of kanamycin. Comparison between cells that were induced with acetosyringone with cells that were not induced.]]<br />
<br />
<br />
<br />
=<partinfo>K389015</partinfo>: VirA/G reporter device with luciferase=<br />
Some important parameters determined by the characterization experiments are shown in tab. X. For more information concerning these experiments click on the corresponding link in tab. X or click here: <br />
<br />
<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389015">Detailed information...</a></div></html><br />
<br />
<br />
<center>Table X: Parameters for <partinfo>K389015</partinfo>. <br />
{|{{Table}}<br />
!Experiment<br />
!Characteristic<br />
!Value<br />
|-<br />
|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Transfer function | Transfer Function]]<br />
|Maximum induction level<br />
|2.2 fold<br />
|-<br />
|Maximum induction level reached<br />
|200 µM acetosyringone<br />
|-<br />
|Hill coefficient<br />
|1.09<br />
|-<br />
|Switch Point<br />
|31.6 µM acetosyringone<br />
|-<br />
|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Growth functions and Luciferase expression for BBa_K389015 | Doubling time / h]]<br />
|without plasmid<br />
|1.98<br />
|-<br />
|carrying K389015<br />
|2.24<br />
|-<br />
|carrying K389015 with 400 µM acetosyringone<br />
|2.67<br />
|-<br />
|rowspan="2"|Response time<br />
|Induction: [[Team:Bielefeld-Germany/Results/Characterization/K389015#Response time | exponential phase]]<br />
|>1 h<br />
|-<br />
|Induction: [[Team:Bielefeld-Germany/Results/Characterization/K389015#Data Analysis | begin of cultivation]]<br />
|max. induction at OD<sub>600</sub> = 1 +/- 0.5<br />
|-<br />
|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Plasmid conformation analysis | Conformation analysis]]<br />
|ratio ccc monomer / %<br />
|91<br />
|-<br />
|ratio ccc dimer / %<br />
|3.7<br />
|-<br />
|ratio oc forms / %<br />
|5.3<br />
|-<br />
|}<br />
</center><br />
<br />
=<partinfo>K389016</partinfo>: VirA/G reporter device with mRFP=<br />
<br />
Protocols for [https://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Cultivation_for_measuring_mRFP_and_Luciferase_expression Cultivation] and [https://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Measuring_of_mRFP Measurement]<br />
<br />
Some important parameters determined by the characterization experiments are shown in tab. X. For more information concerning these experiments click on the corresponding link in tab. X or click here: <br />
<br />
<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389016">Detailed information...</a></div></html><br />
<br />
<br />
<center>Table X: Parameters for <partinfo>K389016</partinfo>. <br />
{|{{Table}}<br />
!Experiment<br />
!Characteristic<br />
!Value<br />
|-<br />
|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Transfer function of BBa_K389016 | Transfer Function]]<br />
|Maximum induction level<br />
|2.6 fold<br />
|-<br />
|Maximum induction level reached<br />
|150 µM acetosyringone<br />
|-<br />
|Hill coefficient<br />
|1.67<br />
|-<br />
|Switch Point<br />
|26.5 µM acetosyringone<br />
|-<br />
|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Growth functions and mRFP expression for BBa_K389016 | Doubling time / h]]<br />
|without plasmid<br />
|1.98<br />
|-<br />
|carrying K389016<br />
|2.57<br />
|-<br />
|carrying K389016 with 150 µM acetosyringone<br />
|2.77<br />
|-<br />
|carrying K389016 with 1000 µM acetosyringone<br />
|3.01<br />
|-<br />
|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Plasmid conformation analysis | Conformation analysis]]<br />
|ratio ccc monomer / %<br />
|91.2<br />
|-<br />
|ratio ccc dimer / %<br />
|3.2<br />
|-<br />
|ratio oc forms / %<br />
|5.6<br />
|-<br />
|rowspan="5"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Different possible inducers | Inducers]]<br />
|Induction by<br />
|Acetosyringone<br />
|-<br />
|rowspan="4"|No Induction by<br />
|Capsaicin<br />
|-<br />
|Dopamine<br />
|-<br />
|Homovanillic acid <br />
|-<br />
|3-Methoxytyramine <br />
|}<br />
</center><br />
<br />
=<partinfo>K389052</partinfo>: Tightly regulated ''lac'' operon with mRFP readout=<br />
This construct was plated for plasmid isolation in a ''lacI<sup>q</sup>'' negative ''E. coli'' strain after assembly - and we have never seen such red plates when working with constructs with mRFP downstream of a promoter. This ''lac'' operon definitely shows a very high basal transcription, so it is not tightly repressed. It seems that the ''lacI'' repressor <partinfo>BBa_C0012</partinfo> is not suitable for this purpose due to its LVA degradation tag or it does not work properly. Another indicator for this assumption is the experience page of <partinfo>C0012</partinfo>.<br />
<br />
=<partinfo>K389421</partinfo>, <partinfo>K389422</partinfo>, <partinfo>K389423</partinfo>: Sensitivity Tuner amplified Vir-test system=<br />
<br />
By self designed PCR-Primer we excluded terminal GFP and the initial promoter pBAD/araC, for replacing our own VirB promotor and reporter gene luc (luciferase). Primers were designed for sensitivity tuner [http://partsregistry.org/Part:BBa_I746370 I746370], [http://partsregistry.org/Part:BBa_I746380 I746380] and [http://partsregistry.org/Part:BBa_I746390 I746390] so that standard assembly would be possible. Assembling of PCR-products took place by Silver Assembly.<br />
<br />
'''Accomplishment'''<br />
<br />
'''PCR-Primer Design'''<br />
<br />
Primer forward activator phage P2:<br />
<br />
5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GTT TCA TTG TCC TTT ATG CC-3`<br />
<br />
Primer forward activator phage PSP3:<br />
<br />
5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GAT GCA CTG CCC GTT ATG- 3`<br />
<br />
Primer forward activator phage phi R73:<br />
<br />
5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GCG CTG CCC TTT CTG-3`<br />
<br />
Primer backward Promotor PF from phage P2:<br />
<br />
5`-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TTC TCC TCT TTC TCT AGT AAG TGG- 3`<br />
<br />
<br />
'''Characterization tests'''<br />
<br />
Cultivation was done by induction with Acetosyringone at 50 µM. Controls were not induced Sensitivity Tuner devices as well as induced and not induced nativ system ([http://partsregistry.org/Part:BBa_K389015 K389015]; without tuning elements). Induction was done upon inoculation. Measuring point for amplification factor calculation was OD 1.0. ([https://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Cultivation_for_measuring_mRFP_and_Luciferase_expression Protocols])<br />
<br />
<br />
'''Results'''<br />
<br />
Three sensitivity tuned Vir-Gen sensing systems were obtained: [http://partsregistry.org/Part:BBa_K389421 K389421], [http://partsregistry.org/Part:BBa_K389422 K389422] and [http://partsregistry.org/Part:BBa_K389423 K389423] distinguishing by the amplification level of luc transcription.<br />
<br />
[[Image:ST Tuner.png|600px|thumb|center| '''Figure 1: Amplification factor of induced, 50 µM Acetosyringone (red) and not induced (green) modified Sensitivity Tuner K389421, K389422 and K389423, Standard deviation shown.''']]<br />
<br />
The amplification factor was received by apply [http://partsregistry.org/Part:BBa_K389015 K389015] as reference. Amplification calculation was done by normalizing relative luminescence units emitted from luciferase per OD.<br />
Output-signal amplification is in the induced contructs (red) [http://partsregistry.org/Part:BBa_K389422 K389422] and [http://partsregistry.org/Part:BBa_K389423 K389423] 100 and respectively 200 fold higher than in not induced controls (green). An exception is K389422 were induced and not indiced system revealed analog results. Corresponding to data of iGEM Team, Cambridge 2009, K389423 (originated from [http://partsregistry.org/Part:BBa_I746390 I746390]) shows the highest amplification rate of all tested Sensitivity Tuners. Our results indicate to higher amplification rate of [http://partsregistry.org/Part:BBa_K389421 K389421] than [http://partsregistry.org/Part:BBa_K389422 K389422] of 100 fold under induced conditions. The controls also show high basal transcription rates.<br />
<br />
Because there is small difference in induced and not induced system visible and basal transcription rates are high, we assume that the sensitivity tuning constructs are not well applicable for luciferase measurements.<br />
<br />
For further theory click [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Read_out_system Read out system]<br />
<br />
=References=<br />
<br />
<br />
<br />
<br />
*Behrens B, Eppendorf AG, Laborpraxis, Nr.20, Reinste Plasmid-DNA in nur 9 Minuten.<br />
<br />
*http://openwetware.org/wiki/CcdB, CcdB (seen on 10.10.10).<br />
<br />
*http://openwetware.org/wiki/E._coli_genotypes, E. coli genotypes (seen on 10.10.10).<br />
<br />
*Metcalf, WW ''et al.'' (1994) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T39-47PNXC3-F3&_user=2459438&_coverDate=01%2F28%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=dfcdeab4c210c1f4ec70de318d013c15&searchtype=a ''Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6Kλ origin plasmids at different copy numbers''], Gene 138(1):1-7.<br />
<br />
*Stadler J, Lemmens R, Nyhammar T 2004, ''Plasmid DNA purification'', The J. of Gene Medicine,Vol.6, pp.54–S66</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/CharacterizationTeam:Bielefeld-Germany/Results/Characterization2010-10-27T22:52:28Z<p>Nkessler: /* K389052: tightly regulated lac operon with mRFP readout */</p>
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<li><a href="/Team:Bielefeld-Germany/Results/Used">Used</a></li><br />
<li><a href="/Team:Bielefeld-Germany/Results/Submitted">Submitted</a></li><br />
<li><a href="/Team:Bielefeld-Germany/Results/Sequencing">Sequencing</a></li><br />
</div><br />
</body><br />
</html><br />
<br />
<br />
<br />
=<partinfo>K238008</partinfo>: ''virA''=<br />
We wanted to use this part in our project, but could only obtain unexpected/faulty restriction patterns. Finally we chose to sequence the part, hoping to find the cause for the maintained restriction patterns. Unfortunately we could not approve the sequence of <partinfo>BBa_K238008</partinfo> deposited in the parts registry so that we chose to design our own VirA BioBrick. I strongly recommend using our VirA since it has been approved by multiple means, e.g. restriction patterns and sequencing (<partinfo>K389001</partinfo>).<br />
<br />
=<partinfo>BBa_K238011</partinfo>: ''vir''-promoter=<br />
We made a restriction analysis and sequenced parts of this BioBrick.<br />
<br />
<br />
=<partinfo>P1010</partinfo>: ''ccdB''-gene=<br />
The ''ccdB'' gene targets the gyrase of ''Escherichia coli'' and is lethal for all ''E. coli'' strains without the gyrase mutation gyrA462 ([http://openwetware.org/wiki/CcdB Openwetware]). The ''ccdB'' BioBrick is used for the 3A-assembly as a positive selection marker. <br />
We transformed this BioBrick into ''E. coli'' JM109, DH5α, TOP10, XL1-Blue, EC100D and DB3.1. ''E. coli'' JM109, XL1-Blue and DH5α seem to be ''ccdB'' resistant because there were as much colonies after P1010 transformation as observed with DB3.1. The P1010 works as expected in ''E. coli'' TOP10, EC100D (no colonies after transformation) and DB3.1 (many colonies after transformation).<br />
<br />
<br />
<center>Table 1: Results of the transformation of the cell-death gene ''ccdB'', BioBrick <partinfo>P1010</partinfo>, into different strains of ''E. coli''. <br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| ''E. coli'' strain<br />
!style="border-style: solid; border-width: 1px"| Resistant to ''ccdB''?<br />
!style="border-style: solid; border-width: 1px"| Expected result?<br />
!style="border-style: solid; border-width: 1px"| Gyrase genotype <br> ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T39-47PNXC3-F3&_user=2459438&_coverDate=01%2F28%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=dfcdeab4c210c1f4ec70de318d013c15&searchtype=a Metcalf ''et al.'', 1994]; [http://openwetware.org/wiki/E._coli_genotypes Openwetware])<br />
|-<br />
|style="border-style: solid; border-width: 1px"| DB3.1<br />
|style="border-style: solid; border-width: 1px"| yes <br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| gyrA462<br />
|-<br />
|style="border-style: solid; border-width: 1px"| DH5α<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| gyrA96<br />
|-<br />
|style="border-style: solid; border-width: 1px"| EC100D<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| WT<br />
|-<br />
|style="border-style: solid; border-width: 1px"| JM109<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| gyrA96<br />
|-<br />
|style="border-style: solid; border-width: 1px"| TOP10<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| WT<br />
|-<br />
|style="border-style: solid; border-width: 1px"| XL1-Blue<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| gyrA96<br />
|-<br />
|}<br />
</center><br />
<br />
<br />
It seems that not only the gyrase mutation gyrA462 is causing a ''ccdB'' resistance. Also the gyrase mutation gyrA96 gives ''E. coli'' a ''ccdB'' resistance. This should be kept in mind when assembling BioBricks with the 3A assembly.<br />
<br />
=<partinfo>K389004</partinfo>: Luciferase from pGL4.10[luc2]=<br />
[[Team:Bielefeld-Germany/Results/Characterization/K389004#mRFP vs. luciferase as reporter gene | For a comparison between mRFP and luciferase as reporter genes click here. ]]<br />
<br />
Some important parameters determined by the characterization experiments are shown in tab. 2. For more information concerning these experiments click on the corresponding link in tab. 2 or click here: <br />
<br />
<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389004">Detailed information...</a></div></html><br />
<br />
<br />
<center>Table 2: Parameters for <partinfo>K389004</partinfo>. <br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Experiment<br />
!style="border-style: solid; border-width: 1px"| Result<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Accumulation of luciferase | Behaviour during cultivation]]<br />
|style="border-style: solid; border-width: 1px"| <br />
* production is growth dependent<br />
* degradation in stationary growth phase<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Kinetic of luciferin conversion | Kinetic of luciferin conversion]]<br />
|style="border-style: solid; border-width: 1px"| max. output between 20 - 40 s<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Sensitivity | Limit of detection (LOD)]]<br />
|style="border-style: solid; border-width: 1px"| 162 RLU ~ 0.3 % of <partinfo>J23103</partinfo> output<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Sensitivity | Limit of quantification (LOQ)]]<br />
|style="border-style: solid; border-width: 1px"| 306 RLU ~ 0.7 % of <partinfo>J23103</partinfo> output<br />
|}<br />
</center><br />
<br />
=<partinfo>K389011</partinfo>: VirA screening device=<br />
<br />
<br />
<br />
<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389011">Detailed information...</a></div></html><br />
<br />
<br />
<br />
[[Image:Bielefeld_LD50_Graph2.jpg|600px|thumb|center|Ratio of surviving colonies of ''E. coli'' EC100D carrying unmutated <partinfo>K389010</partinfo> and <partinfo>K389011</partinfo> plated on PA agar plates with chloramphenicol, ampicillin and different concentrations of kanamycin. Comparison between cells that were induced with acetosyringone with cells that were not induced.]]<br />
<br />
<br />
<br />
=<partinfo>K389015</partinfo>: VirA/G reporter device with luciferase=<br />
Some important parameters determined by the characterization experiments are shown in tab. X. For more information concerning these experiments click on the corresponding link in tab. X or click here: <br />
<br />
<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389015">Detailed information...</a></div></html><br />
<br />
<br />
<center>Table X: Parameters for <partinfo>K389015</partinfo>. <br />
{|{{Table}}<br />
!Experiment<br />
!Characteristic<br />
!Value<br />
|-<br />
|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Transfer function | Transfer Function]]<br />
|Maximum induction level<br />
|2.2 fold<br />
|-<br />
|Maximum induction level reached<br />
|200 µM acetosyringone<br />
|-<br />
|Hill coefficient<br />
|1.09<br />
|-<br />
|Switch Point<br />
|31.6 µM acetosyringone<br />
|-<br />
|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Growth functions and Luciferase expression for BBa_K389015 | Doubling time / h]]<br />
|without plasmid<br />
|1.98<br />
|-<br />
|carrying K389015<br />
|2.24<br />
|-<br />
|carrying K389015 with 400 µM acetosyringone<br />
|2.67<br />
|-<br />
|rowspan="2"|Response time<br />
|Induction: [[Team:Bielefeld-Germany/Results/Characterization/K389015#Response time | exponential phase]]<br />
|>1 h<br />
|-<br />
|Induction: [[Team:Bielefeld-Germany/Results/Characterization/K389015#Data Analysis | begin of cultivation]]<br />
|max. induction at OD<sub>600</sub> = 1 +/- 0.5<br />
|-<br />
|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Plasmid conformation analysis | Conformation analysis]]<br />
|ratio ccc monomer / %<br />
|91<br />
|-<br />
|ratio ccc dimer / %<br />
|3.7<br />
|-<br />
|ratio oc forms / %<br />
|5.3<br />
|-<br />
|}<br />
</center><br />
<br />
=<partinfo>K389016</partinfo>: VirA/G reporter device with mRFP=<br />
<br />
Protocols for [https://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Cultivation_for_measuring_mRFP_and_Luciferase_expression Cultivation] and [https://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Measuring_of_mRFP Measurement]<br />
<br />
Some important parameters determined by the characterization experiments are shown in tab. X. For more information concerning these experiments click on the corresponding link in tab. X or click here: <br />
<br />
<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389016">Detailed information...</a></div></html><br />
<br />
<br />
<center>Table X: Parameters for <partinfo>K389016</partinfo>. <br />
{|{{Table}}<br />
!Experiment<br />
!Characteristic<br />
!Value<br />
|-<br />
|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Transfer function of BBa_K389016 | Transfer Function]]<br />
|Maximum induction level<br />
|2.6 fold<br />
|-<br />
|Maximum induction level reached<br />
|150 µM acetosyringone<br />
|-<br />
|Hill coefficient<br />
|1.67<br />
|-<br />
|Switch Point<br />
|26.5 µM acetosyringone<br />
|-<br />
|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Growth functions and mRFP expression for BBa_K389016 | Doubling time / h]]<br />
|without plasmid<br />
|1.98<br />
|-<br />
|carrying K389016<br />
|2.57<br />
|-<br />
|carrying K389016 with 150 µM acetosyringone<br />
|2.77<br />
|-<br />
|carrying K389016 with 1000 µM acetosyringone<br />
|3.01<br />
|-<br />
|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Plasmid conformation analysis | Conformation analysis]]<br />
|ratio ccc monomer / %<br />
|91.2<br />
|-<br />
|ratio ccc dimer / %<br />
|3.2<br />
|-<br />
|ratio oc forms / %<br />
|5.6<br />
|-<br />
|rowspan="5"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Different possible inducers | Inducers]]<br />
|Induction by<br />
|Acetosyringone<br />
|-<br />
|rowspan="4"|No Induction by<br />
|Capsaicin<br />
|-<br />
|Dopamine<br />
|-<br />
|Homovanillic acid <br />
|-<br />
|3-Methoxytyramine <br />
|}<br />
</center><br />
<br />
=<partinfo>K389052</partinfo>: Tightly regulated ''lac'' operon with mRFP readout=<br />
This construct was plated for plasmid isolation in a ''lacI<sup>q</sup>'' negative ''E. coli'' strain after assembly - and we have never seen such red plates when working with constructs with mRFP downstream of a promoter. This ''lac'' operon definitely shows a very high basal transcription, so it is not tightly repressed. It seems that the ''lacI'' repressor <partinfo>BBa_C0012</partinfo> is not suitable for this purpose due to its LVA degradation tag or it does not work properly. Another indicator for this assumption is the experience page of <partinfo>C0012</partinfo>.<br />
<br />
=<partinfo>K389421</partinfo>, <partinfo>K389422</partinfo>, <partinfo>K389423</partinfo>: Sensitivity Tuner amlified Vir-test system=<br />
<br />
By self designed PCR-Primer we excluded terminal GFP and the initial promoter pBAD/araC, for replacing our own VirB promotor and reporter gene luc (luciferase). Primers were designed for sensitivity tuner [http://partsregistry.org/Part:BBa_I746370 I746370], [http://partsregistry.org/Part:BBa_I746380 I746380] and [http://partsregistry.org/Part:BBa_I746390 I746390] so that standard assembly would be possible. Assembling of PCR-products took place by Silver Assembly.<br />
<br />
'''Accomplishment'''<br />
<br />
'''PCR-Primer Design'''<br />
<br />
Primer forward activator phage P2:<br />
<br />
5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GTT TCA TTG TCC TTT ATG CC-3`<br />
<br />
Primer forward activator phage PSP3:<br />
<br />
5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GAT GCA CTG CCC GTT ATG- 3`<br />
<br />
Primer forward activator phage phi R73:<br />
<br />
5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GCG CTG CCC TTT CTG-3`<br />
<br />
Primer backward Promotor PF from phage P2:<br />
<br />
5`-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TTC TCC TCT TTC TCT AGT AAG TGG- 3`<br />
<br />
<br />
'''Characterization tests'''<br />
<br />
Cultivation was done by induction with Acetosyringone at 50 µM. Controls were not induced Sensitivity Tuner devices as well as induced and not induced nativ system ([http://partsregistry.org/Part:BBa_K389015 K389015]; without tuning elements). Induction was done upon inoculation. Measuring point for amplification factor calculation was OD 1.0. ([https://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Cultivation_for_measuring_mRFP_and_Luciferase_expression Protocols])<br />
<br />
<br />
'''Results'''<br />
<br />
Three sensitivity tuned Vir-Gen sensing systems were obtained: [http://partsregistry.org/Part:BBa_K389421 K389421], [http://partsregistry.org/Part:BBa_K389422 K389422] and [http://partsregistry.org/Part:BBa_K389423 K389423] distinguishing by the amplification level of luc transcription.<br />
<br />
[[Image:ST Tuner.png|600px|thumb|center| '''Figure 1: Amplification factor of induced, 50 µM Acetosyringone (red) and not induced (green) modified Sensitivity Tuner K389421, K389422 and K389423, Standard deviation shown.''']]<br />
<br />
The amplification factor was received by apply [http://partsregistry.org/Part:BBa_K389015 K389015] as reference. Amplification calculation was done by normalizing relative luminescence units emitted from luciferase per OD.<br />
Output-signal amplification is in the induced contructs (red) [http://partsregistry.org/Part:BBa_K389422 K389422] and [http://partsregistry.org/Part:BBa_K389423 K389423] 100 and respectively 200 fold higher than in not induced controls (green). An exception is K389422 were induced and not indiced system revealed analog results. Corresponding to data of iGEM Team, Cambridge 2009, K389423 (originated from [http://partsregistry.org/Part:BBa_I746390 I746390]) shows the highest amplification rate of all tested Sensitivity Tuners. Our results indicate to higher amplification rate of [http://partsregistry.org/Part:BBa_K389421 K389421] than [http://partsregistry.org/Part:BBa_K389422 K389422] of 100 fold under induced conditions. The controls also show high basal transcription rates.<br />
<br />
Because there is small difference in induced and not induced system visible and basal transcription rates are high, we assume that the sensitivity tuning constructs are not well applicable for luciferase measurements.<br />
<br />
For further theory click [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Read_out_system Read out system]<br />
<br />
=References=<br />
<br />
<br />
<br />
<br />
*Behrens B, Eppendorf AG, Laborpraxis, Nr.20, Reinste Plasmid-DNA in nur 9 Minuten.<br />
<br />
*http://openwetware.org/wiki/CcdB, CcdB (seen on 10.10.10).<br />
<br />
*http://openwetware.org/wiki/E._coli_genotypes, E. coli genotypes (seen on 10.10.10).<br />
<br />
*Metcalf, WW ''et al.'' (1994) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T39-47PNXC3-F3&_user=2459438&_coverDate=01%2F28%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=dfcdeab4c210c1f4ec70de318d013c15&searchtype=a ''Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6Kλ origin plasmids at different copy numbers''], Gene 138(1):1-7.<br />
<br />
*Stadler J, Lemmens R, Nyhammar T 2004, ''Plasmid DNA purification'', The J. of Gene Medicine,Vol.6, pp.54–S66</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/CharacterizationTeam:Bielefeld-Germany/Results/Characterization2010-10-27T22:44:46Z<p>Nkessler: /* K389012: VirA reporter system with luciferase */</p>
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<br />
<br />
=<partinfo>K238008</partinfo>: ''virA''=<br />
We wanted to use this part in our project, but could only obtain unexpected/faulty restriction patterns. Finally we chose to sequence the part, hoping to find the cause for the maintained restriction patterns. Unfortunately we could not approve the sequence of <partinfo>BBa_K238008</partinfo> deposited in the parts registry so that we chose to design our own VirA BioBrick. I strongly recommend using our VirA since it has been approved by multiple means, e.g. restriction patterns and sequencing (<partinfo>K389001</partinfo>).<br />
<br />
=<partinfo>BBa_K238011</partinfo>: ''vir''-promoter=<br />
We made a restriction analysis and sequenced parts of this BioBrick.<br />
<br />
<br />
=<partinfo>P1010</partinfo>: ''ccdB''-gene=<br />
The ''ccdB'' gene targets the gyrase of ''Escherichia coli'' and is lethal for all ''E. coli'' strains without the gyrase mutation gyrA462 ([http://openwetware.org/wiki/CcdB Openwetware]). The ''ccdB'' BioBrick is used for the 3A-assembly as a positive selection marker. <br />
We transformed this BioBrick into ''E. coli'' JM109, DH5α, TOP10, XL1-Blue, EC100D and DB3.1. ''E. coli'' JM109, XL1-Blue and DH5α seem to be ''ccdB'' resistant because there were as much colonies after P1010 transformation as observed with DB3.1. The P1010 works as expected in ''E. coli'' TOP10, EC100D (no colonies after transformation) and DB3.1 (many colonies after transformation).<br />
<br />
<br />
<center>Table 1: Results of the transformation of the cell-death gene ''ccdB'', BioBrick <partinfo>P1010</partinfo>, into different strains of ''E. coli''. <br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| ''E. coli'' strain<br />
!style="border-style: solid; border-width: 1px"| Resistant to ''ccdB''?<br />
!style="border-style: solid; border-width: 1px"| Expected result?<br />
!style="border-style: solid; border-width: 1px"| Gyrase genotype <br> ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T39-47PNXC3-F3&_user=2459438&_coverDate=01%2F28%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=dfcdeab4c210c1f4ec70de318d013c15&searchtype=a Metcalf ''et al.'', 1994]; [http://openwetware.org/wiki/E._coli_genotypes Openwetware])<br />
|-<br />
|style="border-style: solid; border-width: 1px"| DB3.1<br />
|style="border-style: solid; border-width: 1px"| yes <br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| gyrA462<br />
|-<br />
|style="border-style: solid; border-width: 1px"| DH5α<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| gyrA96<br />
|-<br />
|style="border-style: solid; border-width: 1px"| EC100D<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| WT<br />
|-<br />
|style="border-style: solid; border-width: 1px"| JM109<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| gyrA96<br />
|-<br />
|style="border-style: solid; border-width: 1px"| TOP10<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| WT<br />
|-<br />
|style="border-style: solid; border-width: 1px"| XL1-Blue<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| gyrA96<br />
|-<br />
|}<br />
</center><br />
<br />
<br />
It seems that not only the gyrase mutation gyrA462 is causing a ''ccdB'' resistance. Also the gyrase mutation gyrA96 gives ''E. coli'' a ''ccdB'' resistance. This should be kept in mind when assembling BioBricks with the 3A assembly.<br />
<br />
=<partinfo>K389004</partinfo>: Luciferase from pGL4.10[luc2]=<br />
[[Team:Bielefeld-Germany/Results/Characterization/K389004#mRFP vs. luciferase as reporter gene | For a comparison between mRFP and luciferase as reporter genes click here. ]]<br />
<br />
Some important parameters determined by the characterization experiments are shown in tab. 2. For more information concerning these experiments click on the corresponding link in tab. 2 or click here: <br />
<br />
<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389004">Detailed information...</a></div></html><br />
<br />
<br />
<center>Table 2: Parameters for <partinfo>K389004</partinfo>. <br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Experiment<br />
!style="border-style: solid; border-width: 1px"| Result<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Accumulation of luciferase | Behaviour during cultivation]]<br />
|style="border-style: solid; border-width: 1px"| <br />
* production is growth dependent<br />
* degradation in stationary growth phase<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Kinetic of luciferin conversion | Kinetic of luciferin conversion]]<br />
|style="border-style: solid; border-width: 1px"| max. output between 20 - 40 s<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Sensitivity | Limit of detection (LOD)]]<br />
|style="border-style: solid; border-width: 1px"| 162 RLU ~ 0.3 % of <partinfo>J23103</partinfo> output<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Sensitivity | Limit of quantification (LOQ)]]<br />
|style="border-style: solid; border-width: 1px"| 306 RLU ~ 0.7 % of <partinfo>J23103</partinfo> output<br />
|}<br />
</center><br />
<br />
=<partinfo>K389011</partinfo>: VirA screening device=<br />
<br />
<br />
<br />
<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389011">Detailed information...</a></div></html><br />
<br />
<br />
<br />
[[Image:Bielefeld_LD50_Graph2.jpg|600px|thumb|center|Ratio of surviving colonies of ''E. coli'' EC100D carrying unmutated <partinfo>K389010</partinfo> and <partinfo>K389011</partinfo> plated on PA agar plates with chloramphenicol, ampicillin and different concentrations of kanamycin. Comparison between cells that were induced with acetosyringone with cells that were not induced.]]<br />
<br />
<br />
<br />
=<partinfo>K389015</partinfo>: VirA/G reporter device with luciferase=<br />
Some important parameters determined by the characterization experiments are shown in tab. X. For more information concerning these experiments click on the corresponding link in tab. X or click here: <br />
<br />
<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389015">Detailed information...</a></div></html><br />
<br />
<br />
<center>Table X: Parameters for <partinfo>K389015</partinfo>. <br />
{|{{Table}}<br />
!Experiment<br />
!Characteristic<br />
!Value<br />
|-<br />
|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Transfer function | Transfer Function]]<br />
|Maximum induction level<br />
|2.2 fold<br />
|-<br />
|Maximum induction level reached<br />
|200 µM acetosyringone<br />
|-<br />
|Hill coefficient<br />
|1.09<br />
|-<br />
|Switch Point<br />
|31.6 µM acetosyringone<br />
|-<br />
|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Growth functions and Luciferase expression for BBa_K389015 | Doubling time / h]]<br />
|without plasmid<br />
|1.98<br />
|-<br />
|carrying K389015<br />
|2.24<br />
|-<br />
|carrying K389015 with 400 µM acetosyringone<br />
|2.67<br />
|-<br />
|rowspan="2"|Response time<br />
|Induction: [[Team:Bielefeld-Germany/Results/Characterization/K389015#Response time | exponential phase]]<br />
|>1 h<br />
|-<br />
|Induction: [[Team:Bielefeld-Germany/Results/Characterization/K389015#Data Analysis | begin of cultivation]]<br />
|max. induction at OD<sub>600</sub> = 1 +/- 0.5<br />
|-<br />
|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Plasmid conformation analysis | Conformation analysis]]<br />
|ratio ccc monomer / %<br />
|91<br />
|-<br />
|ratio ccc dimer / %<br />
|3.7<br />
|-<br />
|ratio oc forms / %<br />
|5.3<br />
|-<br />
|}<br />
</center><br />
<br />
=<partinfo>K389016</partinfo>: VirA/G reporter device with mRFP=<br />
<br />
Protocols for [https://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Cultivation_for_measuring_mRFP_and_Luciferase_expression Cultivation] and [https://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Measuring_of_mRFP Measurement]<br />
<br />
Some important parameters determined by the characterization experiments are shown in tab. X. For more information concerning these experiments click on the corresponding link in tab. X or click here: <br />
<br />
<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389016">Detailed information...</a></div></html><br />
<br />
<br />
<center>Table X: Parameters for <partinfo>K389016</partinfo>. <br />
{|{{Table}}<br />
!Experiment<br />
!Characteristic<br />
!Value<br />
|-<br />
|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Transfer function of BBa_K389016 | Transfer Function]]<br />
|Maximum induction level<br />
|2.6 fold<br />
|-<br />
|Maximum induction level reached<br />
|150 µM acetosyringone<br />
|-<br />
|Hill coefficient<br />
|1.67<br />
|-<br />
|Switch Point<br />
|26.5 µM acetosyringone<br />
|-<br />
|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Growth functions and mRFP expression for BBa_K389016 | Doubling time / h]]<br />
|without plasmid<br />
|1.98<br />
|-<br />
|carrying K389016<br />
|2.57<br />
|-<br />
|carrying K389016 with 150 µM acetosyringone<br />
|2.77<br />
|-<br />
|carrying K389016 with 1000 µM acetosyringone<br />
|3.01<br />
|-<br />
|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Plasmid conformation analysis | Conformation analysis]]<br />
|ratio ccc monomer / %<br />
|91.2<br />
|-<br />
|ratio ccc dimer / %<br />
|3.2<br />
|-<br />
|ratio oc forms / %<br />
|5.6<br />
|-<br />
|rowspan="5"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Different possible inducers | Inducers]]<br />
|Induction by<br />
|Acetosyringone<br />
|-<br />
|rowspan="4"|No Induction by<br />
|Capsaicin<br />
|-<br />
|Dopamine<br />
|-<br />
|Homovanillic acid <br />
|-<br />
|3-Methoxytyramine <br />
|}<br />
</center><br />
<br />
=<partinfo>K389052</partinfo>: tightly regulated ''lac'' operon with mRFP readout=<br />
This construct was plated for plasmid isolation in a ''lacI<sup>q</sup>'' negative ''E. coli'' strain after assembly - and we have never seen such red plates when working with constructs with mRFP downstream of a promoter. This ''lac'' operon definitely shows a very high basal transcription, so it is not tightly repressed. It seems that the ''lacI'' repressor <partinfo>BBa_C0012</partinfo> is not suitable for this purpose due to its LVA degradation tag or it does not work properly. Another indicator for this assumption is the experience page of <partinfo>C0012</partinfo>.<br />
<br />
=<partinfo>K389421</partinfo>, <partinfo>K389422</partinfo>, <partinfo>K389423</partinfo>: Sensitivity Tuner amlified Vir-test system=<br />
<br />
By self designed PCR-Primer we excluded terminal GFP and the initial promoter pBAD/araC, for replacing our own VirB promotor and reporter gene luc (luciferase). Primers were designed for sensitivity tuner [http://partsregistry.org/Part:BBa_I746370 I746370], [http://partsregistry.org/Part:BBa_I746380 I746380] and [http://partsregistry.org/Part:BBa_I746390 I746390] so that standard assembly would be possible. Assembling of PCR-products took place by Silver Assembly.<br />
<br />
'''Accomplishment'''<br />
<br />
'''PCR-Primer Design'''<br />
<br />
Primer forward activator phage P2:<br />
<br />
5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GTT TCA TTG TCC TTT ATG CC-3`<br />
<br />
Primer forward activator phage PSP3:<br />
<br />
5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GAT GCA CTG CCC GTT ATG- 3`<br />
<br />
Primer forward activator phage phi R73:<br />
<br />
5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GCG CTG CCC TTT CTG-3`<br />
<br />
Primer backward Promotor PF from phage P2:<br />
<br />
5`-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TTC TCC TCT TTC TCT AGT AAG TGG- 3`<br />
<br />
<br />
'''Characterization tests'''<br />
<br />
Cultivation was done by induction with Acetosyringone at 50 µM. Controls were not induced Sensitivity Tuner devices as well as induced and not induced nativ system ([http://partsregistry.org/Part:BBa_K389015 K389015]; without tuning elements). Induction was done upon inoculation. Measuring point for amplification factor calculation was OD 1.0. ([https://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Cultivation_for_measuring_mRFP_and_Luciferase_expression Protocols])<br />
<br />
<br />
'''Results'''<br />
<br />
Three sensitivity tuned Vir-Gen sensing systems were obtained: [http://partsregistry.org/Part:BBa_K389421 K389421], [http://partsregistry.org/Part:BBa_K389422 K389422] and [http://partsregistry.org/Part:BBa_K389423 K389423] distinguishing by the amplification level of luc transcription.<br />
<br />
[[Image:ST Tuner.png|600px|thumb|center| '''Figure 1: Amplification factor of induced, 50 µM Acetosyringone (red) and not induced (green) modified Sensitivity Tuner K389421, K389422 and K389423, Standard deviation shown.''']]<br />
<br />
The amplification factor was received by apply [http://partsregistry.org/Part:BBa_K389015 K389015] as reference. Amplification calculation was done by normalizing relative luminescence units emitted from luciferase per OD.<br />
Output-signal amplification is in the induced contructs (red) [http://partsregistry.org/Part:BBa_K389422 K389422] and [http://partsregistry.org/Part:BBa_K389423 K389423] 100 and respectively 200 fold higher than in not induced controls (green). An exception is K389422 were induced and not indiced system revealed analog results. Corresponding to data of iGEM Team, Cambridge 2009, K389423 (originated from [http://partsregistry.org/Part:BBa_I746390 I746390]) shows the highest amplification rate of all tested Sensitivity Tuners. Our results indicate to higher amplification rate of [http://partsregistry.org/Part:BBa_K389421 K389421] than [http://partsregistry.org/Part:BBa_K389422 K389422] of 100 fold under induced conditions. The controls also show high basal transcription rates.<br />
<br />
Because there is small difference in induced and not induced system visible and basal transcription rates are high, we assume that the sensitivity tuning constructs are not well applicable for luciferase measurements.<br />
<br />
For further theory click [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Read_out_system Read out system]<br />
<br />
=References=<br />
<br />
<br />
<br />
<br />
*Behrens B, Eppendorf AG, Laborpraxis, Nr.20, Reinste Plasmid-DNA in nur 9 Minuten.<br />
<br />
*http://openwetware.org/wiki/CcdB, CcdB (seen on 10.10.10).<br />
<br />
*http://openwetware.org/wiki/E._coli_genotypes, E. coli genotypes (seen on 10.10.10).<br />
<br />
*Metcalf, WW ''et al.'' (1994) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T39-47PNXC3-F3&_user=2459438&_coverDate=01%2F28%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=dfcdeab4c210c1f4ec70de318d013c15&searchtype=a ''Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6Kλ origin plasmids at different copy numbers''], Gene 138(1):1-7.<br />
<br />
*Stadler J, Lemmens R, Nyhammar T 2004, ''Plasmid DNA purification'', The J. of Gene Medicine,Vol.6, pp.54–S66</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Judging/BioBricksTeam:Bielefeld-Germany/Judging/BioBricks2010-10-27T22:22:47Z<p>Nkessler: </p>
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= Biobricks =<br />
Biobricks are the central figure of the iGEM competition. It is pretty important to construct functional Biobricks, in order to avoid problems for other teams. Unfortunately we had to handle some problems with Biobricks from the database. Although we create a lot of Biobricks, we therefore focused on creating good and functional working Biobricks. Our best three Biobricks are listed below. The detailed information of the Biobricks can be received by using the links.<br />
<br />
<br />
== K389004 ==<br />
<br />
Luciferase from PGL4.10 [Luc2]:<br />
<br />
Partinfo of the Biobrick <partinfo>K389004</partinfo><br />
<br />
[[Team:Bielefeld-Germany/Results/Characterization#BBa_K389004:_Luciferase_from_pGL4.10.5Bluc2.5D| Characterization of K389004]]<br />
<br />
<br />
== K389015 ==<br />
<br />
Final sensor system with luc readout:<br />
<br />
Partinfo of the Biobrick <partinfo>K389015</partinfo><br />
<br />
[[Team:Bielefeld-Germany/Results/Characterization#BBa_K389015:_VirA.2FG_reporter_device_with_luciferase| Characterization of K389015]]<br />
<br />
== K389016 ==<br />
<br />
Final sensor system with mRFP readout:<br />
<br />
Partinfo of the Biobrick <partinfo>K389016</partinfo><br />
<br />
[[Team:Bielefeld-Germany/Results/Characterization#BBa_K389016:_VirA.2FG_reporter_device_with_mRFP| Characterization of K389016]]</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/UsedTeam:Bielefeld-Germany/Results/Used2010-10-27T21:36:01Z<p>Nkessler: </p>
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<li><a href="/Team:Bielefeld-Germany/Results/Submitted">Submitted</a></li><br />
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= Used BioBricks =<br />
<br />
This is a list of all BioBricks we used from the registry or created by ourselves and a short explanation, why and / or how we used them. The list is sorted alphabetically. <br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| BioBrick (name and partnumber)<br />
!style="border-style: solid; border-width: 1px"| Status<br />
!style="border-style: solid; border-width: 1px"| Comment<br />
|-<br />
|style="border-style: solid; border-width: 1px"| double terminator, <partinfo>B0017</partinfo>: <partinfo>B0017 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a double terminator which contains two <partinfo>B0010</partinfo> terminators. We used this double terminator instead of the commonly used <partinfo>B0015</partinfo> double terminator (which was assembled from the <partinfo>B0010</partinfo> and the <partinfo>B0012</partinfo> terminator) because of problems mentioned in the partsregistry with the <partinfo>B0012</partinfo> terminator. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| RBS, <partinfo>B0034</partinfo>: <partinfo>B0034 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
The strong ribosomal binding site (RBS) AGGAG was always assembled. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''lacI'' gene, <partinfo>C0012</partinfo>: <partinfo>C0012 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a gene for the lac repressor ''lacI'' with a LVA degradation tag. We used this BioBrick to build a tightly regulated ''lac''-operon <partinfo>K389050</partinfo> with low basal transcription. It seems that this ''lacI'' is either degraded too quickly or it does not work properly because it is not possible to repress the ''lac'' operon with this part. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| mRFP, <partinfo>E1010</partinfo>: <partinfo>E1010 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick was used as a reporter gene, ''e.g.'' in the construct <partinfo>K389013</partinfo>. mRFP as a reporter gene has the advantage that it is [[Team:Bielefeld-Germany/Results/Characterization/K389004#mRFP_vs._luciferase_as_reporter_gene | easy to measure and comparatively stable]]. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''lacI<sup>q</sup>'' promoter, <partinfo>I14032</partinfo>: <partinfo>I14032 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This promoter is a strong constitutive promoter which is natively located upstream of the ''lacI'' gene in ''E. coli'' lacI<sup>q</sup> strains. It was used to build a tightly regulated ''lac''-operon <partinfo>K389050</partinfo> in which it was assembled upstream of the ''lacI'' gene from the partsregistry <partinfo>C0012</partinfo>. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| sensitivity tuner, <partinfo>I746370</partinfo>: <partinfo>I746370 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
We used this BioBrick to amplify our read-out signal luciferase in the part <partinfo>K389401</partinfo>. This BioBrick is supposed to amplify the effect of a PoPS device 15 - 20 fold compared to a system without sensitivity tuner ([https://2007.igem.org/Cambridge/Amplifier_project#Results Cambridge, iGEM 2007, amplifier project]). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| sensitivity tuner, <partinfo>I746380</partinfo>: <partinfo>I746380 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
We used this BioBrick to amplify our read-out signal luciferase in the part <partinfo>K389402</partinfo>. This BioBrick is supposed to amplify the effect of a PoPS device 10 fold compared to a system without sensitivity tuner ([https://2007.igem.org/Cambridge/Amplifier_project#Results Cambridge, iGEM 2007, amplifier project]). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| sensitivity tuner, <partinfo>I746390</partinfo>: <partinfo>I746390 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
We used this BioBrick to amplify our read-out signal luciferase in the part <partinfo>K389403</partinfo>. This BioBrick is supposed to amplify the effect of a PoPS device 30 - 35 fold compared to a system without sensitivity tuner ([https://2007.igem.org/Cambridge/Amplifier_project#Results Cambridge, iGEM 2007, amplifier project]). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| mRFP generator, <partinfo>J04450</partinfo>: <partinfo>J04450 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick was used as a visible selection marker for cloning other BioBricks into the <partinfo>pSB1C3</partinfo> plasmid to submit them to the registry. The promoter in this mRFP generator is pretty strong so it is easy to separate colonies which carry this BioBrick from colonies which do not carry this BioBrick anymore. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| strong constitutive promoter, <partinfo>J23102</partinfo>: <partinfo>J23102 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
A strong constitutive promoter which was used to create BioBrick <partinfo>K389318</partinfo> to test the firefly luciferase BioBrick <partinfo>K389004</partinfo> and the luciferase measurement system. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| weak constitutive promoter, <partinfo>J23103</partinfo>: <partinfo>J23103 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
A weak constitutive promoter which was used to create BioBrick <partinfo>K389302</partinfo> to test the firefly luciferase BioBrick <partinfo>K389004</partinfo> and the luciferase measurement system. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| medium strong constitutive promoter, <partinfo>J23110</partinfo>: <partinfo>J23110 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This medium strong constitutive promoter from the registry was assembled before our BioBricks, so they are neither expressed too strong, so the cell suffers from metabolomic stress, nor too weak, so there is no visible effect of our BioBricks. This BioBrick was used ''e.g.'' in <partinfo>K389010</partinfo> or <partinfo>K389011</partinfo>. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| medium strong constitutive promoter, <partinfo>J23115</partinfo>: <partinfo>J23115 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
A medium strong constitutive promoter which was used to create BioBrick <partinfo>K389307</partinfo> to test the firefly luciferase BioBrick <partinfo>K389004</partinfo> and the luciferase measurement system. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| R6K origin, <partinfo>J61001</partinfo>: <partinfo>J61001 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick was used to create a plasmid without ColE1 ori. The R6K ori has a different compatibility class than the ColE1 ori. So it is used for a two plasmid screening system. The R6K ori is a medium copy plasmid in pir+ / pir116 ''E. coli'' strains like EC100D but it does not replicate in pir- strains like ''E. coli'' TOP10. So it is easy to separate a ColE1 ori plasmid from another with R6K ori by simply transforming both to ''e.g. E. coli'' TOP10. <br />
The R6K ori was cloned into the pSB1C3 plasmid. Subsequently, the plasmid containing the R6K ori was digested with Hin6I. The largest fragment was extracted from an agarose gel and ligated with T4 DNA Polymerase. Thereby the ColE1 ori was removed, leaving a small fragment of about 30 bp.<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'', <partinfo>K238008</partinfo>: <partinfo>K238008 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry, fixed<br />
|style="border-style: solid; border-width: 1px"| <br />
We wanted to use this ''virA'' BioBrick from the registry but problems occurred ([[Team:Bielefeld-Germany/Results/Characterization | characterization]]), so we created our own ''virA'' from ''Agrobacterium tumefaciens'' C58 TI-plasmid. Our own (working) ''virA'' has the partnumber <partinfo>K389001</partinfo>. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''vir''-promoter, <partinfo>K238011</partinfo>: <partinfo>K238011 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry, fixed<br />
|style="border-style: solid; border-width: 1px"| <br />
The same problems occurred as with the ''virA'' BioBrick - this one from the registry [[Team:Bielefeld-Germany/Results/Characterization | does not work properly]], so we made a ''vir''-promoter by ourselves (<partinfo>K389003</partinfo>). The part that was sent to us is actually a ''tetR'' gene (<partinfo>C0040</partinfo>) under the control of a constitutive promoter (<partinfo>J23105</partinfo>) before a double terminator (<partinfo>B0015</partinfo>, compare our [[Team:Bielefeld-Germany/Results/Sequencing | sequencing results]]). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'', <partinfo>K389001</partinfo>: <partinfo>K389001 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| new<br />
|style="border-style: solid; border-width: 1px"| <br />
The VirA receptor is used by ''A. tumefaciens'' to detect acetosyringone and other phenolic substances which are secreted by plants after injury. In presence of these substances VirA phosphorylates itself and afterwards VirG, a response regulator which activates ''vir'' promoters. These promoters control genes which are used for infecting the injured plant. <br />
This ''virA'' gene was isolated from the TI-plasmid of ''A. tumefaciens'' C58. An illegal PstI restriction site was removed by site-directed mutagenesis. The functionality of this BioBrick was proved with <partinfo>K389015</partinfo> and <partinfo>K389016</partinfo>. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| mutated ''virG'', <partinfo>K389002</partinfo>: <partinfo>K389002 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| synthesized, new<br />
|style="border-style: solid; border-width: 1px"| <br />
This is the VirG response regulator from the VirA/G receptor system. The VirA/G receptor system is used by ''A. tumefaciens'' to detect phenolic substances secreted by injured plants, ''e.g.'' acetosyringone. In presence of these substances, VirG is activated by the VirA receptor and induces the transcription of genes under the control of a ''vir'' promoter. Normally, VirG needs the ''rpoA'' gene (RNA polymerase subunit) from ''A. tumefaciens'' to work in ''Escherichia coli'' but this ''virG'' BioBrick is mutated, so it works with the ''rpoA'' subunit from ''E. coli''. For this reason the point mutations G56V and I77V were brought into the gene ([http://www.springerlink.com/content/wmq06kua5qkma1au/ YC Jung ''et al.'', 2004]). Because this BioBrick is synthesized (Mr. Gene GmbH), codon usage is optimized for ''E. coli'' and illegal restriction sites were removed. When you use this ''virG'' gene in a VirA/G signaling system you do not need <partinfo>BBa_K238010</partinfo> anymore to get the system working in ''E. coli''.<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''vir''-promoter, <partinfo>K389003</partinfo>: <partinfo>K389003 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| new<br />
|style="border-style: solid; border-width: 1px"| <br />
''Vir''-promoters from ''A. tumefaciens'' are induced by phosphorylated VirG response regulators and control genes for infecting plants in their natural host. They are part of the VirA/G signal transduction system. This is a ''vir'' promoter from ''A. tumefaciens'' C58. It is located on the TI-plasmid upstream the ''virB'' genes.<br />
|-<br />
|style="border-style: solid; border-width: 1px"| firefly luciferase, <partinfo>K389004</partinfo>: <partinfo>K389004 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| new<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a BioBrick for a firefly (''Photinus pyralis'') luciferase. It is a [[Team:Bielefeld-Germany/Results/Characterization/K389004#Sensitivity | sensitive reporter gene]]. It was isolated from Promega's pGL4.10[luc2] plasmid. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| Kanamycin resistance gene, <partinfo>K389005</partinfo>: <partinfo>K389005 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| new / modified<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a kanamycin / neomycin resistance gene without promoter or RBS. It is used for directed evolution of ''virA'' and mutated ''virA'' screenings, respectively. It was isolated from the BioBrick <partinfo>P1003</partinfo> (a kanamycin resistance gene with promoter and RBS). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'' generator, <partinfo>K389010</partinfo>: <partinfo>K389010 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This part is for expressing a VirA receptor. A medium strong constitutive promoter is used, so there is enough VirA receptor expressed but not too much so the cell suffers from the expression (VirA is a membrane protein). This version exists in a <partinfo>pSB1C3</partinfo> and a <partinfo>pSB1AT3</partinfo> vector. We used the BioBrick in the <partinfo>pSB1AT3</partinfo> plasmid for error-prone PCR and later in our two plasmid screening system together with the <partinfo>K389011</partinfo> VirA-screening device. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'' screening device, <partinfo>K389011</partinfo>: <partinfo>K389011 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick contains the new, mutated ''virG'' which works in ''E. coli'' without the ''rpoA'' gene from ''A. tumefaciens'' which is expressed constitutively and a kanamycin resistance gene which is under the control of a ''vir'' promoter. It is used to screen mutants of ''virA'' which are on a different plasmid in a <partinfo>K389010</partinfo>-like part and were created by error-prone PCR. The better the VirA receptor recognizes a substance the more resistant the cell is against kanamycin. This device has to be on a plasmid with different ori than the ''virA'' which should be screened (different compatibility classes). We cloned this BioBrick into a plasmid with R6K ori which only replicates in pir+ or pir116 strains (''e.g.'' ''E. coli'' EC100D), so we can easily seperate the two plasmids by transforming them into strains without Pir protein (''e.g. E. coli'' TOP10). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'' reporter device with luc, <partinfo>K389012</partinfo>: <partinfo>K389012 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick is similar to <partinfo>K389011</partinfo> but with the reporter luciferase instead of an antibiotic resistance gene. With this BioBrick it is possible to measure the activity of the ''vir'' promoter and the VirA receptor, respectively. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'' reporter device with mRFP, <partinfo>K389013</partinfo>: <partinfo>K389013 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick is similar to <partinfo>K389011</partinfo> but with the reporter mRFP instead of an antibiotic resistance gene. With this BioBrick it is possible to measure the activity of the ''vir'' promoter and the VirA receptor, respectively. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'' screening approach test device, <partinfo>K389014</partinfo>: <partinfo>K389014 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick is an assembly of <partinfo>K389010</partinfo> and <partinfo>K389011</partinfo>. It is used for testing the general screening concept of expressing an antibiotic resistance with a ''vir'' promoter after induction with acetosyringone (the natural VirA inductor). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''vir'' promoter characterisation part with luc, <partinfo>K389015</partinfo>: <partinfo>K389015 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick is an assembly of <partinfo>K389010</partinfo> and <partinfo>K389012</partinfo>. It is used for [[Team:Bielefeld-Germany/Results/Characterization/K389015 | characterizing]] the natural VirA/G system and the ''vir'' promoter, respectively. It is also possible to demonstrate the functionality of four new created BioBricks in one device. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''vir'' promoter characterisation part with mRFP, <partinfo>K389016</partinfo>: <partinfo>K389016 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick is an assembly of <partinfo>K389010</partinfo> and <partinfo>K389013</partinfo>. It is used for [[Team:Bielefeld-Germany/Results/Characterization/K389016 | characterizing]] the natural VirA/G system and the ''vir'' promoter, respectively. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''vir'' promoter part, <partinfo>K389017</partinfo>: <partinfo>K389017 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a complete VirA/G signaling system without a reporter gene, so any gene of interest can be assembled to a ''vir'' promoter. We assembled some sensitivity tuners and a luciferase gene behind this BioBrick, so we get an enhanced luciferase signal (''e.g.'' in part <partinfo>K389421</partinfo>). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| tightly controlled ''lac'' operator, <partinfo>K389050</partinfo>: <partinfo>K389050 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
A lac operator is combined with a lacI<sup>q</sup> so there is always enough ''lacI'' to repress the lac operator. The system is still inducible with IPTG but it has a low basal transcription (in theory). This part should be used to test the sensitivity of the luciferase BioBrick <partinfo>K389004</partinfo> and compare it to the sensitivity of the mRFP BioBrick <partinfo>E1010</partinfo>. But the part did not work as expected - it had a very high basal expression. The problem is probably the ''lacI'' gene <partinfo>C0012</partinfo> from the registry which has a degradation tag. Either this tag leads to that quick degradation, that the ''lacI'' repressor cannot work as a repressor anymore or the repressor itself does not work properly. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| tightly controlled ''lac'' operator + luc, <partinfo>K389051</partinfo>: <partinfo>K389051 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
The part <partinfo>K389050</partinfo> is combined with a luciferase gene as reporter. But <partinfo>K389050</partinfo> does not work as expected so this part could not be used for its original purpose (demonstrating the sensitivity of the firefly luciferase). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| tightly controlled ''lac'' operator + mRFP, <partinfo>K389052</partinfo>: <partinfo>K389052 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
The part <partinfo>K389050</partinfo> is combined with a mRFP gene as reporter. But <partinfo>K389050</partinfo> does not work as expected so this part could not be used for its original purpose (comparing the sensitivity of mRFP with the sensitivity of the firefly luciferase). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| weak constitutive promoter + luc, <partinfo>K389302</partinfo>: <partinfo>K389302 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
Luciferase under the control of a weak constitutive promoter. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| medium strong constitutive promoter + luc, <partinfo>K389307</partinfo>: <partinfo>K389307 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
Luciferase under the control of a medium strong constitutive promoter. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| strong constitutive promoter + luc, <partinfo>K389318</partinfo>: <partinfo>K389318 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
Luciferase under the control of a strong constitutive promoter. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| sensitivity tuner no. 1 without promoter + luc readout, <partinfo>K389401</partinfo>: <partinfo>K389401 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| new / modified<br />
|style="border-style: solid; border-width: 1px"| <br />
The <partinfo>I746370</partinfo> sensitivity tuner was modified: the arabinose promoter and the GFP readout were erased from the original part and a firefly luciferase BioBrick was added as a new readout. This part can be used behind a random promoter and gives an enhanced luciferase readout signal. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| sensitivity tuner no. 2 without promoter + luc readout, <partinfo>K389402</partinfo>: <partinfo>K389402 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| new / modified<br />
|style="border-style: solid; border-width: 1px"| <br />
The <partinfo>I746380</partinfo> sensitivity tuner was modified: the arabinose promoter and the GFP readout were erased from the original part and a firefly luciferase BioBrick was added as a new readout. This part can be used behind a random promoter and gives an enhanced luciferase readout signal. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| sensitivity tuner no. 3 without promoter + luc readout, <partinfo>K389403</partinfo>: <partinfo>K389403 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| new / modified<br />
|style="border-style: solid; border-width: 1px"| <br />
The <partinfo>I746390</partinfo> sensitivity tuner was modified: the arabinose promoter and the GFP readout were erased from the original part and a firefly luciferase BioBrick was added as a new readout. This part can be used behind a random promoter and gives an enhanced luciferase readout signal. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'' reporter device with sensitivity tuner no. 1 + luc readout, <partinfo>K389411</partinfo>: <partinfo>K389411 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a modified <partinfo>I746370</partinfo> sensitivity tuner with luciferase readout behind a ''vir'' promoter. This part also contains a ''virG'' gene under the control of a constitutive promoter. The plan is to assemble a mutated ''virA'' from a <partinfo>K389010</partinfo>-like BioBrick in front of this part to get a strong readout signal in order to reach our goal - make spiciness visible. The mutated ''virA'' is generated with error-prone PCR and screened together with the <partinfo>K389011</partinfo> Biobrick. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'' reporter device with sensitivity tuner no. 2 + luc readout, <partinfo>K389412</partinfo>: <partinfo>K389412 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a modified <partinfo>I746380</partinfo> sensitivity tuner with luciferase readout behind a ''vir'' promoter. This part also contains a ''virG'' gene under the control of a constitutive promoter. The plan is to assemble a mutated ''virA'' from a <partinfo>K389010</partinfo>-like BioBrick in front of this part to get a strong readout signal in order to reach our goal - make spiciness visible. The mutated ''virA'' is generated with error-prone PCR and screened together with the <partinfo>K389011</partinfo> Biobrick. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'' reporter device with sensitivity tuner no. 3 + luc readout, <partinfo>K389413</partinfo>: <partinfo>K389413 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a modified <partinfo>I746390</partinfo> sensitivity tuner with luciferase readout behind a ''vir'' promoter. This part also contains a ''virG'' gene under the control of a constitutive promoter. The plan is to assemble a mutated ''virA'' from a <partinfo>K389010</partinfo>-like BioBrick in front of this part to get a strong readout signal in order to reach our goal - make spiciness visible. The mutated ''virA'' is generated with error-prone PCR and screened together with the <partinfo>K389011</partinfo> Biobrick. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''vir'' promoter characterisation part with sensitivity tuner no. 1 + luc readout, <partinfo>K389421</partinfo>: <partinfo>K389421 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a modified <partinfo>I746370</partinfo> sensitivity tuner with luciferase readout behind a ''vir'' promoter characterisation BioBrick, so it also contains a ''virG'' and an unmutated ''virA'' gene under the control of a constitutive promoter. This BioBrick is used to prove the function of the modified sensitivity tuner and to check whether the sensitivity tuner enhances the luciferase signal enough to make it visible. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''vir'' promoter characterisation part with sensitivity tuner no. 2 + luc readout, <partinfo>K389422</partinfo>: <partinfo>K389422 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a modified <partinfo>I746380</partinfo> sensitivity tuner with luciferase readout behind a ''vir'' promoter characterisation BioBrick, so it also contains a ''virG'' and an unmutated ''virA'' gene under the control of a constitutive promoter. This BioBrick is used to prove the function of the modified sensitivity tuner and to check whether the sensitivity tuner enhances the luciferase signal enough to make it visible. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''vir'' promoter characterisation part with sensitivity tuner no. 3 + luc readout, <partinfo>K389423</partinfo>: <partinfo>K389423 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a modified <partinfo>I746370</partinfo> sensitivity tuner with luciferase readout behind a ''vir'' promoter characterisation BioBrick, so it also contains a ''virG'' and an unmutated ''virA'' gene under the control of a constitutive promoter. This BioBrick is used to prove the function of the modified sensitivity tuner and to check whether the sensitivity tuner enhances the luciferase signal enough to make it visible. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| Kanamycin-resistance cassette, <partinfo>P1003</partinfo>: <partinfo>P1003 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This kanamycin resistance cassette BioBrick was used as a template to create <partinfo>K389005</partinfo>. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''ccdB''-gene, <partinfo>P1010</partinfo>: <partinfo>P1010 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
The ''ccdB'' gene targets the gyrase of ''Escherichia coli'' and is lethal for all ''E. coli'' strains without the gyrase mutation gyrA462 ([http://openwetware.org/wiki/CcdB openwetware]). The ''ccdB'' BioBrick is used for the 3A-assembly as a positive selection marker. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| high-copy plasmid with Amp and Tet resistance, <partinfo>pSB1AT3</partinfo>: <partinfo>pSB1AT3 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This plasmid was used in 3A-assemblies and as the backbone of the mutated and unmutated <partinfo>K389010</partinfo> part. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| high-copy plasmid with Cm resistance, <partinfo>pSB1C3</partinfo>: <partinfo>pSB1C3 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This is the plasmid used to submit the BioBricks. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''lac'' operator, <partinfo>R0010</partinfo>: <partinfo>R0010 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a ''lac'' operator which is inducible with IPTG and repressed by ''lacI''. It was used to build a tightly regulated ''lac'' operator (<partinfo>K389050</partinfo>) by adding a ''lacI'' repressor gene under the control of the lacI<sup>q</sup> promoter. <br />
|}<br />
<br />
<br />
=References=<br />
*[https://2007.igem.org/Cambridge/Amplifier_project#Results Cambridge, iGEM 2007, amplifier project].<br />
<br />
*http://openwetware.org/wiki/CcdB, CcdB (seen on 10.10.10).<br />
<br />
*YC Jung ''et al.'' (2004) [http://www.springerlink.com/content/wmq06kua5qkma1au/ ''Mutants of ''Agrobacterium tumefaciens virG'' Gene That Activate Transcription of ''vir'' Promoter in ''Escherichia coli''''], Current Microbiol 49:334-340.</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/UsedTeam:Bielefeld-Germany/Results/Used2010-10-27T21:06:08Z<p>Nkessler: </p>
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= Used BioBricks =<br />
<br />
This is a list of all BioBricks we used from the registry or created by ourselves and a short explanation, why and / or how we used them. The list is sorted alphabetically. <br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| BioBrick (name and partnumber)<br />
!style="border-style: solid; border-width: 1px"| Status<br />
!style="border-style: solid; border-width: 1px"| Comment<br />
|-<br />
|style="border-style: solid; border-width: 1px"| double terminator, <partinfo>B0017</partinfo>: <partinfo>B0017 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a double terminator which contains two <partinfo>B0010</partinfo> terminators. We used this double terminator instead of the commonly used <partinfo>B0015</partinfo> double terminator (which was assembled from the <partinfo>B0010</partinfo> and the <partinfo>B0012</partinfo> terminator) because of problems mentioned in the partsregistry with the <partinfo>B0012</partinfo> terminator. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| RBS, <partinfo>B0034</partinfo>: <partinfo>B0034 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
The strong ribosomal binding site (RBS) AGGAG was always assembled. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''lacI'' gene, <partinfo>C0012</partinfo>: <partinfo>C0012 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a gene for the lac repressor ''lacI'' with a LVA degradation tag. We used this BioBrick to build a tightly regulated ''lac''-operon <partinfo>K389050</partinfo> with low basal transcription. It seems that this ''lacI'' is either degraded too quickly or it does not work properly because it is not possible to repress the ''lac'' operon with this part. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| mRFP, <partinfo>E1010</partinfo>: <partinfo>E1010 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick was used as a reporter gene, ''e.g.'' in the construct <partinfo>K389013</partinfo>. mRFP as a reporter gene has the advantage that it is [[Team:Bielefeld-Germany/Results/Characterization/K389004#mRFP_vs._luciferase_as_reporter_gene | easy to measure and comparatively stable]]. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''lacI<sup>q</sup>'' promoter, <partinfo>I14032</partinfo>: <partinfo>I14032 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This promoter is a strong constitutive promoter which is natively located upstream of the ''lacI'' gene in ''E. coli'' lacI<sup>q</sup> strains. It was used to build a tightly regulated ''lac''-operon <partinfo>K389050</partinfo> in which it was assembled upstream of the ''lacI'' gene from the partsregistry <partinfo>C0012</partinfo>. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| sensitivity tuner, <partinfo>I746370</partinfo>: <partinfo>I746370 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
We used this BioBrick to amplify our read-out signal luciferase in the part <partinfo>K389401</partinfo>. This BioBrick is supposed to amplify the effect of a PoPS device 15 - 20 fold compared to a system without sensitivity tuner ([http://parts.mit.edu/igem07/index.php/Cambridge/Amplifier_project#Results Cambridge, iGEM 2007, amplifier project]). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| sensitivity tuner, <partinfo>I746380</partinfo>: <partinfo>I746380 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
We used this BioBrick to amplify our read-out signal luciferase in the part <partinfo>K389402</partinfo>. This BioBrick is supposed to amplify the effect of a PoPS device 10 fold compared to a system without sensitivity tuner ([http://parts.mit.edu/igem07/index.php/Cambridge/Amplifier_project#Results Cambridge, iGEM 2007, amplifier project]). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| sensitivity tuner, <partinfo>I746390</partinfo>: <partinfo>I746390 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
We used this BioBrick to amplify our read-out signal luciferase in the part <partinfo>K389403</partinfo>. This BioBrick is supposed to amplify the effect of a PoPS device 30 - 35 fold compared to a system without sensitivity tuner ([http://parts.mit.edu/igem07/index.php/Cambridge/Amplifier_project#Results Cambridge, iGEM 2007, amplifier project]). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| mRFP generator, <partinfo>J04450</partinfo>: <partinfo>J04450 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick was used as a visible selection marker for cloning other BioBricks into the <partinfo>pSB1C3</partinfo> plasmid to submit them to the registry. The promoter in this mRFP generator is pretty strong so it is easy to separate colonies which carry this BioBrick from colonies which do not carry this BioBrick anymore. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| strong constitutive promoter, <partinfo>J23102</partinfo>: <partinfo>J23102 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
A strong constitutive promoter which was used to create BioBrick <partinfo>K389318</partinfo> to test the firefly luciferase BioBrick <partinfo>K389004</partinfo> and the luciferase measurement system. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| weak constitutive promoter, <partinfo>J23103</partinfo>: <partinfo>J23103 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
A weak constitutive promoter which was used to create BioBrick <partinfo>K389302</partinfo> to test the firefly luciferase BioBrick <partinfo>K389004</partinfo> and the luciferase measurement system. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| medium strong constitutive promoter, <partinfo>J23110</partinfo>: <partinfo>J23110 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This medium strong constitutive promoter from the registry was assembled before our BioBricks, so they are neither expressed too strong, so the cell suffers from metabolomic stress, nor too weak, so there is no visible effect of our BioBricks. This BioBrick was used ''e.g.'' in <partinfo>K389010</partinfo> or <partinfo>K389011</partinfo>. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| medium strong constitutive promoter, <partinfo>J23115</partinfo>: <partinfo>J23115 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
A medium strong constitutive promoter which was used to create BioBrick <partinfo>K389307</partinfo> to test the firefly luciferase BioBrick <partinfo>K389004</partinfo> and the luciferase measurement system. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| R6K origin, <partinfo>J61001</partinfo>: <partinfo>J61001 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick was used to create a plasmid without ColE1 ori. The R6K ori has a different compatibility class than the ColE1 ori. So it is used for a two plasmid screening system. The R6K ori is a medium copy plasmid in pir+ / pir116 ''E. coli'' strains like EC100D but it does not replicate in pir- strains like ''E. coli'' TOP10. So it is easy to separate a ColE1 ori plasmid from another with R6K ori by simply transforming both to ''e.g. E. coli'' TOP10. <br />
The R6K ori was cloned into the pSB1C3 plasmid. Subsequently, the plasmid containing the R6K ori was digested with Hin6I. The largest fragment was extracted from an agarose gel and ligated with T4 DNA Polymerase. Thereby the ColE1 ori was removed, leaving a small fragment of about 30 bp.<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'', <partinfo>K238008</partinfo>: <partinfo>K238008 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry, fixed<br />
|style="border-style: solid; border-width: 1px"| <br />
We wanted to use this ''virA'' BioBrick from the registry but problems occurred ([[Team:Bielefeld-Germany/Results/Characterization | characterization]]), so we created our own ''virA'' from ''Agrobacterium tumefaciens'' C58 TI-plasmid. Our own (working) ''virA'' has the partnumber <partinfo>K389001</partinfo>. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''vir''-promoter, <partinfo>K238011</partinfo>: <partinfo>K238011 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry, fixed<br />
|style="border-style: solid; border-width: 1px"| <br />
The same problems occurred as with the ''virA'' BioBrick - this one from the registry [[Team:Bielefeld-Germany/Results/Characterization | does not work properly]], so we made a ''vir''-promoter by ourselves (<partinfo>K389003</partinfo>). The part that was sent to us is actually a ''tetR'' gene (<partinfo>C0040</partinfo>) under the control of a constitutive promoter (<partinfo>J23105</partinfo>) before a double terminator (<partinfo>B0015</partinfo>, compare our [[Team:Bielefeld-Germany/Results/Sequencing | sequencing results]]). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'', <partinfo>K389001</partinfo>: <partinfo>K389001 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| new<br />
|style="border-style: solid; border-width: 1px"| <br />
The VirA receptor is used by ''A. tumefaciens'' to detect acetosyringone and other phenolic substances which are secreted by plants after injury. In presence of these substances VirA phosphorylates itself and afterwards VirG, a response regulator which activates ''vir'' promoters. These promoters control genes which are used for infecting the injured plant. <br />
This ''virA'' gene was isolated from the TI-plasmid of ''A. tumefaciens'' C58. An illegal PstI restriction site was removed by site-directed mutagenesis. The functionality of this BioBrick was proved with <partinfo>K389015</partinfo> and <partinfo>K389016</partinfo>. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| mutated ''virG'', <partinfo>K389002</partinfo>: <partinfo>K389002 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| synthesized, new<br />
|style="border-style: solid; border-width: 1px"| <br />
This is the VirG response regulator from the VirA/G receptor system. The VirA/G receptor system is used by ''A. tumefaciens'' to detect phenolic substances secreted by injured plants, ''e.g.'' acetosyringone. In presence of these substances, VirG is activated by the VirA receptor and induces the transcription of genes under the control of a ''vir'' promoter. Normally, VirG needs the ''rpoA'' gene (RNA polymerase subunit) from ''A. tumefaciens'' to work in ''Escherichia coli'' but this ''virG'' BioBrick is mutated, so it works with the ''rpoA'' subunit from ''E. coli''. For this reason the point mutations G56V and I77V were brought into the gene ([http://www.springerlink.com/content/wmq06kua5qkma1au/ YC Jung ''et al.'', 2004]). Because this BioBrick is synthesized (Mr. Gene GmbH), codon usage is optimized for ''E. coli'' and illegal restriction sites were removed. When you use this ''virG'' gene in a VirA/G signaling system you do not need <partinfo>BBa_K238010</partinfo> anymore to get the system working in ''E. coli''.<br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''vir''-promoter, <partinfo>K389003</partinfo>: <partinfo>K389003 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| new<br />
|style="border-style: solid; border-width: 1px"| <br />
''Vir''-promoters from ''A. tumefaciens'' are induced by phosphorylated VirG response regulators and control genes for infecting plants in their natural host. They are part of the VirA/G signal transduction system. This is a ''vir'' promoter from ''A. tumefaciens'' C58. It is located on the TI-plasmid upstream the ''virB'' genes.<br />
|-<br />
|style="border-style: solid; border-width: 1px"| firefly luciferase, <partinfo>K389004</partinfo>: <partinfo>K389004 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| new<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a BioBrick for a firefly (''Photinus pyralis'') luciferase. It is a [[Team:Bielefeld-Germany/Results/Characterization/K389004#Sensitivity | sensitive reporter gene]]. It was isolated from Promega's pGL4.10[luc2] plasmid. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| Kanamycin resistance gene, <partinfo>K389005</partinfo>: <partinfo>K389005 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| new / modified<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a kanamycin / neomycin resistance gene without promoter or RBS. It is used for directed evolution of ''virA'' and mutated ''virA'' screenings, respectively. It was isolated from the BioBrick <partinfo>P1003</partinfo> (a kanamycin resistance gene with promoter and RBS). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'' generator, <partinfo>K389010</partinfo>: <partinfo>K389010 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This part is for expressing a VirA receptor. A medium strong constitutive promoter is used, so there is enough VirA receptor expressed but not too much so the cell suffers from the expression (VirA is a membrane protein). This version exists in a <partinfo>pSB1C3</partinfo> and a <partinfo>pSB1AT3</partinfo> vector. We used the BioBrick in the <partinfo>pSB1AT3</partinfo> plasmid for error-prone PCR and later in our two plasmid screening system together with the <partinfo>K389011</partinfo> VirA-screening device. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'' screening device, <partinfo>K389011</partinfo>: <partinfo>K389011 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick contains the new, mutated ''virG'' which works in ''E. coli'' without the ''rpoA'' gene from ''A. tumefaciens'' which is expressed constitutively and a kanamycin resistance gene which is under the control of a ''vir'' promoter. It is used to screen mutants of ''virA'' which are on a different plasmid in a <partinfo>K389010</partinfo>-like part and were created by error-prone PCR. The better the VirA receptor recognizes a substance the more resistant the cell is against kanamycin. This device has to be on a plasmid with different ori than the ''virA'' which should be screened (different compatibility classes). We cloned this BioBrick into a plasmid with R6K ori which only replicates in pir+ or pir116 strains (''e.g.'' ''E. coli'' EC100D), so we can easily seperate the two plasmids by transforming them into strains without Pir protein (''e.g. E. coli'' TOP10). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'' reporter device with luc, <partinfo>K389012</partinfo>: <partinfo>K389012 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick is similar to <partinfo>K389011</partinfo> but with the reporter luciferase instead of an antibiotic resistance gene. With this BioBrick it is possible to measure the activity of the ''vir'' promoter and the VirA receptor, respectively. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'' reporter device with mRFP, <partinfo>K389013</partinfo>: <partinfo>K389013 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick is similar to <partinfo>K389011</partinfo> but with the reporter mRFP instead of an antibiotic resistance gene. With this BioBrick it is possible to measure the activity of the ''vir'' promoter and the VirA receptor, respectively. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'' screening approach test device, <partinfo>K389014</partinfo>: <partinfo>K389014 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick is an assembly of <partinfo>K389010</partinfo> and <partinfo>K389011</partinfo>. It is used for testing the general screening concept of expressing an antibiotic resistance with a ''vir'' promoter after induction with acetosyringone (the natural VirA inductor). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''vir'' promoter characterisation part with luc, <partinfo>K389015</partinfo>: <partinfo>K389015 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick is an assembly of <partinfo>K389010</partinfo> and <partinfo>K389012</partinfo>. It is used for [[Team:Bielefeld-Germany/Results/Characterization/K389015 | characterizing]] the natural VirA/G system and the ''vir'' promoter, respectively. It is also possible to demonstrate the functionality of four new created BioBricks in one device. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''vir'' promoter characterisation part with mRFP, <partinfo>K389016</partinfo>: <partinfo>K389016 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This BioBrick is an assembly of <partinfo>K389010</partinfo> and <partinfo>K389013</partinfo>. It is used for [[Team:Bielefeld-Germany/Results/Characterization/K389016 | characterizing]] the natural VirA/G system and the ''vir'' promoter, respectively. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''vir'' promoter part, <partinfo>K389017</partinfo>: <partinfo>K389017 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a complete VirA/G signaling system without a reporter gene, so any gene of interest can be assembled to a ''vir'' promoter. We assembled some sensitivity tuners and a luciferase gene behind this BioBrick, so we get an enhanced luciferase signal (''e.g.'' in part <partinfo>K389421</partinfo>). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| tightly controlled ''lac'' operator, <partinfo>K389050</partinfo>: <partinfo>K389050 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
A lac operator is combined with a lacI<sup>q</sup> so there is always enough ''lacI'' to repress the lac operator. The system is still inducible with IPTG but it has a low basal transcription (in theory). This part should be used to test the sensitivity of the luciferase BioBrick <partinfo>K389004</partinfo> and compare it to the sensitivity of the mRFP BioBrick <partinfo>E1010</partinfo>. But the part did not work as expected - it had a very high basal expression. The problem is probably the ''lacI'' gene <partinfo>C0012</partinfo> from the registry which has a degradation tag. Either this tag leads to that quick degradation, that the ''lacI'' repressor cannot work as a repressor anymore or the repressor itself does not work properly. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| tightly controlled ''lac'' operator + luc, <partinfo>K389051</partinfo>: <partinfo>K389051 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
The part <partinfo>K389050</partinfo> is combined with a luciferase gene as reporter. But <partinfo>K389050</partinfo> does not work as expected so this part could not be used for its original purpose (demonstrating the sensitivity of the firefly luciferase). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| tightly controlled ''lac'' operator + mRFP, <partinfo>K389052</partinfo>: <partinfo>K389052 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
The part <partinfo>K389050</partinfo> is combined with a mRFP gene as reporter. But <partinfo>K389050</partinfo> does not work as expected so this part could not be used for its original purpose (comparing the sensitivity of mRFP with the sensitivity of the firefly luciferase). <br />
|-<br />
|style="border-style: solid; border-width: 1px"| weak constitutive promoter + luc, <partinfo>K389302</partinfo>: <partinfo>K389302 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
Luciferase under the control of a weak constitutive promoter. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| medium strong constitutive promoter + luc, <partinfo>K389307</partinfo>: <partinfo>K389307 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
Luciferase under the control of a medium strong constitutive promoter. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| strong constitutive promoter + luc, <partinfo>K389318</partinfo>: <partinfo>K389318 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
Luciferase under the control of a strong constitutive promoter. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| sensitivity tuner no. 1 without promoter + luc readout, <partinfo>K389401</partinfo>: <partinfo>K389401 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| new / modified<br />
|style="border-style: solid; border-width: 1px"| <br />
The <partinfo>I746370</partinfo> sensitivity tuner was modified: the arabinose promoter and the GFP readout were erased from the original part and a firefly luciferase BioBrick was added as a new readout. This part can be used behind a random promoter and gives an enhanced luciferase readout signal. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| sensitivity tuner no. 2 without promoter + luc readout, <partinfo>K389402</partinfo>: <partinfo>K389402 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| new / modified<br />
|style="border-style: solid; border-width: 1px"| <br />
The <partinfo>I746380</partinfo> sensitivity tuner was modified: the arabinose promoter and the GFP readout were erased from the original part and a firefly luciferase BioBrick was added as a new readout. This part can be used behind a random promoter and gives an enhanced luciferase readout signal. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| sensitivity tuner no. 3 without promoter + luc readout, <partinfo>K389403</partinfo>: <partinfo>K389403 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| new / modified<br />
|style="border-style: solid; border-width: 1px"| <br />
The <partinfo>I746390</partinfo> sensitivity tuner was modified: the arabinose promoter and the GFP readout were erased from the original part and a firefly luciferase BioBrick was added as a new readout. This part can be used behind a random promoter and gives an enhanced luciferase readout signal. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'' reporter device with sensitivity tuner no. 1 + luc readout, <partinfo>K389411</partinfo>: <partinfo>K389411 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a modified <partinfo>I746370</partinfo> sensitivity tuner with luciferase readout behind a ''vir'' promoter. This part also contains a ''virG'' gene under the control of a constitutive promoter. The plan is to assemble a mutated ''virA'' from a <partinfo>K389010</partinfo>-like BioBrick in front of this part to get a strong readout signal in order to reach our goal - make spiciness visible. The mutated ''virA'' is generated with error-prone PCR and screened together with the <partinfo>K389011</partinfo> Biobrick. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'' reporter device with sensitivity tuner no. 2 + luc readout, <partinfo>K389412</partinfo>: <partinfo>K389412 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a modified <partinfo>I746380</partinfo> sensitivity tuner with luciferase readout behind a ''vir'' promoter. This part also contains a ''virG'' gene under the control of a constitutive promoter. The plan is to assemble a mutated ''virA'' from a <partinfo>K389010</partinfo>-like BioBrick in front of this part to get a strong readout signal in order to reach our goal - make spiciness visible. The mutated ''virA'' is generated with error-prone PCR and screened together with the <partinfo>K389011</partinfo> Biobrick. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''virA'' reporter device with sensitivity tuner no. 3 + luc readout, <partinfo>K389413</partinfo>: <partinfo>K389413 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a modified <partinfo>I746390</partinfo> sensitivity tuner with luciferase readout behind a ''vir'' promoter. This part also contains a ''virG'' gene under the control of a constitutive promoter. The plan is to assemble a mutated ''virA'' from a <partinfo>K389010</partinfo>-like BioBrick in front of this part to get a strong readout signal in order to reach our goal - make spiciness visible. The mutated ''virA'' is generated with error-prone PCR and screened together with the <partinfo>K389011</partinfo> Biobrick. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''vir'' promoter characterisation part with sensitivity tuner no. 1 + luc readout, <partinfo>K389421</partinfo>: <partinfo>K389421 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a modified <partinfo>I746370</partinfo> sensitivity tuner with luciferase readout behind a ''vir'' promoter characterisation BioBrick, so it also contains a ''virG'' and an unmutated ''virA'' gene under the control of a constitutive promoter. This BioBrick is used to proove the function of the modified sensitivity tuner and to check whether the sensitivity tuner enhances the luciferase signal enough to make it visible. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''vir'' promoter characterisation part with sensitivity tuner no. 2 + luc readout, <partinfo>K389422</partinfo>: <partinfo>K389422 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a modified <partinfo>I746380</partinfo> sensitivity tuner with luciferase readout behind a ''vir'' promoter characterisation BioBrick, so it also contains a ''virG'' and an unmutated ''virA'' gene under the control of a constitutive promoter. This BioBrick is used to proove the function of the modified sensitivity tuner and to check whether the sensitivity tuner enhances the luciferase signal enough to make it visible. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''vir'' promoter characterisation part with sensitivity tuner no. 3 + luc readout, <partinfo>K389423</partinfo>: <partinfo>K389423 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| composite<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a modified <partinfo>I746370</partinfo> sensitivity tuner with luciferase readout behind a ''vir'' promoter characterisation BioBrick, so it also contains a ''virG'' and an unmutated ''virA'' gene under the control of a constitutive promoter. This BioBrick is used to proove the function of the modified sensitivity tuner and to check whether the sensitivity tuner enhances the luciferase signal enough to make it visible. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| Kanamycin-resistance cassette, <partinfo>P1003</partinfo>: <partinfo>P1003 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This kanamycin resistance cassette BioBrick was used as a template to create <partinfo>K389005</partinfo>. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''ccdB''-gene, <partinfo>P1010</partinfo>: <partinfo>P1010 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
The ''ccdB'' gene targets the gyrase of ''Escherichia coli'' and is lethal for all ''E. coli'' strains without the gyrase mutation gyrA462 ([http://openwetware.org/wiki/CcdB openwetware]). The ''ccdB'' BioBrick is used for the 3A-assembly as a positive selection marker. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| high-copy plasmid with Amp and Tet resistance, <partinfo>pSB1AT3</partinfo>: <partinfo>pSB1AT3 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This plasmid was used in 3A-assemblies and as the backbone of the mutated and unmutated <partinfo>K389010</partinfo> part. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| high-copy plasmid with Cm resistance, <partinfo>pSB1C3</partinfo>: <partinfo>pSB1C3 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This is the plasmid used to submit the BioBricks. <br />
|-<br />
|style="border-style: solid; border-width: 1px"| ''lac'' operator, <partinfo>R0010</partinfo>: <partinfo>R0010 SpecifiedComponents</partinfo><br />
|style="border-style: solid; border-width: 1px"| from registry<br />
|style="border-style: solid; border-width: 1px"| <br />
This is a ''lac'' operator which is inducible with IPTG and repressed by ''lacI''. It was used to build a tightly regulated ''lac'' operator (<partinfo>K389050</partinfo>) by adding a ''lacI'' repressor gene under the control of the lacI<sup>q</sup> promoter. <br />
|}<br />
<br />
<br />
=References=<br />
*[http://parts.mit.edu/igem07/index.php/Cambridge/Amplifier_project#Results Cambridge, iGEM 2007, amplifier project].<br />
<br />
*http://openwetware.org/wiki/CcdB, CcdB (seen on 10.10.10).<br />
<br />
*YC Jung ''et al.'' (2004) [http://www.springerlink.com/content/wmq06kua5qkma1au/ ''Mutants of ''Agrobacterium tumefaciens virG'' Gene That Activate Transcription of ''vir'' Promoter in ''Escherichia coli''''], Current Microbiol 49:334-340.</div>Nkesslerhttp://2010.igem.org/File:Bielefeld_luc.jpgFile:Bielefeld luc.jpg2010-10-27T18:16:05Z<p>Nkessler: </p>
<hr />
<div></div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Public_Relations/FotosTeam:Bielefeld-Germany/Public Relations/Fotos2010-10-27T18:07:31Z<p>Nkessler: /* Foto Gallery */</p>
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<li><a href="/Team:Bielefeld-Germany/Public_Relations">Public Relations</a></li><br />
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<li><a href="/Team:Bielefeld-Germany/Public_Relations/Press">Press</a></li><br />
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=Foto Gallery=<br />
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<a class="boxGallery" href="http://www.igem-bielefeld.de/galleries/Waffle1/index.html"><img src="http://www.igem-bielefeld.de/galleries/Waffle1/images/thumb/_6366588556.jpg"></a><br />
<div class="boxHeading">Waffles, the first</div><br />
<div class="boxDescription">Our first Waffle-Baking-Action. The making of :-)<br/><br/>See our second waffle/infopoint in <a class="boxGallery" href="http://www.igem-bielefeld.de/galleries/Waffles/index.html">the other gallery</a>.</div><br />
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<a class="boxGallery" href="http://www.igem-bielefeld.de/galleries/Waffles/index.html"><img src="http://www.igem-bielefeld.de/galleries/Waffles/images/thumb/_5460391312.jpg"></a><br />
<div class="boxHeading">Waffles!</div><br />
<div class="boxDescription">We brought some waffles to the folks of Bielefeld and took the chance to explain our project to interested students, lecturers and other waffle-fans.<br /><br />Plus powdered sugar!<br />Have a look at us feeding Bielefeld.</div><br />
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<a class="boxGallery" href="http://www.igem-bielefeld.de/galleries/ScienceCafe/index.html"><img src="http://www.igem-bielefeld.de/galleries/ScienceCafe/images/thumb/_4221476122.jpg"></a><br />
<div class="boxHeading">Public Discussion in the Science Café</div><br />
<div class="boxDescription">Have a look at the pictures from our talk, where we presented ourselves and our project to the public in a local café. For further reading you might be interested in <a href="/Team:Bielefeld-Germany/Public_Relations/Public_Discussion#Public_Events">a more detailled review</a>.</div><br />
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<a class="boxGallery" href="http://www.igem-bielefeld.de/galleries/Laboratory/index.html"><img src="http://www.igem-bielefeld.de/galleries/Laboratory/images/thumb/_0355605927.jpg"></a><br />
<div class="boxHeading">One day in the lab</div><br />
<div class="boxDescription">Some pictures of our working place and some of our crew.</div><br />
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<a class="boxGallery" href="http://www.igem-bielefeld.de/galleries/BlackWhiteLab/index.html"><img src="http://www.igem-bielefeld.de/galleries/BlackWhiteLab/images/thumb/_3708890847.jpg"></a><br />
<div class="boxHeading">Black and White</div><br />
<div class="boxDescription">Some expressions of our laboratory.</div><br />
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<a class="boxGallery" href="http://www.igem-bielefeld.de/galleries/University/index.html"><img src="http://www.igem-bielefeld.de/galleries/University/images/thumb/_8050695755.jpg"></a><br />
<div class="boxHeading">Some impressions of our university</div><br />
<div class="boxDescription">Bielefeld University as we see it every day. There are some really nice black-and-white fotographs in this gallery. Please have a look!</div><br />
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<a class="boxGallery" href="http://www.igem-bielefeld.de/galleries/University2/index.html"><img src="http://www.igem-bielefeld.de/galleries/University2/images/thumb/_0645898983.jpg"></a><br />
<div class="boxHeading">Road to Bielefeld University</div><br />
<div class="boxDescription">That is what we see, when we arrive at our University every morning.</div><br />
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<a class="boxGallery" href="http://www.igem-bielefeld.de/galleries/Bielefeld/index.html"><img src="http://www.igem-bielefeld.de/galleries/Bielefeld/images/thumb/_0630495766.jpg"></a><br />
<div class="boxHeading">Bielefeld</div><br />
<div class="boxDescription">If you have a minute, go for a walk through Bielefeld, our hometown. You may find some cosy places here and there.<br/><br/>We have removed the too big advertisements in the pictures, just to be sure.</div><br />
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Image:Bielefeld board.JPG<br />
Image:Bielefeld_Musketiere.JPG<br />
Image:Bielefeld Becher.JPG<br />
</gallery></div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Project/OutlookTeam:Bielefeld-Germany/Project/Outlook2010-10-27T17:19:14Z<p>Nkessler: /* Picric acid */</p>
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<li><a href="/Team:Bielefeld-Germany/Project">Project</a></li><br />
<li><a href="/Team:Bielefeld-Germany/Project/Application">Application</a></li><br />
<li><a href="/Team:Bielefeld-Germany/Project/Approach">Approach</a></li><br />
<li><a href="/Team:Bielefeld-Germany/Project/Theory">Theory</a></li><br />
<li><a href="/Team:Bielefeld-Germany/Project/Protocols">Protocols</a></li><br />
<li><a href="/Team:Bielefeld-Germany/Project/Model">Model</a></li><br />
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<br />
= The techniques’ potential =<br />
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<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1548942" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=1548942" width="200"><br />
<h3>Capsaicin</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1548942&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=17198" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=17198" width="200"><br />
<h3>Acetosyringone</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=17198&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
<br />
In our project we modulated the virA-gene from ’’A. tumefaciens’’, which is encoding a receptor for the plant-hormone acetosyringone, via error prone PCR. Slight mutations may alter the receptors conformation or its binding site. Both of these can cause shifts in specificity and sensitivity. This way we created a new receptor, which is capable of sensing capsaicin. The chemical formulas of capsaicin and acetosyringone are quite similar and there are other molecules of interest, that have some of [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Phenolic_Compounds the necessary properties], too. Thus, we suggest that it is possible to modulate VirA receptors in order to make them sense a variety of highly relevant compounds.<br />
<br />
<br />
<br />
<br />
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= Further Possible Substrates =<br />
<br />
As stated, there are other candidates our sensing system may be applied to. All of them have similiar molecular structures as the vir-systems natural inducor acetosyringone. In the following we will present some of the most relevant compounds other than capsaicin, that may be upcoming targets of our research.<br />
<br />
''All of the following images of the compounds molecular structures are linked to [http://pubchem.ncbi.nlm.nih.gov/ '''The PubChem Project'''].''<br />
<br />
== Diagnosis of pheochromocytoma and neuroblastoma (child tumors) ==<br />
<html><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=21100" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=21100" width="200"><br />
<h3>Metanephrine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=21100&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=23615482" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=23615482" width="200"><br />
<h3>Vanilmandelic Acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=23615482&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1738" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=1738" width="200"><br />
<h3>Homovanillic acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1738&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
=== Pheochromocytoma ===<br />
Pheochromocytoma is a rare endocrine tumor originating in the medulla of adrenal glands, localized on top of the kidney. The adrenal glands produce several catecholamines, to which metanephrine and dopamine belong. These hormones regulate responses to stress, heart rate and blood pressure. In patients with pheochromocytoma these hormones are released excessively, potentially causing increased heart rate and blood pressure. Pheochromocytoma may become life threatening when not recognized and treated [http://pheopara.nichd.nih.gov/ (Pheochromocytoma and Paraganglioma website at the NIH)].<br />
<br />
These hormones can be quantified in urine and thus are important compounds of pheochromocytoma diagnostics. [http://jcem.endojournals.org/cgi/content/abstract/92/12/4602 Boyle et al. (2007)] compare different accuracies of diagnostic measures for the tumor and name urinary free metanephrine HPLC-measurement as the most sensitive and specific. Homovanillic acid and vanillyl mandelic acid are measured via HPLC aswell and are indicators for the same tumor.<br />
<br />
=== Neuroblastoma ===<br />
"Neuroblastoma is the most common extracranial solid tumor in infancy. It is an embryonal malignancy of the sympathetic nervous system arising from neuroblasts (pluripotent sympathetic cells). In the developing embryo, these cells invaginate, migrate along the neuraxis, and populate the sympathetic ganglia, adrenal medulla, and other sites. The pattern of distribution of these cells correlates with the sites of primary disease presentation." ([http://emedicine.medscape.com/article/988284-overview Lacayo, Davis 2010])<br />
<br />
Patients with high-risk disease still have very poor outcomes despite intensive therapy.<br />
<br />
"More than 90% of patients have elevated homovanillic acid (HVA) and/or vanillylmandelic acid (VMA) levels detectable in urine."<br />
<br />
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<br />
== 3-Methoxytyramine ==<br />
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<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=681" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=681" width="200"><br />
<h3>Dopamine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=681&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=11957621" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=11957621" width="200"><br />
<h3>3-Methoxytyramine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=11957621&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
<br />
[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0013452 Sotnikova et al. (2010)] suggest, that 3-Methoxytyramine (3-MT) plays an important role as a neuromodulator. 3-MT is a metabolite of Dopamine and is reported to be a potential indicator for dopamine-dependent diseases like the parkinson disease, schizophrenia and dyskinesia. 3-Methoxytyramin as metabolic product of the hormon dopamine can be detected in human urine ([http://edoc.hu-berlin.de/oa/degruyter/cclm.1971.9.6.478.pdf Knoll ''et al.'', 1971]).<br />
Dopamine is known as happiness hormone found in increased levels after taking stimulating substances such as cocaine or amphetamines. The latter is one of the worldwide most used substitutes in sportive competition as doping. Increased levels of 3-MT analyzed in urine taken from sportives can indicate to intake of stimulating substances ([http://journals.lww.com/acsm-msse/Abstract/1980/21000/The_effect_of_amphetamines_on_selected.13.aspx CHANDLER, JOE V. and STEVEN N BLAIR, 1980]).<br />
<br />
== Dopamine ==<br />
<br />
Even though being referred to as "happiness hormone", Dopamine and its receptors are said to play key roles in numerous phsychic disorders and drug addiction. For many years now, the Dopamine hypothesis of Schizophrenia exists and evolves. It states that many symptoms of Schizophrenia correlate with a hyperactive disturbed dopaminergic signal transduction. [http://schizophreniabulletin.oxfordjournals.org/content/early/2009/03/26/schbul.sbp006.abstract Howes and Kapur (2009)] as well as [http://jop.sagepub.com/content/21/4/440.short Stone ''et al.'' (2007)] reviewed the hypothesis and its evolution.<br />
<br />
Concerning drug addiction, Dopamine and the brains reward system are of special interest in research [http://dx.doi.org/10.1016/j.brainresrev.2004.12.033 (Heidbreder, 2005)].<br />
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<br />
== Picric acid==<br />
<html><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=6884" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=6884" width="200"><br />
<h3>Trichloranisol</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=6884&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=6954" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=6954" width="200"><br />
<h3>Picric acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=6954&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
<br />
Nitroaromatic compounds, such as Picrid acid (2,4,6-trinitrophenol), can be used as dyes, explosives, pesticides and energy sources for bacterial growth. The industrial use ([http://www.springerlink.com/content/q82535h870021540/fulltext.pdf Rajan, J 1996]) of picric acid contaminated ground water and is as nitritited aromatic compound a potential explosive ([http://search.barnesandnoble.com/Explosives-Engineering/Paul-W-Cooper/e/9780471186366 Cooper PW, 1997]; [http://onlinelibrary.wiley.com/doi/10.1002/1521-3773%2820010601%2940:11%3C2104::AID-ANIE2104%3E3.0.CO;2-%23/full Sohn H, 2001]). Picric Acid is still a problem by unfound warefare. Existing detection methods are time consuming and even more cost intensive, so that a high selective and sensitiv application by microbial screening would be desirable.<br />
<br />
== Trichloranisol==<br />
<br />
Trichloranisol (2,4,6 Trichloranisol) ist assumingly a compound causing cork taint in wine. The origin is supposed to find in pestizid use for cork oak tree. Further contamination can be found in the barrels used for wine maturation ([http://pubs.acs.org/doi/pdf/10.1021/jf00110a037 Buser ''et al.'', 1982)].<br />
A sommelier could use a pre test system for preventing to serve a contaminated high class wine to a costumer. Our Test system could be adaptable for Trichloranisol, adding, beside Capsaicin, an application in the field of indulgence.<br />
<br />
<br />
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<br />
== Summary ==<br />
The VirA receptor is a great starting point for modulations, in order to address a wide variety of highly relevant compounds. By contructing a Vir-gene based sensing system we present a ''proof of concept'' for a general microbial sensing system adaptable for various other compounds. Since the basic sensing and reporting system was successfully established in ''E. coli'', the next essential steps are to improve the mutagenesis strategy for the receptor, and finally the screening for each candidate compound.<br />
<br />
Using [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Output-signal_amplification_by_Sensitivity_Tuner_implementation a set of sensitivity tuners], the reporter output may then be optimized. Furthermore, the speed of the systems are expected to require refinement.<br />
<br />
As a result, the MARSS has great potential to deliver biological tests for medical diagnosis, drug tests for ''e.g.'' doping conctrols, analysis of food for ''e.g.''allergy triggering compounds and soil contaminating substances such as picric acid.<br />
<br />
=References=<br />
<br />
*Boyle J, Davidson DF, Perry CG and Connell JMC, ''Comparison of Diagnostic Accuracy of Urinary Free Metanephrines, Vanillyl Mandelic Acid, and Catecholamines and Plasma Catecholamines for Diagnosis of Pheochromocytoma'' ,Journal of Clinical Endocrinology & Metabolism, Vol. 92, No. 12, pp. 4602-4608<br />
<br />
*Chandler JV and Blair SN (1980), '' The effect of amphetamines on selected physiological components related to athletic success''. Med. Sci. Sports Exercise, Vol. 12, No. 1, pp. 65-69<br />
<br />
*Cooper PW (1997), ''Explosives Engineering'', Edition 1, Wiley, John & Sons.<br />
<br />
* ''Eunice Kennedy Shriver'' National Institute of ''Child Health and Human Development'', Pheochromocytoma and Paragangliooma, 0-CH-0093<br />
<br />
*Heidbreder CA, Gardner EL, Xi ZX, Thanos PK, Mugnaini M, Hagan JJ, Ashby CR Jr. (2005), ''The role of central dopamine D3 receptors in drug addiction: a review of pharmacological evidence.'', Brain Research Reviews, Vol. 49, pp. 77 – 105 <br />
<br />
*Howes OD and Kapur S (2009), ''The Dopamine Hypothesis of Schizophrenia: Version III—The Final Common Pathway'', Schizophrenia Bulletin, Vol. 35, No. 3, pp.549-62<br />
<br />
*http://pubchem.ncbi.nlm.nih.gov/<br />
<br />
*Knoll E, Wisser H, Stamm D (1971), ''in Verfahren zur Bestimmung der 3-Methoxy-4-hydroxy-phenylessigsäure (Homovanillinsäure) im Harn durch in situ Remissionsmessung nach dünnschichtchromatographischer Trennung'', Z. klin. Chem. u. klin. Biochem., 1971<br />
<br />
*Lacayo NJ (2010), ''Neuroblastoma'', eMedicine from webMD<br />
<br />
*Rajan J,Valli K, Perkins RE, Sariaslani FS, Barns FM, Reysenbach A-L,Rehm S, Ehringer M and Pace NR (1996), ''Mineralization of 2,4,6-trinitrophenol (picric acid): characterization and phylogenetic identification of microbial strains'' Journal of Industrial Microbiology, 16, 319-324<br />
<br />
*Sohn H, Calhoun RM, Sailor MJ, Trogler WC (2001), ''Detection of TNT and Picric Acid on Surfaces and in Seawater by Using Photoluminescent Polysiloles'', Angewandte Chemie, Vol.40, pp.2104–2105<br />
<br />
*Sotnikova TD, Beaulieu J-M, Espinoza S, Masri B, Zhang X, Salahpour A, Barak LS, Caron MG, Gainetdinov RR (2010), ''The Dopamine Metabolite 3-Methoxytyramine Is a Neuromodulator'', PloSONE<br />
<br />
*Stone JM, Morrison PD, Pilowsky LS (2007), ''Review: Glutamate and dopamine dysregulation in schizophrenia — a synthesis and selective review'', Journal of Psychopharmacology, Vol. 21, No. 4, pp. 440-452</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Project/OutlookTeam:Bielefeld-Germany/Project/Outlook2010-10-27T17:18:39Z<p>Nkessler: </p>
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<br />
= The techniques’ potential =<br />
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<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1548942" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=1548942" width="200"><br />
<h3>Capsaicin</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1548942&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=17198" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=17198" width="200"><br />
<h3>Acetosyringone</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=17198&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
<br />
In our project we modulated the virA-gene from ’’A. tumefaciens’’, which is encoding a receptor for the plant-hormone acetosyringone, via error prone PCR. Slight mutations may alter the receptors conformation or its binding site. Both of these can cause shifts in specificity and sensitivity. This way we created a new receptor, which is capable of sensing capsaicin. The chemical formulas of capsaicin and acetosyringone are quite similar and there are other molecules of interest, that have some of [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Phenolic_Compounds the necessary properties], too. Thus, we suggest that it is possible to modulate VirA receptors in order to make them sense a variety of highly relevant compounds.<br />
<br />
<br />
<br />
<br />
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= Further Possible Substrates =<br />
<br />
As stated, there are other candidates our sensing system may be applied to. All of them have similiar molecular structures as the vir-systems natural inducor acetosyringone. In the following we will present some of the most relevant compounds other than capsaicin, that may be upcoming targets of our research.<br />
<br />
''All of the following images of the compounds molecular structures are linked to [http://pubchem.ncbi.nlm.nih.gov/ '''The PubChem Project'''].''<br />
<br />
== Diagnosis of pheochromocytoma and neuroblastoma (child tumors) ==<br />
<html><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=21100" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=21100" width="200"><br />
<h3>Metanephrine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=21100&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=23615482" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=23615482" width="200"><br />
<h3>Vanilmandelic Acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=23615482&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1738" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=1738" width="200"><br />
<h3>Homovanillic acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1738&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
=== Pheochromocytoma ===<br />
Pheochromocytoma is a rare endocrine tumor originating in the medulla of adrenal glands, localized on top of the kidney. The adrenal glands produce several catecholamines, to which metanephrine and dopamine belong. These hormones regulate responses to stress, heart rate and blood pressure. In patients with pheochromocytoma these hormones are released excessively, potentially causing increased heart rate and blood pressure. Pheochromocytoma may become life threatening when not recognized and treated [http://pheopara.nichd.nih.gov/ (Pheochromocytoma and Paraganglioma website at the NIH)].<br />
<br />
These hormones can be quantified in urine and thus are important compounds of pheochromocytoma diagnostics. [http://jcem.endojournals.org/cgi/content/abstract/92/12/4602 Boyle et al. (2007)] compare different accuracies of diagnostic measures for the tumor and name urinary free metanephrine HPLC-measurement as the most sensitive and specific. Homovanillic acid and vanillyl mandelic acid are measured via HPLC aswell and are indicators for the same tumor.<br />
<br />
=== Neuroblastoma ===<br />
"Neuroblastoma is the most common extracranial solid tumor in infancy. It is an embryonal malignancy of the sympathetic nervous system arising from neuroblasts (pluripotent sympathetic cells). In the developing embryo, these cells invaginate, migrate along the neuraxis, and populate the sympathetic ganglia, adrenal medulla, and other sites. The pattern of distribution of these cells correlates with the sites of primary disease presentation." ([http://emedicine.medscape.com/article/988284-overview Lacayo, Davis 2010])<br />
<br />
Patients with high-risk disease still have very poor outcomes despite intensive therapy.<br />
<br />
"More than 90% of patients have elevated homovanillic acid (HVA) and/or vanillylmandelic acid (VMA) levels detectable in urine."<br />
<br />
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<br />
== 3-Methoxytyramine ==<br />
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<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=681" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=681" width="200"><br />
<h3>Dopamine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=681&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=11957621" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=11957621" width="200"><br />
<h3>3-Methoxytyramine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=11957621&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
<br />
[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0013452 Sotnikova et al. (2010)] suggest, that 3-Methoxytyramine (3-MT) plays an important role as a neuromodulator. 3-MT is a metabolite of Dopamine and is reported to be a potential indicator for dopamine-dependent diseases like the parkinson disease, schizophrenia and dyskinesia. 3-Methoxytyramin as metabolic product of the hormon dopamine can be detected in human urine ([http://edoc.hu-berlin.de/oa/degruyter/cclm.1971.9.6.478.pdf Knoll ''et al.'', 1971]).<br />
Dopamine is known as happiness hormone found in increased levels after taking stimulating substances such as cocaine or amphetamines. The latter is one of the worldwide most used substitutes in sportive competition as doping. Increased levels of 3-MT analyzed in urine taken from sportives can indicate to intake of stimulating substances ([http://journals.lww.com/acsm-msse/Abstract/1980/21000/The_effect_of_amphetamines_on_selected.13.aspx CHANDLER, JOE V. and STEVEN N BLAIR, 1980]).<br />
<br />
== Dopamine ==<br />
<br />
Even though being referred to as "happiness hormone", Dopamine and its receptors are said to play key roles in numerous phsychic disorders and drug addiction. For many years now, the Dopamine hypothesis of Schizophrenia exists and evolves. It states that many symptoms of Schizophrenia correlate with a hyperactive disturbed dopaminergic signal transduction. [http://schizophreniabulletin.oxfordjournals.org/content/early/2009/03/26/schbul.sbp006.abstract Howes and Kapur (2009)] as well as [http://jop.sagepub.com/content/21/4/440.short Stone ''et al.'' (2007)] reviewed the hypothesis and its evolution.<br />
<br />
Concerning drug addiction, Dopamine and the brains reward system are of special interest in research [http://dx.doi.org/10.1016/j.brainresrev.2004.12.033 (Heidbreder, 2005)].<br />
<html><div style="clear:both;" /></html><br />
<br />
== Picric acid==<br />
<html><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=6884" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=6884" width="200"><br />
<h3>Trichloranisol</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=6884&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=6954" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=6954" width="200"><br />
<h3>Picric acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=6954&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
Nitroaromatic compounds, such as Picrid acid (2,4,6-trinitrophenol), can be used as dyes, explosives, pesticides and energy sources for bacterial growth. The industrial use ([http://www.springerlink.com/content/q82535h870021540/fulltext.pdf Rajan, J 1996]) of picric acid contaminated ground water and is as nitritited aromatic compound a potential explosive ([http://search.barnesandnoble.com/Explosives-Engineering/Paul-W-Cooper/e/9780471186366 Cooper PW, 1997]; [http://onlinelibrary.wiley.com/doi/10.1002/1521-3773%2820010601%2940:11%3C2104::AID-ANIE2104%3E3.0.CO;2-%23/full Sohn H, 2001]). Picric Acid is still a problem by unfound warefare. Existing detection methods are time consuming and even more cost intensive, so that a high selective and sensitiv application by microbial screening would be desirable.<br />
<br />
== Trichloranisol==<br />
<br />
Trichloranisol (2,4,6 Trichloranisol) ist assumingly a compound causing cork taint in wine. The origin is supposed to find in pestizid use for cork oak tree. Further contamination can be found in the barrels used for wine maturation ([http://pubs.acs.org/doi/pdf/10.1021/jf00110a037 Buser ''et al.'', 1982)].<br />
A sommelier could use a pre test system for preventing to serve a contaminated high class wine to a costumer. Our Test system could be adaptable for Trichloranisol, adding, beside Capsaicin, an application in the field of indulgence.<br />
<br />
<br />
<br />
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<br />
== Summary ==<br />
The VirA receptor is a great starting point for modulations, in order to address a wide variety of highly relevant compounds. By contructing a Vir-gene based sensing system we present a ''proof of concept'' for a general microbial sensing system adaptable for various other compounds. Since the basic sensing and reporting system was successfully established in ''E. coli'', the next essential steps are to improve the mutagenesis strategy for the receptor, and finally the screening for each candidate compound.<br />
<br />
Using [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Output-signal_amplification_by_Sensitivity_Tuner_implementation a set of sensitivity tuners], the reporter output may then be optimized. Furthermore, the speed of the systems are expected to require refinement.<br />
<br />
As a result, the MARSS has great potential to deliver biological tests for medical diagnosis, drug tests for ''e.g.'' doping conctrols, analysis of food for ''e.g.''allergy triggering compounds and soil contaminating substances such as picric acid.<br />
<br />
=References=<br />
<br />
*Boyle J, Davidson DF, Perry CG and Connell JMC, ''Comparison of Diagnostic Accuracy of Urinary Free Metanephrines, Vanillyl Mandelic Acid, and Catecholamines and Plasma Catecholamines for Diagnosis of Pheochromocytoma'' ,Journal of Clinical Endocrinology & Metabolism, Vol. 92, No. 12, pp. 4602-4608<br />
<br />
*Chandler JV and Blair SN (1980), '' The effect of amphetamines on selected physiological components related to athletic success''. Med. Sci. Sports Exercise, Vol. 12, No. 1, pp. 65-69<br />
<br />
*Cooper PW (1997), ''Explosives Engineering'', Edition 1, Wiley, John & Sons.<br />
<br />
* ''Eunice Kennedy Shriver'' National Institute of ''Child Health and Human Development'', Pheochromocytoma and Paragangliooma, 0-CH-0093<br />
<br />
*Heidbreder CA, Gardner EL, Xi ZX, Thanos PK, Mugnaini M, Hagan JJ, Ashby CR Jr. (2005), ''The role of central dopamine D3 receptors in drug addiction: a review of pharmacological evidence.'', Brain Research Reviews, Vol. 49, pp. 77 – 105 <br />
<br />
*Howes OD and Kapur S (2009), ''The Dopamine Hypothesis of Schizophrenia: Version III—The Final Common Pathway'', Schizophrenia Bulletin, Vol. 35, No. 3, pp.549-62<br />
<br />
*http://pubchem.ncbi.nlm.nih.gov/<br />
<br />
*Knoll E, Wisser H, Stamm D (1971), ''in Verfahren zur Bestimmung der 3-Methoxy-4-hydroxy-phenylessigsäure (Homovanillinsäure) im Harn durch in situ Remissionsmessung nach dünnschichtchromatographischer Trennung'', Z. klin. Chem. u. klin. Biochem., 1971<br />
<br />
*Lacayo NJ (2010), ''Neuroblastoma'', eMedicine from webMD<br />
<br />
*Rajan J,Valli K, Perkins RE, Sariaslani FS, Barns FM, Reysenbach A-L,Rehm S, Ehringer M and Pace NR (1996), ''Mineralization of 2,4,6-trinitrophenol (picric acid): characterization and phylogenetic identification of microbial strains'' Journal of Industrial Microbiology, 16, 319-324<br />
<br />
*Sohn H, Calhoun RM, Sailor MJ, Trogler WC (2001), ''Detection of TNT and Picric Acid on Surfaces and in Seawater by Using Photoluminescent Polysiloles'', Angewandte Chemie, Vol.40, pp.2104–2105<br />
<br />
*Sotnikova TD, Beaulieu J-M, Espinoza S, Masri B, Zhang X, Salahpour A, Barak LS, Caron MG, Gainetdinov RR (2010), ''The Dopamine Metabolite 3-Methoxytyramine Is a Neuromodulator'', PloSONE<br />
<br />
*Stone JM, Morrison PD, Pilowsky LS (2007), ''Review: Glutamate and dopamine dysregulation in schizophrenia — a synthesis and selective review'', Journal of Psychopharmacology, Vol. 21, No. 4, pp. 440-452</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Judging/CommentsTeam:Bielefeld-Germany/Judging/Comments2010-10-27T17:05:52Z<p>Nkessler: /* Comments on the iGEM competition */</p>
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= Comments on the iGEM competition =<br />
<br />
First of all we like to state that we had a lot of fun during this project. The idea of a platform to realize own projects sparks our scientific flame. We also greatly enjoyed that the iGEM competition creates a vivid network of students and Ph.D. students not only from all around the world, but also from areas beyond the Life Sciences.<br />
<br />
<br />
We used the competition to acquire interdisciplinary skills and also got in touch with new fields of work, like fundraising and public relations. Moreover we used the chance to get in contact with some really nice students from Syddansk Universitet in Denmark. Together we started an intensive cooperation which lead to a magnificent gathering in Denmark at the end of September. Our whole team is excitedly looking forward to meet more charming and interesting people in Boston during the iGEM Jamborée. <br />
<br />
<br />
Unfortunately, like in every huge project, we also faced some problems. At the beginning we had quite some problems with the design of the registry. A lot of BioBricks could not be found by using the search function and we started to use Google instead since its hit rate was much higher. Moreover we would like to mention that the registry should be reviewed manually. In particular there are various problems concerning some BioBricks that are not properly characterized and/or do not work as expected. This is probably due to inadequate experimental data mining as well as a unreviewed registry entries.</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Project/ModelTeam:Bielefeld-Germany/Project/Model2010-10-27T16:56:02Z<p>Nkessler: </p>
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__NOTOC__<br />
= Construct Maps =<br />
<br />
== Model of <partinfo>K389015</partinfo> ==<br />
[[Image:Bielefeld_k389015.jpg|500px|thumb|center| Main construct of acetosyringone inducible luciferase expression system containing constitutive expression of the two component system receptor system (virA+virG)]]<br />
<br />
== Model of <partinfo>K389016</partinfo> ==<br />
[[Image:Bielefeld_k389016.jpg|500px|thumb|center|Control construct of acetosyringone inducible RFP read out system]] <br />
<br />
== Model of <partinfo>K389014</partinfo> ==<br />
[[Image:Bielefeld_k389014.jpg|500px|thumb|center|Screening construct of acetosyringone inducible Kan read out system]]</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Project/ModelTeam:Bielefeld-Germany/Project/Model2010-10-27T16:54:48Z<p>Nkessler: /* Model of K389015 */</p>
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= Construct Maps =<br />
<br />
== Model of <partinfo>K389015</partinfo> ==<br />
[[Image:Bielefeld_k389015.jpg|500px|thumb|center| Main construct of acetosyringone inducible luciferase expression system containing constitutive expression of the two component system receptor system (virA+virG)]]<br />
<br />
== Model of <partinfo>K389016</partinfo> ==<br />
[[Image:Bielefeld_k389016.jpg|500px|thumb|center|Control construct of acetosyringone inducible RFP read out system]] <br />
<br />
== Model of <partinfo>K389014</partinfo> ==<br />
[[Image:Bielefeld_k389014.jpg|500px|thumb|center|Screening construct of acetosyringone inducible Kan read out system]]</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Project/ModelTeam:Bielefeld-Germany/Project/Model2010-10-27T16:54:05Z<p>Nkessler: </p>
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= Construct Maps =<br />
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== Model of <partinfo>K389015</partinfo> ==<br />
[[Image:Bielefeld_k389015.jpg|500px|thumb|center| Main construct of acetosyringone inducible luciferase expresseion system containing constitutive expression of the two component system receptor system(virA+virG)]] <br />
<br />
== Model of <partinfo>K389016</partinfo> ==<br />
[[Image:Bielefeld_k389016.jpg|500px|thumb|center|Control construct of acetosyringone inducible RFP read out system]] <br />
<br />
== Model of <partinfo>K389014</partinfo> ==<br />
[[Image:Bielefeld_k389014.jpg|500px|thumb|center|Screening construct of acetosyringone inducible Kan read out system]]</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Project/ModelTeam:Bielefeld-Germany/Project/Model2010-10-27T16:53:16Z<p>Nkessler: /* Construct Maps */</p>
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= Construct Maps =<br />
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== <partinfo>Model of K389015</partinfo> ==<br />
[[Image:Bielefeld_k389015.jpg|500px|thumb|center| Main construct of acetosyringone inducible luciferase expresseion system containing constitutive expression of the two component system receptor system(virA+virG)]] <br />
<br />
== <partinfo>Model of K389016</partinfo> ==<br />
[[Image:Bielefeld_k389016.jpg|500px|thumb|center|Control construct of acetosyringone inducible RFP read out system]] <br />
<br />
== <partinfo>Model of K389014</partinfo> ==<br />
[[Image:Bielefeld_k389014.jpg|500px|thumb|center|Screening construct of acetosyringone inducible Kan read out system]]</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Project/ModelTeam:Bielefeld-Germany/Project/Model2010-10-27T16:50:39Z<p>Nkessler: /* Construct Maps */</p>
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= Construct Maps =<br />
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[[Image:Bielefeld_k389015.jpg|500px|thumb|center| Main construct of acetosyringone inducible luciferase expresseion system containing constitutive expression of the two component system receptor system(virA+virG)]] <br />
<br />
[[Image:Bielefeld_k389016.jpg|500px|thumb|center|Control construct of acetosyringone inducible RFP read out system]] <br />
<br />
[[Image:Bielefeld_k389014.jpg|500px|thumb|center|Screening construct of acetosyringone inducible Kan read out system]]</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/SafetyTeam:Bielefeld-Germany/Safety2010-10-27T16:47:23Z<p>Nkessler: /* Referenzen / References */</p>
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<br />
= Biosafety and Biosecurity (English) =<br />
<br />
It is important to discriminate between Biosafety and Biosecurity in the context of working with genetically modified organisms and lab use. Biosecurity comprises the safety of the lab. The [http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_EPR_2006_6.pdf WHO] defines Biosecurity as a task of organisation and administration.The term Biosafety deals with the biological risks of the samples for the user, the enviroment and the lab. In this article, we are mostly summarizing the German law on Biosecurity and Biosafety regarding to the [http://www.bvl.bund.de/nn_495478/DE/06__Gentechnik/093__ZKBS/gentechnik__zkbs__node.html__nnn=true ZKBS (zentrale Kommission für Biologische Sicherheit)] and [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf their lists of unhazardous organisms] plus the comment on safety of the [http://www.dfg.de/download/pdf/dfg_im_profil/reden_stellungnahmen/2009/stellungnahme_synthetische_biologie.pdf DFG (Deutsche Forschungsgemeinschaft)].<br />
Furthermore we crosslinked the laws of the United States of America and the European law of Biosecurity and Biosafety in this article. We marked this statement in the text, so the reader can discrimante between the law systems. This kind of modulation makes sense, because our experimental work was done in Germany, shipped in europe (SDU Denmark) and used as a biobrick in United States of America.<br />
<br />
== Biosecurity ==<br />
[[Image:Safe labwork.jpg|200px|thumb|right|Picture 2: We are using a cleanbench to avoid any contamination of our samples. A proper use of the cleanbench can only be achieved by placing all the big things in the rear of the bench, by not disturbing the air circulation and by avoiding any kind of aerosoles. Use gloves for your own protection.]]<br />
<br />
<br />
In the context of "genetically engineered" it is quite important to keep unqualified personnel and trespassers away from your laboratory and your samples. In our case we secure our lab with a keypad. Every staff member needs a key to open the laboratory door. Nobody is able to open the door without the proper keys. Staff members receive a key, after beeing briefed by the safety officer on safety issues. You can only get a key for the safety stage you are working in. The labs with higher safety stages are cut off from the lower ones. Staff members need a continuative safety guide to receive a key for the higher security levels. Concludingly no unqualified personel can pass the door to the laboratory.<br />
The picture 1 shows the keypad in front of the lab.<br />
<br />
The laboratories, which are in use for our iGEM project are security stage 1 (S1). For the safety of the staff our labs are equiped with fire extinguisher, emergency showers and a heart defibrillator.<br />
<br />
== Biosafety ==<br />
[[Image:Keypad.JPG|200px|thumb|left| Picture 1: Security entrance to the lab. Every Member of the Team needs a key-token in order to get acces to the lab]]<br />
<br />
The term "biological safety" defines the effort to reduce or eliminate any potential risks caused by biotechnolgy or genetical engineering. The laboratories are scaled into four different saftey stages. The stage "S1" defines working with organsims and methods, which do no prooved harm neither to the enviroment nor mankind. The stages rise up to biosafety stage four, which defines working with prooved humanpathogenous organisms. The WHO defines for the United states the same safety stages, which are called risk groups [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf Manual of Biosecurity, WHO, page 1].<br />
<br />
The Biosafety is ruled by the "Zentrale Kommission für Biologische Sicherheit" (ZKBS) in Germany. The ZKBS reviewed and verified all commercial, research or privat laboratories about their safety issues. The iGEM Team Bielefeld is currently working with the organisms ''Agrobacterium tumefaciens'' and a derivate of ''E. coli'' K12 in a ZKBS reviewed S1-lab. The organisms strains are declared as riskfactor 1 by the german act of genetics (GenTSV, § 5; 15.06.2010). The act contains that the organisms (abstract):<br />
<br />
1.<br />
<br />
: - are no proved human-, phyto- or animalpathogen<br />
: - do not contain or release organism containing to a higher risk stage<br />
: - are prooved by experiments or long term evaluation or do not proliferate in the enviroment because of biological implanted boundaries<br />
<br />
<br />
2.<br />
Organisms, which confirm the paragraph 1, are defined by the $6 of the act of genetics (GenTSV):<br />
<br />
: a) as biological safe organisms<br />
: b) organims or strains, which are contaminated by organisms or strains of a higher security level<br />
: c) organisms defined by the risk level S1 (list of organisms used for genetically engineering research)<br />
<br />
<br />
The [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf list of organisms] categorize the strains ''E. coli'' K12 and ''Agrobacterium tumefaciens'' as biological safety risk 1 (S1). The strain [http://www.bvl.bund.de/cln_007/nn_520774/DE/06__Gentechnik/093__ZKBS/01__Allg__Stellungnahmen/02__bakterien/zkbs__bakterien__agrobacterium__tumefaciens,templateId=raw,property=publicationFile.pdf/zkbs_bakterien_agrobacterium_tumefaciens.pdf ''Agrobacterium tumefaciens''] is a known phytopathogen. Because of its frequent appearence in the earth (500 bacteria in 1g earth) it is generally regarded as safe.<br />
<br />
The WHO defines "security assesments" has the highest priority for the work with organisms. This includes a proper theoretically background of the organism. Further they defined rules for good molecular practice and proper lab security [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf WHO Manual Biosafety, page 9 following]. They note that most of the security guidelines are not quite neccessary for organism of risk stage 1. Although we play by the rules and work as carefully as possible (see, what we do to protect).<br />
<br />
= Biosafety und Biosecurity (German)=<br />
<br />
Es ist bei der Inbetriebnahme und Nutzung einer Gentechnischen Anlagen darauf zu achten, dass Vorkehrungen sowohl für den Betriebsschutz (Biosecurity) als auch für die biologische Sicherheit (Biosafety) getroffen werden. Biosecurity wird von der [http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_EPR_2006_6.pdf WHO] als Aufgabe der Adminstrative des Labors definiert.<br />
<br />
== Biosecurity ==<br />
Der Biosecurity wird Sorge getragen, in dem kein unbefugter Zugang zum Laborplatz hat. Die Türen sind nur per Schlüssel zu öffnen. Jeder Mitarbeiten muss eine Sicherheitsbelehrung beim Sicherheitsbeauftragten ablegen ehe er Zugang zu den Laboratorien bekommt. Die Labore sind je nach Sicherheitsgefährdung gekennzeichnet und verschlossen, so dass keine unterqualifizierte Person in ein höheres Sicherheitslevel eindringen kann. In dem vom iGEM genutzten Labor handelt es sich um die Sicherheitstufe S1. Die WHO definiert die gleichen Sicherheitsstufen, hier "risk group" genannt. [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf Manual of Biosecurity, WHO, page 1].<br />
<br />
== Biosafety ==<br />
[[Image:Bielefeld Sign.JPG|200px|thumb|left| Picture 3: The Sign says: No Trespassing, Security stage 1.]]<br />
<br />
Als Biologische Sicherheit (Biosafety) wird der Versuch zur Reduzierung bzw. Eleminierung potentieller Gefahren durch Biotechnologie definiert. In Deutschland werden die unterschiedlichen Organismen in vier Sicherheitstufen von S1 für keine bekannte Gefährdung bis S4 für erwiesene humanpathogene Gefährdung eingeteilt. Die zentrale Kommussion für Biologische Sicherheit (ZKBS) überprüft alle Laboratorien, ob privat, gewerblich oder für die Forschung, vor in Betriebnahme auf Sicherheitsmängel und Einhaltung der rechtlichen Grundlagen. Das iGEM Team Bielefeld nutzt die Stämme ''Agrobacterium tumefaciens'' und einen Ableger des ''E. coli'' K12 in einem ZKBS geprüften Labor der Sicherheitstufe S1 für gentechnische Arbeiten. Die Stämme sind nach dem Gentechnik Gesetz (GenTSV) § 5 Absatz 1 Satz 1 und AnhangI Nummer 1 GenTSV der Risikogruppe 1 zu zuordnen (Stand 15.06.2010). Das beinhaltet, dass die Organismen (Auszug):<br />
<br />
1.<br />
<br />
: - weder ein human-, pflanzen- noch tierpathogen sind<br />
: - keine Organismen höhere Risikogruppen abgeben<br />
: - sich durch experimentell erwiesene oder langfristig erprobte Anwendung auszeichnen oder eingebaute biologische Schranken […] die Überlebens- bzw. Vermehrungsfähigkeit in der Umwelt begrenzen<br />
<br />
2.<br />
Organismen, die die Punkte unter 1 erfüllen, sind gemäß §6 Abs. 1 in Verbindung mit Anhang II Teil A des GenTVS<br />
<br />
: a) Organismen, die als biologische Sicherheitsmaßnahme anerkannt sind (§6 Abs. 3 GenTSV)<br />
: b) Zellen oder Zelllinien, die nicht von außen mit Organismen höherer Risikogruppe (2-4) kontaminiert sind<br />
: c) Organismen der Risikogruppe 1 nach der Organismenliste (Spender- und Empfängerorganismen für gentechnische Arbeiten zu Forschungszwecken<br />
<br />
Die verwendeten Stämme ''E. coli'' K12 und ''Agrobacterium tumefaciens'' werden nach [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf dieser Organismenliste] als Risikogruppe 1 klassifiziert. [http://www.bvl.bund.de/cln_007/nn_520774/DE/06__Gentechnik/093__ZKBS/01__Allg__Stellungnahmen/02__bakterien/zkbs__bakterien__agrobacterium__tumefaciens,templateId=raw,property=publicationFile.pdf/zkbs_bakterien_agrobacterium_tumefaciens.pdf ''Agrobacterium tumefaciens''] ist zwar als phytopathogen vermerkt, ist jedoch ubiquitär, d.h. weltweit im Boden (ca. 500 Bakterien / 1 g Boden) verbreitet und wurde aus diesem Grund der Risikogruppe 1 zugeordnet. <br />
<br />
Zur Sicherheit des Personals und der Umwelt herrscht ein konstanter leichter Unterdruck in den Laboratorien und die Fenster sind geschlossen zu halten, so dass weder Organismen weder hinaus noch hineingelangen können. Zudem wird der anfallende Organismen-Müll in spezillen Säcken gesammelt, autoklaviert und entsorgt. Die Organismen werden abgetötet. Zudem werden benutzen Flächen vor und nach der Arbeit desinfiziert.<br />
<br />
= Risk Assesment =<br />
<br />
== What we do to protect ==<br />
<br />
:*Every single person in the lab is trained on their work<br />
:*Every working person in the lab is trained on safety issues<br />
:*We got a safety and a disinfection officer at our lab <br />
:*No unqualified personal has access to the lab<br />
:*No public access to the lab<br />
:*We reduced the risk of contamination of the environment or staff members by disinfection, autoclavation and using of protecting clothes<br />
:*In case of emergency there are telephones, fire extinguisher, defibrillator and alarm buttons around the lab<br />
:*We only used organism of the risk stage 1, which do not harm neither mankind nor the environment<br />
:*We modify a unharzadous organism with soecific, identified and characterized biobricks. So we can appraise the risk causing by the modified organisms<br />
:*Constantly low pressure, no open windows => no organism can get in or out<br />
:*Desinfection after work of all working places (everyday)<br />
:*Autoclavation of waste (solid/liquid)<br />
:*No pipetting with the mouth<br />
<br />
== Unsere Schutzmaßnahmen ==<br />
<br />
:*Jede Person, die sich im Labor aufhält und arbeitet ist in ihrer Arbeit ausgebildet und/oder unterwiesen<br />
:*Jede Person, die sich im Labor aufhält oder arbeitet hat eine Sicherheitsbelehrung unterlaufen<br />
:*Es gibt einen Sicherheits- und eine Desinfektionsfachkraft in unserem Labor<br />
:*Kein unqualifiziertes Personal gelangt ins Labor<br />
:*Kein unqualifiziertes Personal gelangt in eine höhere Sicherheitsstufe als ihr erlaubt<br />
:*Kein öffentlicher Zugang zum Labor möglich<br />
:*We reduzieren das Kontaminationsrisiko für Arbeiter und Umwelt durch Autoklavieren, Desinfizierren und das Nutzen von protektiver Arbeitskleidung auf ein Minimum<br />
:*Im Falle eines Notfalls gibt es ein Telefon zum Hilferuf, Feuerlöscherr, Defibrilatoren und Feueralarmschalter in Reichweite<br />
:*Wir benutzen ausschließlich Organismen der Sicherheitsstufe 1<br />
<br />
= Short Summary =<br />
: 1'''. Would any of your project ideas raise safety issues ?'''<br />
<br />
:The bacteria we are currently working with are defined as bio security stage 1 (S1). Our produced GMOs (genetically modified organisms) are therefore no proofed harm neither to mankind nor to the environment. Thus they are GRAS - generally regarded as safe.<br />
:We are working in a bio security laboratory of the stage 1 (S1). There is no possible access for the public. Every person working in the lab is trained and instructed by the safety rules for laboratories S1. <br />
<br />
: '''2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?''' <br />
:No. All GMOs are S1 biosafety level. We did a risk assesement and know what kind of modilation we did on the GMOs. There is no risk of hazard or biological safety issues to mankind.<br />
<br />
: ''' 3. Is there a local biosafety group, committee, or review board at your institution?'''<br />
:Yes. There is a safety and a desinfection officer at our institution.<br />
<br />
: '''4.Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?''' <br />
: Every team must be aware of the risk that might be based in the organisms they use and they modify. Maybe a general safety instruction, which can be downlaoded from the igem.org page will be helpful to secure the research done in this competition. Moreover a list of risk organisms, with easy acces on the igem.safety page, would be a great benefit. Additional the safety.page could be tidied up to ease the acces to the key facts. Hence a small safety summary would be usefull. Some kind of a checklist.<br />
Certainly, it has to be said that the safety of the project must be reviewed by the team performing the experiments.<br />
<br />
= Referenzen / References =<br />
<br />
<br />
*Comment on synthetic biology of the DFG (German Research Association)/pdf, German<br />
<br />
*[http://daccess-dds-ny.un.org/doc/UNDOC/GEN/G08/625/32/PDF/G0862532.pdf?OpenElement Comments on Safety UNOG Genova, 2008 / pdf, English]<br />
<br />
*WHO; Laboratory Biosafety Manual (English)<br />
<br />
*WHO; Laboratory biosecurity guidance (English, 2008)<br />
<br />
*Stellungnahme der ZKBS zur Einstufung von ''Agrobacterium tumefaciens'' (Deutsch) / Statement of the ZKBS concerning the classification of ''Agrobacterium tumefaciens'' (German)<br />
<br />
*Kategorisierte Organismenliste (Deutsch) der ZKBS / List of categorized organisms (German) of the ZKBS<br />
<br />
*Merkblatt zum Transportieren von GVOs / ZKBS<br />
<br />
*ZKBS, 2010</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/CharacterizationTeam:Bielefeld-Germany/Results/Characterization2010-10-27T16:03:52Z<p>Nkessler: /* References */</p>
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<br />
<br />
=<partinfo>K238008</partinfo>: ''virA''=<br />
We wanted to use this part in our project, but could only obtain unexpected/faulty restriction patterns. Finally we chose to sequence the part, hoping to find the cause for the maintained restriction patterns. Unfortunately we could not approve the sequence of <partinfo>BBa_K238008</partinfo> deposited in the parts registry so that we chose to design our own VirA BioBrick. I strongly recommend using our VirA since it has been approved by multiple means, e.g. restriction patterns and sequencing (<partinfo>K389001</partinfo>).<br />
<br />
=<partinfo>BBa_K238011</partinfo>: ''vir''-promoter=<br />
We made a restriction analysis and sequenced parts of this BioBrick.<br />
<br />
<br />
=<partinfo>P1010</partinfo>: ''ccdB''-gene=<br />
The ''ccdB'' gene targets the gyrase of ''Escherichia coli'' and is lethal for all ''E. coli'' strains without the gyrase mutation gyrA462 ([http://openwetware.org/wiki/CcdB Openwetware]). The ''ccdB'' BioBrick is used for the 3A-assembly as a positive selection marker. <br />
We transformed this BioBrick into ''E. coli'' JM109, DH5α, TOP10, XL1-Blue, EC100D and DB3.1. ''E. coli'' JM109, XL1-Blue and DH5α seem to be ''ccdB'' resistant because there were as much colonies after P1010 transformation as observed with DB3.1. The P1010 works as expected in ''E. coli'' TOP10, EC100D (no colonies after transformation) and DB3.1 (many colonies after transformation).<br />
<br />
<br />
<center>Table 1: Results of the transformation of the cell-death gene ''ccdB'', BioBrick <partinfo>P1010</partinfo>, into different strains of ''E. coli''. <br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| ''E. coli'' strain<br />
!style="border-style: solid; border-width: 1px"| Resistant to ''ccdB''?<br />
!style="border-style: solid; border-width: 1px"| Expected result?<br />
!style="border-style: solid; border-width: 1px"| Gyrase genotype <br> ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T39-47PNXC3-F3&_user=2459438&_coverDate=01%2F28%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=dfcdeab4c210c1f4ec70de318d013c15&searchtype=a Metcalf ''et al.'', 1994]; [http://openwetware.org/wiki/E._coli_genotypes Openwetware])<br />
|-<br />
|style="border-style: solid; border-width: 1px"| DB3.1<br />
|style="border-style: solid; border-width: 1px"| yes <br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| gyrA462<br />
|-<br />
|style="border-style: solid; border-width: 1px"| DH5α<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| gyrA96<br />
|-<br />
|style="border-style: solid; border-width: 1px"| EC100D<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| WT<br />
|-<br />
|style="border-style: solid; border-width: 1px"| JM109<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| gyrA96<br />
|-<br />
|style="border-style: solid; border-width: 1px"| TOP10<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| WT<br />
|-<br />
|style="border-style: solid; border-width: 1px"| XL1-Blue<br />
|style="border-style: solid; border-width: 1px"| yes<br />
|style="border-style: solid; border-width: 1px"| no<br />
|style="border-style: solid; border-width: 1px"| gyrA96<br />
|-<br />
|}<br />
</center><br />
<br />
<br />
It seems that not only the gyrase mutation gyrA462 is causing a ''ccdB'' resistance. Also the gyrase mutation gyrA96 gives ''E. coli'' a ''ccdB'' resistance. This should be kept in mind when assembling BioBricks with the 3A assembly.<br />
<br />
=<partinfo>K389004</partinfo>: Luciferase from pGL4.10[luc2]=<br />
[[Team:Bielefeld-Germany/Results/Characterization/K389004#mRFP vs. luciferase as reporter gene | For a comparison between mRFP and luciferase as reporter genes click here. ]]<br />
<br />
Some important parameters determined by the characterization experiments are shown in tab. 2. For more information concerning these experiments click on the corresponding link in tab. 2 or click here: <br />
<br />
<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389004">Detailed information...</a></div></html><br />
<br />
<br />
<center>Table 2: Parameters for <partinfo>K389004</partinfo>. <br />
<br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| Experiment<br />
!style="border-style: solid; border-width: 1px"| Result<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Accumulation of luciferase | Behaviour during cultivation]]<br />
|style="border-style: solid; border-width: 1px"| <br />
* production is growth dependent<br />
* degradation in stationary growth phase<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Kinetic of luciferin conversion | Kinetic of luciferin conversion]]<br />
|style="border-style: solid; border-width: 1px"| max. output between 20 - 40 s<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Sensitivity | Limit of detection (LOD)]]<br />
|style="border-style: solid; border-width: 1px"| 162 RLU ~ 0.3 % of <partinfo>J23103</partinfo> output<br />
|-<br />
|style="border-style: solid; border-width: 1px"| [[Team:Bielefeld-Germany/Results/Characterization/K389004#Sensitivity | Limit of quantification (LOQ)]]<br />
|style="border-style: solid; border-width: 1px"| 306 RLU ~ 0.7 % of <partinfo>J23103</partinfo> output<br />
|}<br />
</center><br />
<br />
=<partinfo>K389011</partinfo>: VirA screening device=<br />
[[Image:Bielefeld_LD50_Graph2.jpg|600px|thumb|center|Ratio of surviving colonies of ''E. coli'' EC100D carrying unmutated <partinfo>K389010</partinfo> and <partinfo>K389011</partinfo> plated on PA agar plates with chloramphenicol, ampicillin and different concentrations of kanamycin. Comparison between cells that were induced with acetosyringone with cells that were not induced.]]<br />
<br />
The ratio of surviving colonies ϕS was calculated like<br />
<br />
[[Image:IGEM-Bielefeld-formel-LD50.jpg|100px|center]]<br />
<br />
with the number of colony forming units CFU, the concentration of kanamycin on the considered plate KanX and no kanamycin on the plate Kan0.<br />
<br />
=<partinfo>K389012</partinfo>: VirA reporter system with luciferase=<br />
coming more soon<br />
<br />
<br />
=<partinfo>K389015</partinfo>: VirA/G reporter device with luciferase=<br />
Some important parameters determined by the characterization experiments are shown in tab. X. For more information concerning these experiments click on the corresponding link in tab. X or click here: <br />
<br />
<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389015">Detailed information...</a></div></html><br />
<br />
<br />
<center>Table X: Parameters for <partinfo>K389015</partinfo>. <br />
{|{{Table}}<br />
!Experiment<br />
!Characteristic<br />
!Value<br />
|-<br />
|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Transfer function | Transfer Function]]<br />
|Maximum induction level<br />
|2.2 fold<br />
|-<br />
|Maximum induction level reached<br />
|200 µM acetosyringone<br />
|-<br />
|Hill coefficient<br />
|1.09<br />
|-<br />
|Switch Point<br />
|31.6 µM acetosyringone<br />
|-<br />
|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Growth functions and Luciferase expression for BBa_K389015 | Doubling time / h]]<br />
|without plasmid<br />
|1.98<br />
|-<br />
|carrying K389015<br />
|2.24<br />
|-<br />
|carrying K389015 with 400 µM acetosyringone<br />
|2.67<br />
|-<br />
|rowspan="2"|Response time<br />
|Induction: [[Team:Bielefeld-Germany/Results/Characterization/K389015#Response time | exponential phase]]<br />
|>1 h<br />
|-<br />
|Induction: [[Team:Bielefeld-Germany/Results/Characterization/K389015#Data Analysis | begin of cultivation]]<br />
|max. induction at OD<sub>600</sub> = 1 +/- 0.5<br />
|-<br />
|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389015#Plasmid conformation analysis | Conformation analysis]]<br />
|ratio ccc monomer / %<br />
|91<br />
|-<br />
|ratio ccc dimer / %<br />
|3.7<br />
|-<br />
|ratio oc forms / %<br />
|5.3<br />
|-<br />
|}<br />
</center><br />
<br />
=<partinfo>K389016</partinfo>: VirA/G reporter device with mRFP=<br />
<br />
Protocols for [https://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Cultivation_for_measuring_mRFP_and_Luciferase_expression Cultivation] and [https://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Measuring_of_mRFP Measurement]<br />
<br />
Some important parameters determined by the characterization experiments are shown in tab. X. For more information concerning these experiments click on the corresponding link in tab. X or click here: <br />
<br />
<html><div style="font-size:20px; text-align:center; font-weight:bold;"><a href="https://2010.igem.org/Team:Bielefeld-Germany/Results/Characterization/K389016">Detailed information...</a></div></html><br />
<br />
<br />
<center>Table X: Parameters for <partinfo>K389016</partinfo>. <br />
{|{{Table}}<br />
!Experiment<br />
!Characteristic<br />
!Value<br />
|-<br />
|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Transfer function of BBa_K389016 | Transfer Function]]<br />
|Maximum induction level<br />
|2.6 fold<br />
|-<br />
|Maximum induction level reached<br />
|150 µM acetosyringone<br />
|-<br />
|Hill coefficient<br />
|1.67<br />
|-<br />
|Switch Point<br />
|26.5 µM acetosyringone<br />
|-<br />
|rowspan="4"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Growth functions and mRFP expression for BBa_K389016 | Doubling time / h]]<br />
|without plasmid<br />
|1.98<br />
|-<br />
|carrying K389016<br />
|2.57<br />
|-<br />
|carrying K389016 with 150 µM acetosyringone<br />
|2.77<br />
|-<br />
|carrying K389016 with 1000 µM acetosyringone<br />
|3.01<br />
|-<br />
|rowspan="3"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Plasmid conformation analysis | Conformation analysis]]<br />
|ratio ccc monomer / %<br />
|91.2<br />
|-<br />
|ratio ccc dimer / %<br />
|3.2<br />
|-<br />
|ratio oc forms / %<br />
|5.6<br />
|-<br />
|rowspan="5"|[[Team:Bielefeld-Germany/Results/Characterization/K389016#Different possible inducers | Inducers]]<br />
|Induction by<br />
|Acetosyringone<br />
|-<br />
|rowspan="4"|No Induction by<br />
|Capsaicin<br />
|-<br />
|Dopamine<br />
|-<br />
|Homovanillic acid <br />
|-<br />
|3-Methoxytyramine <br />
|}<br />
</center><br />
<br />
=<partinfo>K389052</partinfo>: tightly regulated ''lac'' operon with mRFP readout=<br />
This construct was plated for plasmid isolation in a ''lacI<sup>q</sup>'' negative ''E. coli'' strain after assembly - and we have never seen such red plates when working with constructs with mRFP downstream of a promoter. This ''lac'' operon definitely shows a very high basal transcription, so it is not tightly repressed. It seems that the ''lacI'' repressor <partinfo>BBa_C0012</partinfo> is not suitable for this purpose due to its LVA degradation tag or it does not work properly. Another indicator for this assumption is the experience page of <partinfo>C0012</partinfo>.<br />
<br />
=<partinfo>K389421</partinfo>, <partinfo>K389422</partinfo>, <partinfo>K389423</partinfo>: Sensitivity Tuner amlified Vir-test system=<br />
<br />
By self designed PCR-Primer we excluded terminal GFP and the initial promoter pBAD/araC, for replacing our own VirB promotor and reporter gene luc (luciferase). Primers were designed for sensitivity tuner [http://partsregistry.org/Part:BBa_I746370 I746370], [http://partsregistry.org/Part:BBa_I746380 I746380] and [http://partsregistry.org/Part:BBa_I746390 I746390] so that standard assembly would be possible. Assembling of PCR-products took place by Silver Assembly.<br />
<br />
'''Accomplishment'''<br />
<br />
'''PCR-Primer Design'''<br />
<br />
Primer forward activator phage P2:<br />
<br />
5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GTT TCA TTG TCC TTT ATG CC-3`<br />
<br />
Primer forward activator phage PSP3:<br />
<br />
5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GAT GCA CTG CCC GTT ATG- 3`<br />
<br />
Primer forward activator phage phi R73:<br />
<br />
5`-GTT TCT TCG AAT TCG CGG CCG CTT CTA GAT GCG CTG CCC TTT CTG-3`<br />
<br />
Primer backward Promotor PF from phage P2:<br />
<br />
5`-GTT TCT TCC TGC AGC GGC CGC TAC TAG TAT TTC TCC TCT TTC TCT AGT AAG TGG- 3`<br />
<br />
<br />
'''Characterization tests'''<br />
<br />
Cultivation was done by induction with Acetosyringone at 50 µM. Controls were not induced Sensitivity Tuner devices as well as induced and not induced nativ system ([http://partsregistry.org/Part:BBa_K389015 K389015]; without tuning elements). Induction was done upon inoculation. Measuring point for amplification factor calculation was OD 1.0. ([https://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#Cultivation_for_measuring_mRFP_and_Luciferase_expression Protocols])<br />
<br />
<br />
'''Results'''<br />
<br />
Three sensitivity tuned Vir-Gen sensing systems were obtained: [http://partsregistry.org/Part:BBa_K389421 K389421], [http://partsregistry.org/Part:BBa_K389422 K389422] and [http://partsregistry.org/Part:BBa_K389423 K389423] distinguishing by the amplification level of luc transcription.<br />
<br />
[[Image:ST Tuner.png|600px|thumb|center| '''Figure 1: Amplification factor of induced, 50 µM Acetosyringone (red) and not induced (green) modified Sensitivity Tuner K389421, K389422 and K389423, Standard deviation shown.''']]<br />
<br />
The amplification factor was received by apply [http://partsregistry.org/Part:BBa_K389015 K389015] as reference. Amplification calculation was done by normalizing relative luminescence units emitted from luciferase per OD.<br />
Output-signal amplification is in the induced contructs (red) [http://partsregistry.org/Part:BBa_K389422 K389422] and [http://partsregistry.org/Part:BBa_K389423 K389423] 100 and respectively 200 fold higher than in not induced controls (green). An exception is K389422 were induced and not indiced system revealed analog results. Corresponding to data of iGEM Team, Cambridge 2009, K389423 (originated from [http://partsregistry.org/Part:BBa_I746390 I746390]) shows the highest amplification rate of all tested Sensitivity Tuners. Our results indicate to higher amplification rate of [http://partsregistry.org/Part:BBa_K389421 K389421] than [http://partsregistry.org/Part:BBa_K389422 K389422] of 100 fold under induced conditions. The controls also show high basal transcription rates.<br />
<br />
Because there is small difference in induced and not induced system visible and basal transcription rates are high, we assume that the sensitivity tuning constructs are not well applicable for luciferase measurements.<br />
<br />
For further theory click [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Read_out_system Read out system]<br />
<br />
=References=<br />
*http://openwetware.org/wiki/CcdB, CcdB (seen on 10.10.10).<br />
<br />
*http://openwetware.org/wiki/E._coli_genotypes, E. coli genotypes (seen on 10.10.10).<br />
<br />
*Metcalf, WW ''et al.'' (1994) [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T39-47PNXC3-F3&_user=2459438&_coverDate=01%2F28%2F1994&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=dfcdeab4c210c1f4ec70de318d013c15&searchtype=a ''Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6Kλ origin plasmids at different copy numbers''], Gene 138(1):1-7.<br />
<br />
*[4] Stadler J, Lemmens R, Nyhammar T 2004, ''Plasmid DNA purification'', The J. of Gene Medicine,Vol.6, pp.54–S66<br />
<br />
*[5] Behrens B, Eppendorf AG, Laborpraxis, Nr.20, Reinste Plasmid-DNA in nur 9 Minuten.</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Results/SubmittedTeam:Bielefeld-Germany/Results/Submitted2010-10-27T16:02:46Z<p>Nkessler: /* References */</p>
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= Submitted BioBricks =<br />
<br />
We have submitted the following [[Team:Bielefeld-Germany/Results/Characterization | working]] BioBricks. For a more detailed theoretical background to these BioBricks check our [[Team:Bielefeld-Germany/Project/Theory | theory pages]], too. <br />
<br />
===VirA receptor===<br />
The VirA receptor is used by ''A. tumefaciens'' to detect acetosyringone and other phenolic substances which are secreted by plants after injury. In presence of these substances VirA phosphorylates itself and afterwards VirG, a response regulator which activates ''vir'' promoters. These promoters control genes which are used for infecting the injured plant. <br />
Actually we wanted to use the VirA receptor already existing in the partsregistry (<partinfo>K238008</partinfo>). But due to some problems (compare results in [[Team:Bielefeld-Germany/Results/Characterization | characterization]]) we decided to isolate the ''virA'' gene from the TI-plasmid of ''A. tumefaciens'' C58 ourselves and bring it into a BioBrick compatible form. We removed an illegal ''PstI'' restriction site in the ''virA'' gene by site-directed mutagenesis. <br />
<br />
You can find this BioBrick here: <partinfo>K389001</partinfo><br />
<br />
You can find this BioBrick under the control of a constitutive promoter (<partinfo>J23110</partinfo>) here: <partinfo>K389010</partinfo>.<br />
<br />
<br />
===Mutated ''virG''===<br />
Phosphorylated VirG binds to ''vir'' promoters and activates them. VirG is activated by the acetosyringone receptor VirA. <br />
This version of VirG activates ''vir'' promoters in ''Escherichia coli'' without the ''rpoA''-gene from ''Agrobacterium tumefaciens''. For this reason the point mutations G56V and I77V are brought into the molecule ([http://www.springerlink.com/content/wmq06kua5qkma1au/ YC Jung ''et al.'', 2004]). Because this BioBrick is synthesized (Mr. Gene GmbH), codon usage is optimized for ''E. coli'' and illegal restriction sites were removed. When you use this ''virG'' gene in a VirA/G signaling system you do not need <partinfo>K238010</partinfo> anymore to get the system working in ''E. coli''. <br />
<br />
You can find this BioBrick here: <partinfo>K389002</partinfo><br />
<br />
===''virB''-promoter===<br />
''Vir''-promoters from ''A. tumefaciens'' are induced by phosphorylated VirG response regulators and control genes for infecting plants in their natural host. They are part of the VirA/G signal transduction system. <br />
We wanted to use the ''vir''-promoter from the partsregistry (<partinfo>K238011</partinfo>) but the same problems occurred like with the use of the VirA receptor BioBrick from the partsregistry. So we also have to create a new ''vir''-promoter BioBrick (again from TI-plasmid of ''A. tumefaciens'' C58).<br />
<br />
You can find this BioBrick here: <partinfo>K389003</partinfo><br />
<br />
<br />
===Firefly luciferase===<br />
Bringing the firefly luciferase gene from Promega's pGL4.10[luc2] vector into a BioBrick compatible form as a sensitive reporter gene. To amplify the signal of the luciferase three different sensitivity tuners are assembled before the luciferase gene. The sensitivity tuners were created in 2007 by the iGEM team from Cambridge and amplify the read-out signal. We also assembled the firefly luciferase behind three different strong constitutive promoters for gathering additional information about this reporter gene ([http://parts.mit.edu/igem07/index.php/Cambridge/Amplifier_project#Results Cambridge, 2007]). <br />
<br />
You can find this BioBrick here: <partinfo>K389004</partinfo><br />
<br />
You can find this BioBrick with sensitivity tuner 1 here: <partinfo>K389401</partinfo><br />
<br />
You can find this BioBrick with sensitivity tuner 2 here: <partinfo>K389402</partinfo><br />
<br />
You can find this BioBrick with sensitivity tuner 3 here: <partinfo>K389403</partinfo><br />
<br />
You can find this BioBrick under the control of a weak constitutive promoter here: <partinfo>K389302</partinfo><br />
<br />
You can find this BioBrick under the control of a medium strong constitutive promoter here: <partinfo>K389307</partinfo><br />
<br />
You can find this BioBrick under the control of a strong constitutive promoter here: <partinfo>K389318</partinfo><br />
<br />
===Neomycin / kanamycin resistance===<br />
A neomycin / kanamycin resistance gene without promoter is isolated and brought into a BioBrick compatible form. We will use the BioBrick <partinfo>P1003</partinfo> as source for the kanamycin resistance gene. <br />
<br />
You can find this BioBrick here: <partinfo>K389005</partinfo><br />
<br />
<br />
===BioBrick for ''virA''-screenings===<br />
This part contains our mutated ''virG'' BioBrick under the control of a constitutive promoter (<partinfo>J23110</partinfo>) and an antibiotic resistance (<partinfo>K389005</partinfo>) under the control of the ''virB'' promoter (<partinfo>K389003</partinfo>). The better the VirA receptor recognizes a substance the stronger will the antibiotic resistance be expressed. <br />
<br />
You can find this BioBrick here: <partinfo>K389011</partinfo><br />
<br />
<br />
===Reporter constructs===<br />
The reporter constructs are similar to the ''virA'' screening construct but instead of the antibiotic resistance they carry a reporter gene. The amount of produced reporter shows the activity of the VirA receptor and the ''vir'' promoter, respectively. If the original ''vir'' promoter is too weak, we will use Cambridge's sensitivity tuners to increase the output signal of our biosensor ([http://parts.mit.edu/igem07/index.php/Cambridge/Amplifier_project#Results Cambridge, 2007]). <br />
<br />
You can find this BioBrick with luciferase read-out here: <partinfo>K389012</partinfo><br />
<br />
You can find this BioBrick with mRFP read-out here: <partinfo>K389013</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 1 here: <partinfo>K389411</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 2 here: <partinfo>K389412</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 3 here: <partinfo>K389413</partinfo><br />
<br />
<br />
===Characterization constructs===<br />
The characterization constructs are like the reporter constructs but a ''virA'' gene under the control of a constitutive promoter is assembled before them. These constructs are used to characterize the natural, unmutated VirA/G signaling system and the ''vir'' promoter, respectively. The natural system is induced with acetosyringone and the read-out is measured to determine the behaviour of the VirA/G signaling system. <br />
<br />
You can find this BioBrick with luciferase read-out here: <partinfo>K389015</partinfo><br />
<br />
You can find this BioBrick with mRFP read-out here: <partinfo>K389016</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 1 here: <partinfo>K389421</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 2 here: <partinfo>K389422</partinfo><br />
<br />
You can find this BioBrick with amplified luciferase read-out No. 3 here: <partinfo>K389423</partinfo><br />
<br />
<br />
===VirA/G signaling system===<br />
A complete VirA/G signaling system without read-out was submitted. The system contains a ''virA'' and our mutated ''virG'' gene under the control of a constitutive promoter and a ''vir'' promoter without reporter gene. <br />
<br />
You can find this BioBrick here: <partinfo>K389017</partinfo><br />
<br />
=References=<br />
*[http://parts.mit.edu/igem07/index.php/Cambridge/Amplifier_project Cambridge iGEM Team wiki 2007, amplifier project]<br />
<br />
*YC Jung ''et al.'' (2004) [http://www.springerlink.com/content/wmq06kua5qkma1au/ ''Mutants of ''Agrobacterium tumefaciens virG'' Gene That Activate Transcription of ''vir'' Promoter in ''Escherichia coli''''], Current Microbiol 49:334-340.</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Project/ProtocolsTeam:Bielefeld-Germany/Project/Protocols2010-10-27T16:02:19Z<p>Nkessler: /* References */</p>
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= Lab protocols =<br />
<br />
<br />
== Preparation of electrocompetent ''E.coli'' cells ==<br />
<br />
modified from [http://openwetware.org/wiki/Quint_Lab:electrocompetent_cells_e.coli Quint Lab]<br />
<br />
<br />
Materials: <br />
* 10 mL LB-Medium (1 % Bacto Trypton; 0.5 % Yeast Extract; 0.5 % NaCl) <br />
* 2 L cooled bidest H<sub>2</sub>O <br />
* 200 mL cooled, sterile-filtered 10 % Glycerol <br />
* Box with ice-water for 2-litre-flask <br />
* 4 pre-cooled 250 mL (or 2x500 mL) bins for centrifugation <br />
* 2 pre-cooled 50 mL Falcons <br />
* Centrifuge pre-cooled to 2°C (max. 4°C) <br />
<br />
<br />
Protocol<br />
* Inoculate 2x10 mL LB with bacterial stock; incubate over night at 37°C and 200 rpm <br />
* Inoculate 2x250 mL LB in 1-litre-flask with OD<sub>600</sub>=0.1 @37°C or @ 19°C over night<br />
* Incubate until OD<sub>600</sub> 0.4-0.6 (~5 h)<br />
* From now on everything is done at 2-4°C (best in a cold room) <br />
<br />
* Cool 1L-culture 10-15 minutes in ice water (shake sometimes) <br />
* Divide culture into 2x5 cooled 50 mL Falcon-tubes for centrifugation <br />
* Centrifuge 10min @ 4°C, 4000 rcf <br />
* Discard supernatant <br />
* Resuspend pellet in 5 mL bidest H2O <br />
* Add bidest. H<sub>2</sub>O up to 50 mL <br />
* Centrifuge 10min @ 4°C, 4000 rcf <br />
* Discard supernatant immediately <br />
* Resuspend pellet in residual supernatant <br />
* Add bidest H<sub>2</sub>O up to 50 mL <br />
* Centrifuge 10min @ 4°C, 4000 rcf <br />
* Discard supernatant immediately <br />
* Resuspend pellet in residual supernatant <br />
* Transfer suspension in 2x50 mL Falcons <br />
* Add 10% Glycerol up to 50 mL <br />
* Centrifuge 5 min @ 4°C, 4000 rcf <br />
* Discard supernatant <br />
* Estimate volume of the pellet; fill up with equal volume of 10% Glycerol <br />
* Resuspend pellet on ice; ‘’’don´t vortex!!’’’ (just shake cautiously) <br />
* Divide cells into 150 µL Alicuots (use 1.5 mL Eppis) <br />
* Freeze in liquid N<sub>2</sub> or dry-ice <br />
* Store @ -80°C<br />
<br />
== Preparation of heat shock competent ''E.coli'' cells ==<br />
<br />
<br />
* Inoculate 2x5 mL LB with bacterial stock; incubate over night at 37°C and 200 rpm <br />
* Inoculate 2x250 mL SOB in 1-litre-flasks with OD<sub>600</sub>=0.1<br />
* Incubate @19°C until OD<sub>600</sub>=0.4-0.6 (20h)<br />
* From now on everything is done at 2-4°C (best in a cold room) <br />
<br />
* Cool 1L-culture 10-15 minutes in ice water (shake sometimes) <br />
* Divide culture into 2x5 cooled 50 mL Falcon-tubes for centrifugation <br />
* Centrifuge 10min @ 4°C, 4000 rcf <br />
* Discard supernatant<br />
* Resuspend cells in 80 mL TB buffer<br />
* Cool cells for 10 min on ice<br />
* Centrifuge 10min @ 4°C, 2500 rcf<br />
* Discard supernatant<br />
* Resuspend cells in 20 mL TB buffer<br />
* Add 1.5 mL Dimethyl sulfoxide (DMSO) for each 20 mL cells (DMSO concentration = 7%) <br />
* Cool cells for 10 min on ice<br />
* Divide cells into 150 µL Alicuots (use 1.5 mL Eppis)<br />
* Freeze in liquid N<sub>2</sub> or dry-ice <br />
* Store @ -80°C<br />
<br />
== Transformation via electroporation ==<br />
<br />
* Thaw 50 µL competent ''E.coli'' cells on ice, dilute with icecold 50 µL Glycerin (10 %) if necessary<br />
* Add 0.5-5 µL plasmid to 50 µl electrocompetent cells<br />
* Store cells on ice for 1 minute <br />
* Electroporate at U= 2.5 kV, C= 25 µF, R = 200 Ώ <br />
* Transfer transformation reaction to 450 µL SOC-Medium and shake 1 h at 37°C <br />
* Centrifuge 2 min @ 800 rpm and plate on selective LB-Medium<br />
<br />
== Heat shock transformation ==<br />
<br />
* Thaw 150 µL competent ''E. coli'' cells on ice<br />
<br />
* Add max. 10 µL DNA (the less the better your transformation works but at least about 50 ng vector)<br />
<br />
* Incubate 30 min on ice<br />
<br />
* Heatshock: 42 °C, 45 s (water bath because of quick heat transfer)<br />
<br />
* 1 min on ice<br />
<br />
* Add 1 mL prewarmed SOC-medium<br />
<br />
* Incubate: 45 - 60 min, 37 °C<br />
<br />
* Plate 100 µL<br />
<br />
* Spin down the remaining cells (2 min, 5000 g), discard most of the supernatant, resuspend the cells in the remaining medium and plate them<br />
<br />
== Silver BioBrick Assembly ==<br />
<br />
modified from [http://openwetware.org/wiki/Silver:_BB_Strategy Silver lab]:<br />
<br />
This assembly method can be used for BioBricks which are bigger than 150 bp. The BioBrick should be at least 500 bp bigger or smaller than the backbone. The BioBrick, which complies with these conditions, is used as the insert and is assembled into the prefix or suffix of the other used BioBrick, called vector. So you have to differentiate between a prefix and a suffix insertion.<br />
<br />
[[Image:Bielefeld_Silver_1.png|300px|thumb|right|Silver Suffix Insertion]]<br />
[[Image:Bielefeld_Silver_2.png|300px|thumb|right|Silver Prefix Insertion]]<br />
<br />
=== Suffix Insertion ===<br />
<br />
* Digestion of insert: at least 700 ng DNA / 10 µL volume, 1 µL 10x Tango buffer, 0.5 µL XbaI, 1 µL PstI. Digest for 2 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel try to avoid staining or exposure to ultraviolet light of the insert. <br />
<br />
* Digestion of vector about 700 ng DNA / 10 µL volume, 1 µL 10x orange buffer, 0.5 µL SpeI, 0.5 µL PstI. Digest for 2h at 37 °C, afterwards inactivation for 20 min at 80 °C. Add 1 µL SAP (shrimp alcaline phosphatase) and 1.2 µL 10 x SAP buffer, incubate for 1 h at 37 °C. Clean up the vector with a PCR clean-up kit. <br />
<br />
* Ligation: after digestion and clean-up: 50 - 200 ng of vector, 3 - 10 fold molar access of insert, 20 µL ligation volume, 2 µL T4-Ligase-Buffer, 1 µL T4-Ligase. Incubate for 1 h at 37 °C, afterwards inactivation for 5 min at 70 °C. Then: store at -20 °C or transform.<br />
<br />
<br />
<br />
<br />
=== Prefix Insertion ===<br />
<br />
* Digestion of insert: at least 700 ng DNA / 10 µL volume, 1 µL 10x BamHI buffer, 0.5 µL EcoRI, 0.5 µL SpeI. Digest for 2 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel try to avoid staining or exposure to ultraviolet light of the insert. <br />
<br />
* Digestion of vector about 700 ng DNA / 10 µL volume, 1 µL 10 x Tango buffer, 0.5 µL EcoRI, 0.5 µL XbaI. Digest for 2h at 37 °C, afterwards inactivation for 20 min at 80 °C. Add 1 µL SAP (shrimp alcaline phosphatase) and 1.2 µL 10 x SAP buffer, incubate for 1 h at 37 °C. Clean up the vector with a PCR clean-up kit. <br />
<br />
* Ligation: after digestion and clean-up: 50 - 200 ng of vector, 3 - 10 fold molar access of insert, 20 µL ligation volume, 2 µL T4-Ligase-Buffer, 1 µL T4-Ligase. Incubate for 1 h at 37 °C, afterwards inactivation for 5 min at 70 °C. Then: store at -20 °C or transform.<br />
<br />
<br />
<br />
=== Variations ===<br />
<br />
* A digestion over night is possible. If you digest over night use only 0.1 µL restriction enzyme. <br />
<br />
* It is also possible to use PCR product as insert. Digest after PCR with corresponding restriction enzymes and clean up with PCR clean-up kit. This could lead to higher yields of insert DNA because a lot of DNA gets lost during the gel electrophoresis clean up. <br />
<br />
* Sometimes some BioBricks are hard to assemble. Then you have to clean up the vector by gel electrophoresis as well.<br />
<br />
<br />
==3A assembly==<br />
Modified from [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf BioBrick Assembly Manual by Ginkgo BioWorks]<br />
<br />
<br />
===Digestion===<br />
* Thaw DNA from upstream and downstream part and the destination plasmid on ice. <br />
<br />
** Destination plasmid has to carry the ''ccdB'' gene <partinfo>P1010</partinfo> as insert and has to have a different antibiotic resistance than the plasmids carrying the upstream and downstream parts<br />
<br />
** DNA has to be cleaned (by MiniPrep or after a PCR)<br />
<br />
* 500 ng DNA / digestion mix for upstream and downstream part, 150 ng DNA / digestion mix for destination plasmid (total volume of mix 10 µl, dilute with ddH<sub>2</sub>0 if necessary)<br />
<br />
* Add 1 µl of buffer and restriction enzymes as shown in the following table: <br />
<br />
<center><br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 1px; border-style: solid; border-color: #000"<br />
|-<br />
!style="border-style: solid; border-width: 1px"| <br />
!style="border-style: solid; border-width: 1px"| Upstream part<br />
!style="border-style: solid; border-width: 1px"| Downstream part<br />
!style="border-style: solid; border-width: 1px"| Destination plasmid<br />
|-<br />
|style="border-style: solid; border-width: 1px"| enzyme 1<br />
|style="border-style: solid; border-width: 1px"| 1 µl EcoRI<br />
|style="border-style: solid; border-width: 1px"| 0.5 µl XbaI<br />
|style="border-style: solid; border-width: 1px"| 0.5 µl EcoRI<br />
|-<br />
|style="border-style: solid; border-width: 1px"| enzyme 2<br />
|style="border-style: solid; border-width: 1px"| 1 µl SpeI<br />
|style="border-style: solid; border-width: 1px"| 1 µl PstI<br />
|style="border-style: solid; border-width: 1px"| 0.5 µl PstI<br />
|-<br />
|style="border-style: solid; border-width: 1px"| buffer<br />
|style="border-style: solid; border-width: 1px"| BamHI<br />
|style="border-style: solid; border-width: 1px"| Tango<br />
|style="border-style: solid; border-width: 1px"| Orange<br />
|}<br />
</center><br />
<br />
<br />
* Incubation of the digestion mixes for 2 h at 37 °C, afterwards heat inactivation for 20 min at 80 °C<br />
<br />
* Continue with ligation or freeze the mixes<br />
<br />
<br />
===Ligation===<br />
* Ligation mix: <br />
<br />
** 2 µl ddH<sub>2</sub>O<br />
<br />
** 5 µl of every digestion mix (so 15 µl in total)<br />
<br />
** 2 µl T4-DNA-ligase buffer (thaw on ice!)<br />
<br />
** 1 µl T4-DNA-ligase<br />
<br />
* Incubate 1 h at 37 °C, afterwards heat inactivation for 5 min at 70 °C<br />
<br />
* Freeze ligation mix or continue with transformation (heatshock or electroporation)<br />
<br />
== Phusion PCR ==<br />
<br />
* Perform a Phusion PCR for amplifying BioBricks or gaining new BioBricks<br />
<br />
* For one PCR-Reaction mix:<br />
** 10 µL 5x HF-Buffer<br />
** 4 µL dNTPs (2.5 mM each)<br />
** 1.5 µL DMSO<br />
** 31.5 µL H<sub>2</sub>O<br />
** 1 µL Primer Mix<br />
** 1 µL Template<br />
** 1 µL Phusion DNA-Polymerase<br />
<br />
* PCR program: <br />
** Start: 30 sec, 98 °C<br />
** 35 cycles of: <br />
*** 10 sec, 98 °C<br />
*** 30 sec, 58-66 °C (depending on primers)<br />
*** 15-30 sec / 1 kb template, 72 °C<br />
** 10 min, 72 °C<br />
** 4 °C until further processing<br />
<br />
* Perform a gel electrophoresis:<br />
** Check the fragment size<br />
** Depending whether there are fragments with wrong size perform a clean up via gel electrophoresis or just with a PCR clean-up kit<br />
<br />
== Colony PCR ==<br />
* Pick one colony with a sterile tip and elute it in 100 µl ddH<sub>2</sub>0 or medium<br />
<br />
* Store the colony in 4°C while colony PCR is running<br />
<br />
* One reaction mix contains: <br />
** 2.5 µl 10x buffer<br />
** 0.75 µl MgCl<sub>2</sub><br />
** 1 µl dNTPs<br />
** 0.5 µl primer mix (prefix/suffix primers or sequencing primers)<br />
** 19.25 µl ddH<sub>2</sub>O<br />
** 0.5 µl taq-polymerase<br />
** 0.5 µl template<br />
<br />
* PCR program: <br />
** Start: 8 min, 98 °C<br />
** 30 cycles of: <br />
*** 30 s, 98 °C<br />
*** 30 s, 60 °C<br />
*** 30 s / 1 kb template, 72 °C<br />
** Finish: 5 min, 72 °C<br />
<br />
* Gel electrophoresis: check the fragment size<br />
<br />
* Plate the correct colony<br />
<br />
==Construction of a plasmid with R6K origin of replication (ori)==<br />
<br />
<br />
==Determination of minimal inhibitory concentration (MIC) of kanamycin==<br />
<br />
* Transform the plasmid K389212 (native virA) in bacteria which are already containing K389101 (kanamycin resistance read out) using [[#Transformation via electroporation |electroporation]].<br />
* Plate 100 µL of the transformed bacteria in different steps of dilution (10<sup>-1</sup> to 10<sup>-6</sup>) on LB-Agar with ampicillin (100 µg mL<sup>-1</sup>) and chloramphenicol (10 µg mL<sup>-1</sup>).<br />
* Incubate the plates upside down at 37 °C for 16 – 20 h.<br />
* Compare the plates of different dilution steps and select those, showing a high but countable number of colonies (aprox. 100 – 500).<br />
<br />
<br />
In the following steps the colonies are transferred to LB-Agar with raising concentrations of kanamycin either in the presence of acetosyringone or without any inductor.<br />
<br />
* Transfer the colonies of the selected plates to LB-Agars described below using replica plating. Take care to operate from lowest up to highest concentration of kanamycin. You must also use a fresh and sterile stamp every time you start a new sequence of plating.<br />
<br />
* Composition of the LB-Agars (plating sequence from top to bottom)<br />
<br />
** A) Uninduced state (no acetosyringone)<br />
<center><br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 0.5px; border-style: solid; border-color: #000"<br />
|-<br />
|style="border-style: solid; border-width: 0.5px"| Positive Control<br />
|style="border-style: solid; border-width: 0.5px"| Ampicillin (100 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Chloramphenicol (10 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Acetosyringone ('''0 µM''')<br />
|style="border-style: solid; border-width: 0.5px"| Kanamycin ('''0 µg mL<sup>-1</sup>''')<br />
|-<br />
|style="border-style: solid; border-width: 0.5px"|<br />
|style="border-style: solid; border-width: 0.5px"| Ampicillin (100 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Chloramphenicol (10 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Acetosyringone ('''0 µM''')<br />
|style="border-style: solid; border-width: 0.5px"| Kanamycin ('''10 µg mL<sup>-1</sup>''')<br />
|-<br />
|style="border-style: solid; border-width: 0.5px"|<br />
|style="border-style: solid; border-width: 0.5px"| Ampicillin (100 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Chloramphenicol (10 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Acetosyringone ('''0 µM''')<br />
|style="border-style: solid; border-width: 0.5px"| Kanamycin ('''25 µg mL<sup>-1</sup>''')<br />
|-<br />
|style="border-style: solid; border-width: 0.5px"|<br />
|style="border-style: solid; border-width: 0.5px"| Ampicillin (100 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Chloramphenicol (10 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Acetosyringone ('''0 µM''')<br />
|style="border-style: solid; border-width: 0.5px"| Kanamycin ('''50 µg mL<sup>-1</sup>''')<br />
|-<br />
|style="border-style: solid; border-width: 0.5px"|<br />
|style="border-style: solid; border-width: 0.5px"| Ampicillin (100 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Chloramphenicol (10 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Acetosyringone ('''0 µM''')<br />
|style="border-style: solid; border-width: 0.5px"| Kanamycin ('''100 µg mL<sup>-1</sup>''')<br />
|-<br />
|style="border-style: solid; border-width: 0.5px"|<br />
|style="border-style: solid; border-width: 0.5px"| Ampicillin (100 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Chloramphenicol (10 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Acetosyringone ('''0 µM''')<br />
|style="border-style: solid; border-width: 0.5px"| Kanamycin ('''200 µg mL<sup>-1</sup>''')<br />
|}<br />
</center><br />
<br />
<br />
** B) Induced state (200 µM acetosyringone)<br />
<center><br />
{|cellpadding="10" style="border-collapse: collapse; border-width: 0.5px; border-style: solid; border-color: #000"<br />
|-<br />
|style="border-style: solid; border-width: 0.5px"| Positive Control<br />
|style="border-style: solid; border-width: 0.5px"| Ampicillin (100 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Chloramphenicol (10 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Acetosyringone ('''200 µM''')<br />
|style="border-style: solid; border-width: 0.5px"| Kanamycin ('''0 µg mL<sup>-1</sup>''')<br />
|-<br />
|style="border-style: solid; border-width: 0.5px"|<br />
|style="border-style: solid; border-width: 0.5px"| Ampicillin (100 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Chloramphenicol (10 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Acetosyringone ('''200 µM''')<br />
|style="border-style: solid; border-width: 0.5px"| Kanamycin ('''10 µg mL<sup>-1</sup>''')<br />
|-<br />
|style="border-style: solid; border-width: 0.5px"|<br />
|style="border-style: solid; border-width: 0.5px"| Ampicillin (100 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Chloramphenicol (10 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Acetosyringone ('''200 µM''')<br />
|style="border-style: solid; border-width: 0.5px"| Kanamycin ('''25 µg mL<sup>-1</sup>''')<br />
|-<br />
|style="border-style: solid; border-width: 0.5px"|<br />
|style="border-style: solid; border-width: 0.5px"| Ampicillin (100 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Chloramphenicol (10 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Acetosyringone ('''200 µM''')<br />
|style="border-style: solid; border-width: 0.5px"| Kanamycin ('''50 µg mL<sup>-1</sup>''')<br />
|-<br />
|style="border-style: solid; border-width: 0.5px"|<br />
|style="border-style: solid; border-width: 0.5px"| Ampicillin (100 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Chloramphenicol (10 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Acetosyringone ('''200 µM''')<br />
|style="border-style: solid; border-width: 0.5px"| Kanamycin ('''100 µg mL<sup>-1</sup>''')<br />
|-<br />
|style="border-style: solid; border-width: 0.5px"|<br />
|style="border-style: solid; border-width: 0.5px"| Ampicillin (100 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Chloramphenicol (10 µg mL<sup>-1</sup>)<br />
|style="border-style: solid; border-width: 0.5px"| Acetosyringone ('''200 µM''')<br />
|style="border-style: solid; border-width: 0.5px"| Kanamycin ('''200 µg mL<sup>-1</sup>''')<br />
|}<br />
</center><br />
<br />
<br />
* Incubate the plates upside down at 37 °C for 16 – 20 h.<br />
* Count the number of colonies on each plate.<br />
* Calculate the ration of colonies grown on each plate, compared to the number of colonies on the first plate of each sequence (positive control).<br />
<br />
==Directed mutagenesis – Error-prone PCR and screening system==<br />
<br />
===1. Error PCR Step===<br />
Mix:<br />
* 10 µL 100 mM Tris-HCL, pH 8.3<br />
* 2.5 µL 2M KCL<br />
* 3.5 µL 200 mM MgCL<sub>2</sub><br />
* 2 µL 25 mM MnCL<sub>2</sub> (Add immediately before initiation of the PCR)<br />
* 4 µL 25 mM dCTP<br />
* 4 µL 25 mM dTTP<br />
* 4 µL 5 mM dATP<br />
* 4 µL 5 mM dGTP<br />
* 0.5 µL 100 µM 5´primer (equals 50 pmol)<br />
* 0.5 µL 100 µM 3´primer (equals 50 pmol)<br />
* x µL Template K389212 – total amount of 1.5 pmol<br />
* 5 µL 1 U/µL Taq DNA polymerase<br />
* Add Milli Q water to 100 µL total volume<br />
<br />
* PCR program:<br />
** Start: 8 min, 98 °C (melt)<br />
** 24 cycles of:<br />
*** 30 s, 98 °C (melt)<br />
*** 30 s, 60 °C (anneal)<br />
*** 30 s / 1 kb template, 72 °C (extension)<br />
** Final extension: 5 min, 72 °C <br />
** End: Keep at 4 °C<br />
<br />
===2. Cloning of virA variants===<br />
Clone the randomly mutated virA sequences (2.5 kB) under the control of the promoter J23110 and the RBS B0034 in the pSB1AT2 backbone.<br />
<br />
(See above for [[#Silver BioBrick Assembly |protocol of Prefix- / Suffix-Insertion]])<br />
<br />
===3. Primary selection of novel virA variants===<br />
Transform the libraries of virA variants into electro-competent E. coli, in which K389101 and thereby the read out system via kanamycin resistanc is already present.<br />
<br />
(See above for [[#Transformation via electroporation |protocol of transformation]])<br />
<br />
Plate the transformed bacteria on selective LB-Agar. When producing the plates add the following mixture of substances to the hand-warm LB-Agar:<br />
<br />
* 1 mL Ampicillin stock solution (final concentration 100 µg mL<sup>-1</sup>)<br />
* 286 µL Chloramphenicol stock solution (final concentration 10 µg mL<sup>-1</sup>)<br />
** These antibiotica are used to ensure stability of both plasmids<br />
<br />
* 50 mL substance-mix – including capsaicin, dopamine, homovanillic acid and 3-O methyldopamine (all final concentration of 200 µM)<br />
** Any bacteria with a virA variation that is activated by at least one of these novel inductors will express the kanamycin resistance<br />
<br />
* 2 mL Kanamycin stock solution (final: 100 µg mL<sup>-1</sup>)<br />
** Compareable high kanamycin concentration to allow only these bacteria to grow which show a strong expression o the kanamycin resistance. (This optimal concentration was previously determined in MIC analysis.)<br />
<br />
Incubate plates upside down at 37 °C for 16 – 20 h.<br />
The grown colonies will include a virA variant that is either constitutively active or had been induced by at least one of the tested substances.<br />
<br />
===4. Quantitative analysis of virA variants after induction with novel substances===<br />
To quantify the induction profile of the selected virA variants it is appropriate to change the read out system from kanamycin resistence to luciferase expression. This complex and time consuming task can easily be achieved without any cloning step, when using the advantage of our two plasmid system:<br />
<br />
* Pick a colony of interest and plate it on a fresh LB-Agar, containing Amp (100 µg mL<sup>-1</sup>) and Cm (10 µg mL<sup>-1</sup>)<br />
* Incubate upside down at 37 °C for 16 – 20 h.<br />
* Isolate plasmids from the bacterial lawn with a miniprep.<br />
** In this step two plasmids are isolated – One includes the virA variants in a normal pSB1AT3 backbone. The second type with the kanamycin read out has the special R6K ori, which can only amplify in certain E. coli strains.<br />
<br />
* Transform the isolated plasmids to a strain which is not capable to amplify the R6K ori (e.g. TOP10)<br />
* Plate the transformants on LB-Agar with ampicillin (100 µg mL<sup>-1</sup>) and incubate upside down at 37 °C for 16 – 20 h.<br />
* Isolate plasmids from the bacterial lawn with a miniprep.<br />
** Here you are only harvesting the plasmid including the virA variant in psB1AT3, since the other plasmid was not amplified in the grown E. coli.<br />
<br />
* Transform the plasmid with the virA variant into competent cells, already including a second plasmid with a read out system luciferase or mRFP.<br />
<br />
* Grow the transformants in shake flaks in LB-Media containing ampicillin (100 µg mL<sup>-1</sup>), chloramphenicol (10 µg mL<sup>-1</sup>) and 200 µM of one testing substance (e.g. capsaicin). As a control you should also grow the same bacteria without any inductor and with the native inductor acetosyringone.<br />
* Take samples in certain intervals (e.g. 1 h) and [[#Measuring of mRFP |measure the activity of the luciferase or mRFP]].<br />
<br />
== Restriction analysis ==<br />
<br />
* Digest BioBrick of interest: about 400 ng DNA / 10 µL volume, 1 µL 10x orange buffer, 0.5 µL NotI or PstI. Digest for 2 h at 37 °C. NotI is used to determine the length of the BioBrick and the plasmid backbone, PstI ist used to determine the length of the BioBrick in the plasmid backbone. <br />
<br />
* Gel electrophoresis: add 2 µL loading buffer to every digestion mix, apply about 100 - 200 ng DNA / pocket in gel. Don't forget to apply the uncut BioBrick as well. A good agarose concentration for BioBricks between 0.2 and 3 kb is 1.5 %. The smaller your BioBrick of interest is the higher the agarose concentration should be and vice versa. The gel electrophoresis is made with TAE-buffer. Be sure that you melt your agarose gel in the same buffer you use for the electrophoresis later. <br />
<br />
<br />
== Cultivation for measuring mRFP and Luciferase expression ==<br />
* Inoculate 10 mL LB containing desired Antibiotic with glyccerol stock<br />
* Cultivate over night at 37 °C and 175 rpm<br />
* Measure the OD<sub>600</sub><br />
* Prepare shake flasks with LB, antibiotic and inducer<br />
** For Luciferase Measurement at least 10 mL starting volume<br />
** For mRFP Measurement at least 20 mL starting volume<br />
* Inoculate the main culture with a starting OD<sub>600</sub> of 0.1<br />
* Cultivate at 37 °C and 175 rpm<br />
* Take a sample at least every hour and measure the OD<sub>600</sub><br />
** The sample handling depends on the readout strategy and is described in the corresponding measuring protocol ([[#Measuring_of_mRFP| mRFP]] or [[#Measuring_of_Luciferase| Luciferase]])<br />
<br />
== Measuring of mRFP ==<br />
* Take at least 500 µL sample for each measurement (200 µL is needed for one measurement) so you can perform a repeat determination<br />
* Freeze samples at -80 °C for storage<br />
* To measure the samples thaw at room temperature and fill 200 µL of each sample in one well of a black, flat bottom 96 well microtiter plate (perform at least a repeat determination)<br />
* Measure the Fluorescence in a platereader (we used a Tecan Infinite® m200platereader ) with following settings<br />
** 20 sec orbital shaking (1 mm amplitude with a frequenzy of 87.6 rpm)<br />
** Measurement mode: Top<br />
** Excitation: 584 nm<br />
** Emission: 620 nm<br />
** Number of reads: 25<br />
** Manual gain: 150<br />
** Integration time: 20 µs<br />
<br />
== Measuring of Luciferase ==<br />
For the luciferase detection we used a [http://www.promega.com/tbs/tb281/tb281.pdf Promega Luciferase Assay System], containing a Cell Culture Lysis Reagent, Luciferase Assay Substrate and Luciferase Assay Buffer<br />
<br />
<br />
Protocol:<br />
* Prepare reaction tubes with 10 µL of high salt buffer (1M K<sub>2</sub>HPO<sub>4</sub>, 20mM EDTA, pH 7.8)<br />
* Add 90 µL sample, mix and freeze at -80 °C<br />
* For the measurement thaw by placing the tubes in room temperature water<br />
* Add 300 µL of freshly prepared lysis mix ([[#Cell_Culture_Lysis_Reagent| 1X Cell Culture Lysis Reagent]], 1.25 mg/mL lysozyme, 2.5 mg/mL BSA, add Water for desired Volume)<br />
* Mix and incubate the cells for 10 minutes at room temperature<br />
* Prepare the Luciferase Assay Reagent, by adding 10 mL of Luciferase Assay Buffer to the vial containing the Luciferase Assay Substrate<br />
* Fill each well of a white, flat bottom 96 well microtiter plate with 20 µL of cell lysate<br />
* For the Detection of Luciferase use a plate reading luminometer with Injector for the Luciferase Assay Reagent and following settings (we used a Promega GloMax®-Multi Detection System with dual injector):<br />
** Injection volume of Luciferase Assay Reagent: 100 µL<br />
** Delay: 20 secs<br />
** Integration: 3 secs<br />
<br />
== Chemicals, material etc. ==<br />
===Enzymes===<br />
<br />
{| class="wikitable" style="text-align:left"<br />
<br />
|-<br />
! Enzyme !! style="padding-left:5px;" |Producer<br />
|-<br />
| Pfu DNA-polymerase || style="padding-left:5px;" |Promega<br />
|-<br />
| Phusion DNA-polymerase || style="padding-left:5px;" |Finnzymes<br />
|- <br />
| Restriction enzymes || style="padding-left:5px;" |Fermentas<br />
|-<br />
| Shrimp alcaline phosphatase || style="padding-left:5px;" |Fermentas<br />
|- <br />
| T4-DNA-Ligase || style="padding-left:5px;" |Fermentas<br />
|-<br />
| taq-DNA-polymerase || style="padding-left:5px;" |Bioline<br />
|-<br />
|}<br />
<br />
<br />
===Kits===<br />
<br />
{| class="wikitable" style="text-align:left"<br />
|-<br />
! Function !! style="padding-left:5px;" | Name<br />
|-<br />
| Plasmid purification || style="padding-left:5px;" | Fermentas GeneJET™ Plasmid Miniprep Kit<br />
|-<br />
| PCR Cleanup || style="padding-left:5px;" | Macherey Nagel NucleoSpin® Extract II<br />
|-<br />
|}<br />
<br />
<br />
=== Inducer Stock Solutions ===<br />
* Acetosyringone: 20 mM solved in 10 % (v/v) DMSO and 90 % (v/v) H<sub>2</sub>O<br />
* Capsaicin: 20 mM solved in Methanol<br />
* Dopamine: 20 mM solved in H<sub>2</sub>O<br />
* Homovanillic acid: 20 mM solved in H<sub>2</sub>O<br />
* 3-Methoxytyramine: 20 mM solved in H<sub>2</sub>O<br />
<br />
<br />
===TAE buffer===<br />
For 1 L of 50 x TAE buffer you need:<br />
<br />
* 242.48 g Tris<br />
<br />
* 41.02 g Sodiumacetate<br />
<br />
* 18.612 g EDTA<br />
<br />
* Adjust pH to 7.8<br />
<br />
* Solve in dH<sub>2</sub>O<br />
<br />
20 mL of the stock is diluted in 1 L dH<sub>2</sub>O for the gel electrophoresis.<br />
<br />
<br />
===DNA loading buffer===<br />
* 50 % (v/v) Glycerine<br />
<br />
* 1 mM EDTA<br />
<br />
* 0.1 % (w/v) Bromphenol blue<br />
<br />
* Solve in ddH20<br />
<br />
<br />
===LB medium===<br />
For 1 L of LB medium you need: <br />
<br />
* 10 g Trypton<br />
<br />
* 5 g yeast extract<br />
<br />
* 10 g NaCl<br />
<br />
* 12 g Agar-Agar (for plates)<br />
<br />
* Adjust pH to 7.0<br />
<br />
<br />
===Cell Culture Lysis Reagent===<br />
* 25 mM Tris-phosphate (pH 7.8)<br />
* 2 mM DTT<br />
* 2 mM 1,2-diaminocyclohexane-N,N,N´,N´-tetraacetic acid<br />
* 10 % glycerol<br />
* 1 % Triton® X-100<br />
<br />
= References =<br />
<br />
<br />
*Openwetware: Quint Lab: electrocompetent cells e.coli, [http://openwetware.org/wiki/Quint_Lab:electrocompetent_cells_e.coli http://openwetware.org/wiki/Quint_Lab:electrocompetent_cells_e.coli], October 24th 2010.<br />
<br />
*Openwetware: Silver: BB Strategy, [http://openwetware.org/wiki/Silver:_BB_Strategy http://openwetware.org/wiki/Silver:_BB_Strategy], October 24th 2010.<br />
<br />
*BioBrick Assembly Manual, Ginkgo BioWorks, [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf], October 24th 2010.<br />
<br />
*''Luciferase Assay System Technical Bulletin'' #TB281, Promega Cooperation, [http://www.promega.com/tbs/tb281/tb281.pdf http://www.promega.com/tbs/tb281/tb281.pdf], October 24th 2010.</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Project/TheoryTeam:Bielefeld-Germany/Project/Theory2010-10-27T15:59:24Z<p>Nkessler: /* Weblinks */</p>
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<br />
= Introduction =<br />
<br />
Our iGEM project is to create an ''E. coli'' cell which is capable to sense Capsaicin in a complex sample and report the concentration with a luciferase light signal. The native receptor senses Acetosyringone. We used directed evolution to modify the binding region of the native receptor to generate a new capsaicin receptor. We established a screening system based on antibiotic concentration gradients to screen the newly generated receptors. In our ''E. coli'' Acetosyringone sensing system several parts driving from different organisms were assemlbed. The native receptor was subcloned from ''Agrobacterium tumefaciens'' to create a read out system with firefly luciferase.<br />
<br />
= ''Agrobacterium tumefaciens'' =<br />
<br />
[[Image:Bielefeld_Agrobacterium_tumefaciens_attached_to_a_plant_cell_Image_by_Martha_Hawe.jpg|500px|right|thumb|right|Image by Martha Hawe]] <br />
<br />
<br />
The model organism ''Agrobacterium tumefaciens'' is a soil bacterium and can be found at nearly every point of the world Agrobacterium became known as a phyto-pathogen leading to the crown gall disease in dicotyledonous species ([http://www.springerlink.com/content/hm17520m287ht766/ DeCleene M, DeLay J, 1976]). The infection is actually caused by a gene transfer system located on an extrachromosomal element, the Ti-plasmid. The infection can be divided into several steps. Predominantely ''A. tumefaciens'' senses phenolic compunds from hurted plants, but also aldose monosaccharides, low pH and low phosphate ([http://www.annualreviews.org/doi/abs/10.1146/annurev.phyto.41.052002.095701?journalCode=phyto Palmer AG. ''et al.''2004]), ([http://mmbr.asm.org/cgi/content/abstract/69/1/155 Brencic A and Winans SC, 2005]). When ''A. tumefaciens'' recognizes phenols with the virA receptor, a signal transduction cascade is initiated leadig to the expression of virulence genes. The next step is a physical interaction with the host plant. A type three secretion system is responsible for the DNA transfer of the Ti-plasmid from the bacterium into the host. The DNA is translocated to the nucleus, leading to the gene expression and the production of opin. ''A. tumefaciens'' uses the reprogrammed plant cells for metabolite production and therefore as a nutrient supplier.<br />
<br />
For biotechnology purpose the Ti-plasmid was disharmed. Instead the native transfer region (T-region) and a gene of interest could be easily introduced into the Ti-plasmid. Agrobacterium-mediated DNA transfer is one of the most commonly used techniques of plant transformation ([http://people.uleth.ca/~alicja.ziemienowicz/extra/Ziemienowicz_ABP2001.pdf Ziemienowicz A, 2001]).<br />
<br />
= Native receptor =<br />
<br />
To gain an evolutionary advance, ''A. tumefaciens'' needs a precise recognition system for potential hosts. The native sensing system is a two-component phospho-relay<br />
system in which VirA is an transmembrane-bound sensor while VirG is the intracellular response regulator ([http://www.biomedcentral.com/content/pdf/gb-2002-3-10-reviews3013.pdf Wolanin PM ''et al.'', 2002]). The two genes for the sensing system are virA and virG ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1166964/ Stachel SE and Nester EW, 1986]) which are constitutively expressed at a basal level. VirA is a hitstidine kinase and after sensing the phenol 3,5-Dimethoxyacetophenone, Acetosyringone an autophosphorylation occurs at the His-474 residue ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC208549/?pageindex=1&tool=pmcentrez Huang Y ''et al.'', 1990]),([http://jb.asm.org/cgi/content/abstract/172/2/531 Jin SG ''et al.'', 1990]). Later in the signal transduction cascade, the phosphorylated VirA leads to the transfer of the phosphate to Asp-52 residue of VirG ([http://jb.asm.org/cgi/content/abstract/172/2/531 Jin SG ''et al.'', 1990]), ([http://jb.asm.org/cgi/content/abstract/172/9/4945 Jin SG ''et al.'', 1990]), ([http://nar.oxfordjournals.org/content/18/23/6909.short Pazour GJ and Das A, 1990]). VirG is the response regulator of the two-component system (Brencic A, Winans SC, 2005). VirG acts as a transcription factor and binds to virulence box (vir Box) containing promotors, for example virB ([http://jb.asm.org/cgi/content/abstract/172/2/531 in SG ''et al.'', 1990]), ([http://jb.asm.org/cgi/content/abstract/172/3/1241 Pazour GJ and Das A, 1990]).<br />
<br />
== VirA receptor structure ==<br />
<br />
The virA receptor consists of 829 amino acids and is a transmembrane protein in the inner menbrane of ''A. tumefaciens'' ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC401051/ Melchers LS, 1989]). VirA spans the inner membrane, with two transmembrane domains, a large periplasmic region, and a large C-terminal cytoplasmic domain ([http://jb.asm.org/cgi/content/abstract/176/11/3242 Banta LM, 1994]). VirA directly senses the phenolic compounds for vir activation ([http://linkinghub.elsevier.com/retrieve/pii/S0378111996003289 Lee YW ''et al.'', 1996]) Therefore the linker domain is essential for induction by phenolic compounds ([http://jb.asm.org/cgi/content/abstract/174/21/7033 Chang CH and Winans SC., 1992]). The linker region is located in the cytosolic site at position 280 to 414 ([http://linkinghub.elsevier.com/retrieve/pii/S0378111996003289 Lee YW ''et al.'', 1996]). This region between aa 283 and 304 was highly conserved in four different strains of Agrobacterium, and therefore likely to serve as the receptor region for the phenolic inducers which are common to all four strains ([http://www.springerlink.com/index/G548R7745685341P.pdf Turk SC ''et al.'', 1994]).<br />
<br />
<br />
<br />
[[Image:Bielefeld_Melcher_1989_VirA_structure.jpg|300px|left|thumb|right| Melchers et al., 1989 ]] <br />
<br />
[[Image:Bielefeld_VirA_structure_Lee_1996.jpg|300px|center|thumb|right|Lee et al., 1996 ]] <br />
<br />
<br />
Chang and Winnans revealed in their studies, the parts of the VirA receptor essential for the signal transduction ([http://jb.asm.org/cgi/content/abstract/174/21/7033 Chang CH and Winans SC., 1992]). A structured model for different inducing conditions are shown in the figure below.<br />
<br />
[[Image:Bielefeld_Chang_1992.jpg|300px|center|thumb|right|Chang, Winans, 1992 ]]<br />
<br />
<html><div style="font-size:16px; font-weight:bold;"><a href="/Team:Bielefeld-Germany/Project/Approach">For information about modulation strategy click here</a></div></html><br />
<br />
== Phenolic Compounds == <br />
<br />
The Ligand receptor interaction between Acetosyringone and VirA is based on the interaction of several chemical groups. First of all, the hydroxylated aromat is essential. Metoxy groups in ortho position of the phenol play also crucial role in the signaling. Dimetoxy compounds have a higher activity than monometoxy compounds. The acetyl and alkyl groups in para position enhancing the binding affinity. VirA activating compounds must have two met-oxy groups in the ortho position and an additional carbonyl group on the R3 chain. The potential capacity of the group para to the phenolic hydroxyl group is associated with higher activities and chirality at this carbon center is critical for inducing activity ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC372852/?pageindex=1&tool=pmcentrez Winans SC, 1992]).<br />
Regarding to the proton transfer model of Hess et al 1996 the VirA activator transfers a proton to the basic area receptor binding site. The allosteric change leads to the phosphotransfer and the signaltransduction ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC52402/?pageindex=1&tool=pmcentrez Hess KM ''et al.'', 1991]).<br />
<br />
[[Image:Bielefeld_benzol_structure.jpg|400px|left|thumb|right|Chemical structure of phenol]] <br />
[[Image:Bielefeld_proton_transfer_model.jpg|400px|center|thumb|right|Hess et al., 1991]]<br />
[[Image:Bielefeld_Winans_1992.jpg|400px|center|thumb|right|Winans, 1992]]<br />
<br />
<html><div style="font-size:16px; font-weight:bold;"><a href="/Team:Bielefeld-Germany/Project/Outlook">See more possible compounds in future by clicking here</a></div></html><br />
<br />
== Inducing enhancers ==<br />
<br />
The sensitivity of this system is highly enhanced when additional aldose monosacchardic suggars occur in the environment of Agrobacterium. The sugar binding protein ChvE interact with the VirA receptor, leading to a much stronger vir gene expression ([http://jb.asm.org/cgi/content/abstract/176/11/3242 Banta LM, ''et al.'', 1994]).<br />
<br />
== Subcloning into ''E. coli'' and receptor function in new host ==<br />
<br />
In our project we decided to work with ''E. coli'' instead of ''A. tumefaciens''. The transcription procedure in ''E. coli'' is very similar to ''A. tumefaciens'' but not complete homolog. In ''E. coli'' the rpoA gene - encoding the α-subunit of RNA polymerase in ''A. tumefaciens'' is not present but essential for the transcription of a virB promoter-driven genes ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC103583/?report=abstract&tool=pmcentrez Lohrke SM ''et al.'', 1990]) For this reason it was necessary to subclone a modified virG gene that is capable to be trancribed by the ''E. coli'' expression system.<br />
<br />
Yong-Chul described VirG conderived mutants that are capable of expressing the virB promoter-driven genes in ''E. coli'' without the requirement for the RpoA from ''A. tumefaciens'', suggesting that the virG mutants are able to interact with the transcription system of ''E. coli'' ([http://www.springerlink.com/content/wmq06kua5qkma1au/fulltext.pdf Yong-Chul J ''et al.'', 2004]).<br />
In VirG the amino acid at position 56 is likely to play a key role in the interaction with the RpoA of ''E. coli''. <br />
Regarding to Yong-Chul J ''et al.'', 2004 we used virG mutants, with amino acid substitutions of G56V and I77V that are capable of activating vir genes in ''E. coli'' in response to inducer acetosyringone in a virA-dependent manner.<br />
<br />
= Read out system =<br />
<br />
==Output-signal amplification by Sensitivity Tuner implementation==<br />
<br />
Using an standard, inducible promoter/reporter system, often results in weak reporter expression and so on in difficulties in quantification. An amplification of transcription rate of desired genes can be realized using so called ''sensitivity tuner'' devices. Amplification takes place as promoter induction upregulates a phage activator, which binds to a phage promoter upstream of a reporter. As result a PoPs input (Inducer) generate a PoPs output at higher signal. PoPs is equivalent to the flow of RNA polymerase molecules along DNA ([http://jb.asm.org/cgi/content/abstract/178/19/5668 Julien and Calendar, 1996]),([http://parts.mit.edu/igem07/index.php/Cambridge/Amplifier_project iGEM Team Cambridge, 2009]).<br />
<br />
[[Image:Bielefeld readout.png|600px|center|thumb| Figure 1: Gene sequence of final test contruct including Sensitivity Tuner elements.]]<br />
<br />
----<br />
<br />
'''Purpose of Sensitivity Tuner application'''<br />
<br />
We presumed weak expression rates of our reporter luciferase indicated by pretesting the native system [http://partsregistry.org/Part:BBa_K389015 K389015]. For having a broader range of quantification for our prototype test system, an amplification device was implemented. <br />
For amplifying the output signal of luciferase induced by acetosyringone, three sensitivity tuner distinguished by the amplification factor were combined with our detection system. To modify the sensitivity tuner for our purpose we took BioBricks with amplification factors from 15 ([http://partsregistry.org/Part:BBa_I746370 I746370]), 10 ([http://partsregistry.org/Part:BBa_I746370 I746380]) and 35 ([http://partsregistry.org/Part:BBa_I746370 I746390]) removed pBAD/araC promotor ([http://partsregistry.org/Part:BBa_I0500 I0500]) and GFP ([http://partsregistry.org/Part:BBa_E0040 E0040]) by self- designed primer PCR and replaced it upstream by a VirA/G response regulater [http://partsregistry.org/Part:BBa_K389015 K389015] and Virb promoter element [http://partsregistry.org/Part:BBa_K389003 K389003] and downstream by the reporter [http://partsregistry.org/Part:BBa_K389004 K389004], a luciferase (Figure 1). The benefits of luciferase reporter instead of GFP are a broader range of measurement, higher sensitivity and low half-live making cinetic tests possible ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4F031H9-30&_user=10&_coverDate=01%2F31%2F1989&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1514624813&_rerunOrigin=google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=ee400628b119490fcdc44ccdd856c4e8&searchtype=a Williams ''et al.''1989]).<br />
<br />
<br />
<br />
<br />
<br />
For test results click [https://2010.igem.org/Team:Bielefeld-Germany/Results/Tests#BBa_K389421.2C_BBa_K389422.2C_BBa_K389423:_Sensitivity_Tuner_amlified_Vir-test_system Sensitivity Tuner amlified Vir-test system]<br />
<br />
= Receptor modification strategy =<br />
<br />
The smartest way of receptor-modification is initiated by a silico approach, based on a 3D structure of the native receptor. Followed by primer mutagenesis of the computational gained results a precise adapted peptide emerges. This concept has been proofen by [http://www.nature.com/nature/journal/v423/n6936/abs/nature01556.html Looger et al.,2003].<br />
<br />
Because of time limitations within the iGEM competition and a leck of biological data in literature - no x-ray tructure data for VirA linker region available- this strategy was not applicable. <br />
Therefore we developed two different stragies in our MARSS project:<br />
<br />
The first strategy was an ''error prone PCR'' approach by building a mutant data base for selection. The second strategy was a ''primer mutagenesis based'' approach.<br />
<br />
== Random mutagenesis by error-prone PCR (EP-PCR)==<br />
<br />
In order to detect novel substances (e.g. capsaicin) with the virA receptor, the first step was to create a mutagenised library of virA variants, which could subsequently be screened for new binding characteristics. Plenty of different strategies for mutagenesis of DNA are known, including the use nucleotide analogues, bacteria containing mutator genes, the mutagenesis with UV light or chemicals and inaccurate PCR ([http://genome.cshlp.org/content/2/1/28.short Cadwell RC and Joyce GF, 1992]).<br />
<br />
When designing our strategy we rejected the use of bacteria strains with high mutation rates, since the changes in base sequence would occur all over the transformed plasmids. Thereby some mutations would also take place in the backbone of the plasmid, our might even been found in the standardized BB prefix and suffix. We also excluded the possibility of using UV light or mutagenic chemicals, due to reasons of safety and minimizing the exposure toxic substances.<br />
<br />
In our experiment we wanted to alter only the part of the plasmid coding for the virA receptor, while using a not harmful and thereby safe technique. Thus, our method of choice was inaccurate PCR that allows the exclusive variations of a distinct region of a plasmid, which is defined by the location of the upstream and downstream primers. This mutagenic method of PCR, called error-prone PCR (EP-PCR) has been described and improved a lot in scientific community ([http://www.springerlink.com/content/r62q360t82764508/#section=746282&page=1 McCullum EO ''et al.'', 2010]).<br />
<br />
The basic principle of this technique uses the natural high infidelity of the Taq DNA polymerase, which can even be increased by special changes in buffer conditions compared to standard PCR. These alterations may include the unequal distribution of dNTPs (5 mM purines, 25 mM pyrimidines) as well as an increased amount of MgCl<sub>2</sub> and the addition of MnCl<sub>2</sub>. The total rate of base exchange can be adjusted by the number of PCR cycles, since mutations will accumulate during the exponential amplification of the sequence ([http://onlinelibrary.wiley.com/doi/10.1002/0471142727.mb0803s51/full Wilson DS and Keefe AD, 2000]). The experimental conditions of the performed error-prone PCR are described in the section “protocols”.<br />
<br />
== Directed mutagenesis ==<br />
<br />
After the identification of the 100 amino acids linker region responsible of VirA ligand binding ([[Team:Bielefeld-Germany/Project/Theory#VirA_receptor_structure|Compare VirA-Recptor]]) we compared this region with well characterized capsacicin receptors derived from animal model organisms (TRPV1). The conserved receptor region is shown in the figure beneath.<br />
<br />
The species-specific sensitivity of TRPV1 can be ascribed to about eight amino acids in the vicinity of TM3 ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4C5H6M5-F&_user=2459438&_coverDate=02%2F08%2F2002&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=93a9e63f48d785a0b400131a058ee269&searchtype=a Jordt, 2002])<br />
<br />
[[Image:Bielefeld_VirA_conserved_sequence.jpg|600px|center|thumb|Molecular determinants of species-specific vanilloid sensitivity. Sequence alignment of rat (top), human (middle), and chicken (bottom) VR1 within the TM3-4 region is shown. Conserved residues are indicated by black background. The chimera V3/C contains a minimal segment of rat VR1 that is sufficient to confer vanilloid sensitivity ([http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WSN-4C5H6M5-F&_user=2459438&_coverDate=02%2F08%2F2002&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000057302&_version=1&_urlVersion=0&_userid=2459438&md5=93a9e63f48d785a0b400131a058ee269&searchtype=a Jordt, 2002]).]] <br />
<br />
We further aligned the TM3 region of the TRPV1 receptor to the ligand binding region of the native VirA receptor from ''Agrobacterium'' by the use of the tool MUltiple Sequence Comparison by Log-Expectation ([http://www.ebi.ac.uk/Tools/muscle/index.html Muscle]). <br />
<br />
=== VirA_mut1 ===<br />
Regarding to the results from Muscle, we used site directed mutagenises (for Protocols see: ([http://openwetware.org/wiki/Site-directed_mutagenesis OpenWet Ware])) to change VirA protein sequence leucin 293 to tyrosine (L293Y = mut1).<br />
<br />
=== VirA_mut2 ===<br />
After VirA_mut1 with its single mutation failed working in our screening system, we decided to mutagenize some further amino acids.<br />
<br />
= Screening system =<br />
<br />
==Development of a high-troughput screening==<br />
<br />
The screening of randomly mutagenised genes for a desired function or application is always a very time-consuming procedure ([http://www.ncbi.nlm.nih.gov/pubmed/1496376 Beaudry and Joyce, 1992]). It requires a huge amount of material and might takes several months or even years to result in a promising new version of a gene ([http://mbe.oxfordjournals.org/content/17/7/1050.long Hanczyc and Dorit, 2000]). As we faced the challenge to modify the virA receptor in only few weeks we designed a strategy for a fast high-throughput screening, by using subsequent steps of different read out systems and a strategy with two different plasmids.<br />
<br />
In the first step after mutagenesis of virA it is necessary to separate thousands of transformants with minor or unwanted changes in the virA gene, from few bacteria that included interesting virA variants. Thus, we wanted to construct our system to lead in the expression of a kanamycin resistance after the induction of the virA receptor, enabling the quick exclusion of all unwanted virA variants.<br />
<br />
For that purpose a kanamycin resistance cassette should be set under control of the virB promotor, leading in the expression of aminoglycoside phosphotransferase (APH) that can inactivate kanamycin. The mode of inactivation is the transfer of the y-phosphate from ATP to the hydroxyl group at C3 of the antibiotic ([http://www.bioscience.org/1999/v4/d/wright/wright.pdf Wright and Thompson, 1999]). This phosphorylation results in the loss of binding capacity of the aminoglycoside to the 30S subunit of bacterial ribosomes, which would lead to inhibition of protein synthesis without the presence of the APH ([http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1365070/pdf/brjclinpharm00008-0014.pdf Begg EJ and Barclay ML, 1995]).<br />
<br />
As indicated by the first results, the reporter genes under control of the virB promoter showed a slight but measureable expression without any induction of virA with acetosyringone. This basal transcription resulted in the growth of bacteria without induction of virA at normal working concentrations of kanamycin of 25 to 50 µg mL<sup>-1</sup> ([http://books.google.de/books?hl=de&lr=&id=9mO2Fx0CuEYC&oi=fnd&pg=PA4&dq=Molecular+Cloning:+A+Laboratory+Manual,+Vol+1.+&ots=Ctw-SlcSLm&sig=FvweaKb_qiUxClNNiRfdwpmbqdo#v=onepage&q&f=false Sambrook J and Russell DW, 2001]).<br />
<br />
Concludingly, prior to the screening experiments it was necessary to adjust the concentration of kanamycin, which inhibits the growth of uninduced bacteria, while allowing bacteria to grow when an appropriate inductor was present and able to activate virA. This analysis was performed using the method of determination of minimal inhibitory concentrations (MIC) as described below.<br />
<br />
==Determination of minimal inhibitory concentration (MIC) of kanamycin==<br />
<br />
There are several ways to investigate the susceptibility of bacteria to inhibiting drugs like antibiotics. Nevertheless, the result of all this tests is the amount of an assayed substance that inhibits visible growth of the bacteria, called the minimal inhibitory concentration (MIC). The most common way of determination is to grow bacteria in liquids with several concentrations of the inhibiting drug. This procedure is chosen mostly, since many different conditions can be measured at the same time when using microtiterplates ([http://www.nature.com/nprot/journal/v3/n2/abs/nprot.2007.521.html Wiegand I ''et al.'', 2008]).<br />
<br />
As we planned to use the kanamycin not in liquid culture but in LB-Agar, we chose to determine the MIC at the same conditions as the desired experiment. Therefore, we planned to construct ''E. coli'' inhabiting the native virA system and a KanR read out and plated a small volume in different dilutions on LB-Agar without kanamycin. The grown colonies could then be transferred to agar plates with rising concentrations of kanamycin using replica plating. By counting the colonies it should be possible to calculate the percentage of colonies that could withstand each kanamycin concentration. This experiment should be carried out with and without acetosyringone to determine a kanamycin concentration induced ''E. coli'' could withstand, while the same bacteria die without the presence of acetosyringone.<br />
<br />
==Primary selection of virA variants with novel binding properties==<br />
After the determination of the MIC the screening for virA variants with new binding properties could be started. The aim of this screening is to find versions of virA that can be induced by one of the tested substances capsaicin, homovanillic acid, dopamine and 3-O methyldopamine.<br />
<br />
For that purpose one should transform the mutagenised variants of virA to ''E. coli'' and plate the bacteria on LB-agar with the determined kanamycin concentration. At the same time a mixture of all mentioned substances should be present in the agar. All bacteria including a virA variant that is activated by at least one of the substances will grow on the selective agar, since it expresses the kanamycin resistance. With this step it is thereby possible to select thousands of bacteria with unwanted versions of virA from few individuals with wanted binding properties.<br />
<br />
At this point it must be mentioned that some grown colonies might still be non-induced and false positive results. As the virA gene has been randomly changed before, it is possible that some variants occur where the receptor is always active. This would lead to a constitutive expression of the reporter gene and thereby a high level of kanamycin resistance.<br />
To exclude those false positive clones, the bacteria should be tested whether they are only resistance to the MIC of kanamycin when one of the tested substances is present. Every colony that can withstand the high kanamycin concentration without any inductor includes a constitutive version of virA and should be discarded in futher analysis.<br />
<br />
==Quantitative analysis of virA variants after induction with novel substances==<br />
Should we find some bacteria that respond to the presence of the tested substances (capsaicin, homovanillic acid, dopamine and 3-O methyldopamine) by growing on the MIC of kanamycin, it is desirable to quantify the induction. For that purpose it it is appropriate to change the read out system from kanamycin resistence to luciferase expression. This complex and time consuming task can easily be achieved without any cloning step, when using the advantage of our two plasmid system.<br />
<br />
After primary selection each bacteria includes two plasmids with different origins of replication (oris). The plasmid with the virA is in a common psB1AT3 backbone with a ColE1 ori. Contrary to that the read out plasmid with KanR has a special ori, named R6K, which can only amplify in ''E. coli'' strains that express the gene pir to produce the so called pi protein. Most of the strains used in laboratory are pir<sup>-</sup> but few (e.g EC100D) are pri<sup>+</sup> ([http://www.ncbi.nlm.nih.gov/pubmed/17383678 Bowers et al., 2007]).<br />
<br />
The setup of the different oris was chosen to separate both plamids at this experimental stage. To change the read out system from KanR to luciferase, one just needs to perform two transformations and isolations of plasmids. In the first step plasmids are isolated from colonies with positive binding properties to one of the tested substances. This mixture of plasmids with ColE1 and R6K oris is then transformed to a pir<sup>-</sup> strain (e.g. TOP10). In the following only the plasmid with ColE1 ori will be amplified during the growth of the transformants, leading to pure plasmids with virA when plasmids are isolated for a second time. In the last step this isolated DNA can be transformed to bacteria including another read out plasmid (e.g. with luciferase).<br />
<br />
<br />
= References =<br />
<br />
*Banta LM, Joerger RD, Howitz VR, Campbell AM, Binns AN., 1994, Glu-255 outside the predicted ChvE binding site in VirA is crucial for sugar enhancement of acetosyringone perception by Agrobacterium tumefaciens., J Bacteriol. 176(11):3242-9. <br />
<br />
*Brencic A, Winans SC, 2005, Detection of and response to signals involved in host-microbe interactions by plant-associated bacteria., Microbiol Mol Biol Rev 69: 155–194. <br />
<br />
*Chang CH, Winans SC., 1992, Functional roles assigned to the periplasmic, linker, and receiver domains of the Agrobacterium tumefaciens VirA protein., J Bacteriol. 174(21):7033-9. <br />
<br />
*DeCleene M, DeLay J, 1976, The host range of crown gall, Bot Rev 42: 389–466 <br />
<br />
*Hess KM, Dudley MW, Lynn DG, Joerger RD, Binns AN., 1991, Mechanism of phenolic activation of Agrobacterium virulence genes: development of a specific inhibitor of bacterial sensor/response systems., Proc. Natl. Acad. Sci. USA 88:7854–58.<br />
<br />
*Huang Y, Morel P, Powell B, Cado CI, 1990, VirA, a corregulator of Ti-specific virulence genes, is phosphorylated in vitro., J Bacteriol 172:1142–1144. <br />
<br />
*Jin S, Roitsch T, Ankenbauer RG, Gordon MP, Nester EW, 1990, The VirA protein of Agrobacterium tumefaciens is autophosphorylated and is essential for vir gene regulation., J Bacteriol 172: 525–530. <br />
<br />
*Jin SG, Prusti RK, Roitsch T, Ankenbauer RG, Nester EW, 1990, Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: Essential role in biological activity of VirG., J Bacteriol 172:4945–4950. <br />
<br />
*Jin SG, Roitsch T, Christie PJ, Nester EW, 1990, The regulatory VirG protein specifically binds to a cis-acting regulatory sequence involved in transcriptional activation of Agrobacterium tumefaciens virulence genes., J Bacteriol 172:531–537 <br />
<br />
*Jin SG, Roitsch T, Christie PJ, Nester EW, 1990, The regulatory VirG protein specifically binds to a cis-acting regulatory sequence involved in transcriptional activation of Agrobacterium tumefaciens virulence genes., J Bacteriol 172:531–537. <br />
<br />
*Kyunghee Lee, 1996, A structure-based activation model of phenol-receptor protein interactions. <br />
*Lee YW, Jin S, Sim WS, Nester EW, 1996, The sensing of plant signal molecules by Agrobacterium: genetic evidence for direct recognition of phenolic inducers by the VirA protein., Gene. 179(1):83-8. <br />
<br />
*Lohrke SM, Nechaev S, Yang H, Severinov K, Jin SJ, 1999, Transcriptional activation of Agrobacterium tumefaciens virulence gene promoters in Escherichia coli requires the A. tumefaciens RpoA gene, encoding the alpha subunit of RNA polymerase., J Bacteriol 181:4533–4539. <br />
<br />
*McCullen CA., Binns AN. , 2006, Agrobacterium tumefaciens and Plant Cell Interactions and Activities Required for Interkingdom Macromolecular Transfer, Annu. Rev. Cell Dev. Biol. 22:101-127 <br />
<br />
*Melchers LS, 1989, Membrane topology and functional analysis of the sensory protein VirA of Agrobacterium tumefaciens., The EMBO Journal vol.8 no.7 pp.1919- 1925. <br />
<br />
*Palmer AG, Gao R, Maresh J, Erbil WK, Lynn DG, 2004 Chemical biology of multi-host/pathogen interactions: chemical perception and metabolic complementation, Annu Rev Phytopathol 42: 439–464. <br />
<br />
*Pazour GJ, Das A, 1990, Characterization of the VirG binding site of Agrobacterium tumefaciens., Nucleic Acids Res 18:6909–6913. <br />
<br />
*Pazour GJ, Das A, 1990, virG, an Agrobacterium tumefaciens transcriptional activator, initiates translation at a UUG codon and is a sequence-specific DNA-binding protein., J Bacteriol 172:1241– 1249. <br />
<br />
*Scott M., Lohrke, 2001, Reconstitution of Acetosyringone-Mediated Agrobacterium tumefaciens Virulence Gene Expression in the Heterologous Host Escherichia coli ., J. of Bacteriology, p. 3704–3711 Vol. 183, No. 12. <br />
<br />
*Shimoda N,Toyoda-Yamamoto A, Shinsuke S, Machica Y., 1993, Genetic evidence for an interaction between the VirA sensor protein and the ChvE sugar-binding protein of Agrobacterium. J. Biol. Chem. 268:26552–58 <br />
<br />
*Stachel SE, Nester EW, 1986, The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens., EMBO J 5: 1445–1454. <br />
<br />
*Turk SC, van Lange RP, Regensburg-Tuïnk TJ, Hooykaas PJ., 1994, Localization of the VirA domain involved in acetosyringone-mediated vir gene induction in Agrobacterium tumefaciens., Plant Mol Biol. 25(5):899-907. <br />
<br />
*Williams TM, Burlein JE, Ogden S, Kricka LJ and Kant JA, 1989 ''Advantages of firefly luciferase as a reporter gene: Application to the interleukin-2 gene promoter'', Analytical Biochemistry, Vol. 176, pp. 28-32 <br />
*Winans SC., 1992, Two-way chemical signaling in Agrobacterium-plant interactions., Microbiol Rev. 56(1):12-31. <br />
<br />
*Wolanin PM, Thomason PA, Stock J.B., 2002, Histidine protein kinases: key signal transducers outside the animal kingdom., Genome Biol 3: REVIEWS3013. <br />
<br />
*Yi Han Linn et al., 2008, Capturing the VirA/VirG TCS of Agrobacterium tumefaciens., Adv Exp Med Biol. 631:161-77. <br />
<br />
*Yi-Han Lin et al., 2007, The initial steps in Agrobacterium tumefaciens pathogenesis: chemical biology of host recognition, Agrobacterium: From Biology to Biotechnology,2008, pp. 221-241. <br />
<br />
*Yong-Chul J., Yunrong G., Donghai W., Shouguang J., 2004, Mutants of Agrobacterium tumefaciens virG Gene That Activate Transcription of vir Promoter in Escherichia coli, Current Microbiology Vol. 49, pp. 334–340. <br />
<br />
*Ziemienowicz A., 2001, Odyssey of agrobacterium T-DNA,. Acta Biochim Pol. 2001;48(3):623-35. <br />
<br />
<br />
<br />
=== Literature of error-prone PCR and screening ===<br />
<br />
<br />
*Beaudry AA, Joyce GF (1992) ''Directed evolution of an RNA enzyme'', Science, 257(5070):635-41.<br />
<br />
*Begg EJ, Barclay ML (1995) ''Aminoglycosides – 50 years on'', Br J clin Pharmac, 39, 597-603.<br />
<br />
*Bowers LM, Krüger R, Filutowicz M (2007) ''Mechanism of origin activation by monomers of R6K-encoded pi protein'', J Mol Biol, 368(4), 928-938.<br />
<br />
*Cadwell RC and Joyce GF (1992) ''Randomization of genes by PCR mutagenesis'', Genome Res., 28-33.<br />
<br />
*Hanczyz MM, Dortit RL (2000) ''Replicability and Recurrence in the Experimental Evolution of a Group I Ribozyme'', Mol Biol Evol, 17 (7), 1050-1060.<br />
<br />
*Jordt SE, Julius D (2002) ''Molecular Basis for Species-Specific Sensitivity to „Hot“ Chili Peppers'', Cell, Vol. 108, 421-430. <br />
<br />
*McCullum EO, Willliams BAR, Zhang J, Chaput JC (2010) ''Random Mutagenesis by Error-Prone PCR, In vitro mutagenesis protocols'', Methods in Molecular Biology, Vol 634, 103-109.<br />
<br />
*MUltiple Sequence Comparison by Log-Expectation. MUSCLE http://www.ebi.ac.uk/Tools/muscle/index.html<br />
<br />
*Looger et al., (2003), ''Computational design of receptor and sensor proteins with novel functions'', Nature 423, 185-190<br />
<br />
*Sambrook J, Russel DW (2001) ''Molecular Cloning – A Laboratory Manual'', Cold Spring Harbor Laboratory Press, Vol 1.<br />
<br />
*Wiegand I, Hilpert K, Hancok REW (2008) ''Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances'', Nature Protocols, 3 , 163-175.<br />
<br />
*Wilson DS, Keefe AD (2000) ''Random Mutagenesis by PCR'', Current Protocols in Molecular Biology, 8.3.1-8.3.9.<br />
<br />
*Wright GD, Thompson PR (1999) ''Aminoglycoside phosphotransferases: Proteins, Structure and Mechanism'', Frontiers in Bioscience 4, d9-21.</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Project/OutlookTeam:Bielefeld-Germany/Project/Outlook2010-10-27T15:58:54Z<p>Nkessler: /* Weblinks */</p>
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<br />
= The techniques’ potential =<br />
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<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1548942" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=1548942" width="200"><br />
<h3>Capsaicin</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1548942&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=17198" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=17198" width="200"><br />
<h3>Acetosyringone</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=17198&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
<br />
In our project we modulated the virA-gene from ’’A. tumefaciens’’, which is encoding a receptor for the plant-hormone acetosyringone, via error prone PCR. Slight mutations may alter the receptors conformation or its binding site. Both of these can cause shifts in specificity and sensitivity. This way we created a new receptor, which is capable of sensing capsaicin. The chemical formulas of capsaicin and acetosyringone are quite similar and there are other molecules of interest, that have some of [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Phenolic_Compounds the necessary properties], too. Thus, we suggest that it is possible to modulate VirA receptors in order to make them sense a variety of highly relevant compounds.<br />
<br />
<br />
<br />
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= Further Possible Substrates =<br />
<br />
As stated, there are other candidates our sensing system may be applied to. All of them have similiar molecular structures as the vir-systems natural inducor acetosyringone. In the following we will present some of the most relevant compounds other than capsaicin, that may be upcoming targets of our research.<br />
<br />
''All of the following images of the compounds molecular structures are linked to [http://pubchem.ncbi.nlm.nih.gov/ '''The PubChem Project'''].''<br />
<br />
== Diagnosis of pheochromocytoma and neuroblastoma (child tumors) ==<br />
<html><br />
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<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=21100" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=21100" width="200"><br />
<h3>Metanephrine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=21100&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=23615482" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=23615482" width="200"><br />
<h3>Vanilmandelic Acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=23615482&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1738" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=1738" width="200"><br />
<h3>Homovanillic acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1738&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
=== Pheochromocytoma ===<br />
Pheochromocytoma is a rare endocrine tumor originating in the medulla of adrenal glands, localized on top of the kidney. The adrenal glands produce several catecholamines, to which metanephrine and dopamine belong. These hormones regulate responses to stress, heart rate and blood pressure. In patients with pheochromocytoma these hormones are released excessively, potentially causing increased heart rate and blood pressure. Pheochromocytoma may become life threatening when not recognized and treated [http://pheopara.nichd.nih.gov/ (Pheochromocytoma and Paraganglioma website at the NIH)].<br />
<br />
These hormones can be quantified in urine and thus are important compounds of pheochromocytoma diagnostics. [http://jcem.endojournals.org/cgi/content/abstract/92/12/4602 Boyle et al. (2007)] compare different accuracies of diagnostic measures for the tumor and name urinary free metanephrine HPLC-measurement as the most sensitive and specific. Homovanillic acid and vanillyl mandelic acid are measured via HPLC aswell and are indicators for the same tumor.<br />
<br />
=== Neuroblastoma ===<br />
"Neuroblastoma is the most common extracranial solid tumor in infancy. It is an embryonal malignancy of the sympathetic nervous system arising from neuroblasts (pluripotent sympathetic cells). In the developing embryo, these cells invaginate, migrate along the neuraxis, and populate the sympathetic ganglia, adrenal medulla, and other sites. The pattern of distribution of these cells correlates with the sites of primary disease presentation." ([http://emedicine.medscape.com/article/988284-overview Lacayo, Davis 2010])<br />
<br />
Patients with high-risk disease still have very poor outcomes despite intensive therapy.<br />
<br />
"More than 90% of patients have elevated homovanillic acid (HVA) and/or vanillylmandelic acid (VMA) levels detectable in urine."<br />
<br />
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<br />
== 3-Methoxytyramine ==<br />
<html><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=681" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=681" width="200"><br />
<h3>Dopamine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=681&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=11957621" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=11957621" width="200"><br />
<h3>3-Methoxytyramine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=11957621&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
<br />
[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0013452 Sotnikova et al. (2010)] suggest, that 3-Methoxytyramine (3-MT) plays an important role as a neuromodulator. 3-MT is a metabolite of Dopamine and is reported to be a potential indicator for dopamine-dependent diseases like the parkinson disease, schizophrenia and dyskinesia. 3-Methoxytyramin as metabolic product of the hormon dopamine can be detected in human urine ([http://edoc.hu-berlin.de/oa/degruyter/cclm.1971.9.6.478.pdf Knoll ''et al.'', 1971]).<br />
Dopamine is known as happiness hormone found in increased levels after taking stimulating substances such as cocaine or amphetamines. The latter is one of the worldwide most used substitutes in sportive competition as doping. Increased levels of 3-MT analyzed in urine taken from sportives can indicate to intake of stimulating substances ([http://journals.lww.com/acsm-msse/Abstract/1980/21000/The_effect_of_amphetamines_on_selected.13.aspx CHANDLER, JOE V. and STEVEN N BLAIR, 1980]).<br />
<br />
== Dopamine ==<br />
<br />
Even though being referred to as "happiness hormone", Dopamine and its receptors are said to play key roles in numerous phsychic disorders and drug addiction. For many years now, the Dopamine hypothesis of Schizophrenia exists and evolves. It states that many symptoms of Schizophrenia correlate with a hyperactive disturbed dopaminergic signal transduction. [http://schizophreniabulletin.oxfordjournals.org/content/early/2009/03/26/schbul.sbp006.abstract Howes and Kapur (2009)] as well as [http://jop.sagepub.com/content/21/4/440.short Stone ''et al.'' (2007)] reviewed the hypothesis and its evolution.<br />
<br />
Concerning drug addiction, Dopamine and the brains reward system are of special interest in research [http://dx.doi.org/10.1016/j.brainresrev.2004.12.033 (Heidbreder, 2005)].<br />
<html><div style="clear:both;" /></html><br />
<br />
== Picric acid==<br />
<html><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=6954" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=6954" width="200"><br />
<h3>Picric acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=6954&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
Nitroaromatic compounds, such as Picrid acid (2,4,6-trinitrophenol), can be used as dyes, explosives, pesticides and energy sources for bacterial growth. The industrial use ([http://www.springerlink.com/content/q82535h870021540/fulltext.pdf Rajan, J 1996]) of picric acid contaminated ground water and is as nitritited aromatic compound a potential explosive ([http://search.barnesandnoble.com/Explosives-Engineering/Paul-W-Cooper/e/9780471186366 Cooper PW, 1997]; [http://onlinelibrary.wiley.com/doi/10.1002/1521-3773%2820010601%2940:11%3C2104::AID-ANIE2104%3E3.0.CO;2-%23/full Sohn H, 2001]). Picric Acid is still a problem by unfound warefare. Existing detection methods are time consuming and even more cost intensive, so that a high selective and sensitiv application by microbial screening would be desirable.<br />
<br />
<br />
<html><div style="clear:both;" /></html><br />
<br />
== Summary ==<br />
The VirA receptor is a great starting point for modulations, in order to address a wide variety of highly relevant compounds. By contructing a Vir-gene based sensing system we present a ''proof of concept'' for a general microbial sensing system adaptable for various other compounds. Since the basic sensing and reporting system was successfully established in ''E. coli'', the next essential steps are to improve the mutagenesis strategy for the receptor, and finally the screening for each candidate compound.<br />
<br />
Using [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Output-signal_amplification_by_Sensitivity_Tuner_implementation a set of sensitivity tuners], the reporter output may then be optimized. Furthermore, the speed of the systems are expected to require refinement.<br />
<br />
As a result, the MARSS has great potential to deliver biological tests for medical diagnosis, drug tests for ''e.g.'' doping conctrols, analysis of food for ''e.g.''allergy triggering compounds and soil contaminating substances such as picric acid.<br />
<br />
=References=<br />
<br />
*Boyle J, Davidson DF, Perry CG and Connell JMC, ''Comparison of Diagnostic Accuracy of Urinary Free Metanephrines, Vanillyl Mandelic Acid, and Catecholamines and Plasma Catecholamines for Diagnosis of Pheochromocytoma'' ,Journal of Clinical Endocrinology & Metabolism, Vol. 92, No. 12, pp. 4602-4608<br />
<br />
*Chandler JV and Blair SN (1980), '' The effect of amphetamines on selected physiological components related to athletic success''. Med. Sci. Sports Exercise, Vol. 12, No. 1, pp. 65-69<br />
<br />
*Cooper PW (1997), ''Explosives Engineering'', Edition 1, Wiley, John & Sons.<br />
<br />
* ''Eunice Kennedy Shriver'' National Institute of ''Child Health and Human Development'', Pheochromocytoma and Paragangliooma, 0-CH-0093<br />
<br />
*Heidbreder CA, Gardner EL, Xi ZX, Thanos PK, Mugnaini M, Hagan JJ, Ashby CR Jr. (2005), ''The role of central dopamine D3 receptors in drug addiction: a review of pharmacological evidence.'', Brain Research Reviews, Vol. 49, pp. 77 – 105 <br />
<br />
*Howes OD and Kapur S (2009), ''The Dopamine Hypothesis of Schizophrenia: Version III—The Final Common Pathway'', Schizophrenia Bulletin, Vol. 35, No. 3, pp.549-62<br />
<br />
*http://pubchem.ncbi.nlm.nih.gov/<br />
<br />
*Knoll E, Wisser H, Stamm D (1971), ''in Verfahren zur Bestimmung der 3-Methoxy-4-hydroxy-phenylessigsäure (Homovanillinsäure) im Harn durch in situ Remissionsmessung nach dünnschichtchromatographischer Trennung'', Z. klin. Chem. u. klin. Biochem., 1971<br />
<br />
*Lacayo NJ (2010), ''Neuroblastoma'', eMedicine from webMD<br />
<br />
*Rajan J,Valli K, Perkins RE, Sariaslani FS, Barns FM, Reysenbach A-L,Rehm S, Ehringer M and Pace NR (1996), ''Mineralization of 2,4,6-trinitrophenol (picric acid): characterization and phylogenetic identification of microbial strains'' Journal of Industrial Microbiology, 16, 319-324<br />
<br />
*Sohn H, Calhoun RM, Sailor MJ, Trogler WC (2001), ''Detection of TNT and Picric Acid on Surfaces and in Seawater by Using Photoluminescent Polysiloles'', Angewandte Chemie, Vol.40, pp.2104–2105<br />
<br />
*Sotnikova TD, Beaulieu J-M, Espinoza S, Masri B, Zhang X, Salahpour A, Barak LS, Caron MG, Gainetdinov RR (2010), ''The Dopamine Metabolite 3-Methoxytyramine Is a Neuromodulator'', PloSONE<br />
<br />
*Stone JM, Morrison PD, Pilowsky LS (2007), ''Review: Glutamate and dopamine dysregulation in schizophrenia — a synthesis and selective review'', Journal of Psychopharmacology, Vol. 21, No. 4, pp. 440-452</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Project/OutlookTeam:Bielefeld-Germany/Project/Outlook2010-10-27T15:58:07Z<p>Nkessler: /* Weblinks */</p>
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= The techniques’ potential =<br />
<html><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1548942" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=1548942" width="200"><br />
<h3>Capsaicin</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1548942&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=17198" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=17198" width="200"><br />
<h3>Acetosyringone</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=17198&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
<br />
In our project we modulated the virA-gene from ’’A. tumefaciens’’, which is encoding a receptor for the plant-hormone acetosyringone, via error prone PCR. Slight mutations may alter the receptors conformation or its binding site. Both of these can cause shifts in specificity and sensitivity. This way we created a new receptor, which is capable of sensing capsaicin. The chemical formulas of capsaicin and acetosyringone are quite similar and there are other molecules of interest, that have some of [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Phenolic_Compounds the necessary properties], too. Thus, we suggest that it is possible to modulate VirA receptors in order to make them sense a variety of highly relevant compounds.<br />
<br />
<br />
<br />
<br />
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= Further Possible Substrates =<br />
<br />
As stated, there are other candidates our sensing system may be applied to. All of them have similiar molecular structures as the vir-systems natural inducor acetosyringone. In the following we will present some of the most relevant compounds other than capsaicin, that may be upcoming targets of our research.<br />
<br />
''All of the following images of the compounds molecular structures are linked to [http://pubchem.ncbi.nlm.nih.gov/ '''The PubChem Project'''].''<br />
<br />
== Diagnosis of pheochromocytoma and neuroblastoma (child tumors) ==<br />
<html><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=21100" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=21100" width="200"><br />
<h3>Metanephrine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=21100&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=23615482" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=23615482" width="200"><br />
<h3>Vanilmandelic Acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=23615482&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1738" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=1738" width="200"><br />
<h3>Homovanillic acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1738&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
=== Pheochromocytoma ===<br />
Pheochromocytoma is a rare endocrine tumor originating in the medulla of adrenal glands, localized on top of the kidney. The adrenal glands produce several catecholamines, to which metanephrine and dopamine belong. These hormones regulate responses to stress, heart rate and blood pressure. In patients with pheochromocytoma these hormones are released excessively, potentially causing increased heart rate and blood pressure. Pheochromocytoma may become life threatening when not recognized and treated [http://pheopara.nichd.nih.gov/ (Pheochromocytoma and Paraganglioma website at the NIH)].<br />
<br />
These hormones can be quantified in urine and thus are important compounds of pheochromocytoma diagnostics. [http://jcem.endojournals.org/cgi/content/abstract/92/12/4602 Boyle et al. (2007)] compare different accuracies of diagnostic measures for the tumor and name urinary free metanephrine HPLC-measurement as the most sensitive and specific. Homovanillic acid and vanillyl mandelic acid are measured via HPLC aswell and are indicators for the same tumor.<br />
<br />
=== Neuroblastoma ===<br />
"Neuroblastoma is the most common extracranial solid tumor in infancy. It is an embryonal malignancy of the sympathetic nervous system arising from neuroblasts (pluripotent sympathetic cells). In the developing embryo, these cells invaginate, migrate along the neuraxis, and populate the sympathetic ganglia, adrenal medulla, and other sites. The pattern of distribution of these cells correlates with the sites of primary disease presentation." ([http://emedicine.medscape.com/article/988284-overview Lacayo, Davis 2010])<br />
<br />
Patients with high-risk disease still have very poor outcomes despite intensive therapy.<br />
<br />
"More than 90% of patients have elevated homovanillic acid (HVA) and/or vanillylmandelic acid (VMA) levels detectable in urine."<br />
<br />
<html><div style="clear:both;" /></html><br />
<br />
== 3-Methoxytyramine ==<br />
<html><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=681" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=681" width="200"><br />
<h3>Dopamine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=681&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=11957621" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=11957621" width="200"><br />
<h3>3-Methoxytyramine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=11957621&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
<br />
[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0013452 Sotnikova et al. (2010)] suggest, that 3-Methoxytyramine (3-MT) plays an important role as a neuromodulator. 3-MT is a metabolite of Dopamine and is reported to be a potential indicator for dopamine-dependent diseases like the parkinson disease, schizophrenia and dyskinesia. 3-Methoxytyramin as metabolic product of the hormon dopamine can be detected in human urine ([http://edoc.hu-berlin.de/oa/degruyter/cclm.1971.9.6.478.pdf Knoll ''et al.'', 1971]).<br />
Dopamine is known as happiness hormone found in increased levels after taking stimulating substances such as cocaine or amphetamines. The latter is one of the worldwide most used substitutes in sportive competition as doping. Increased levels of 3-MT analyzed in urine taken from sportives can indicate to intake of stimulating substances ([http://journals.lww.com/acsm-msse/Abstract/1980/21000/The_effect_of_amphetamines_on_selected.13.aspx CHANDLER, JOE V. and STEVEN N BLAIR, 1980]).<br />
<br />
== Dopamine ==<br />
<br />
Even though being referred to as "happiness hormone", Dopamine and its receptors are said to play key roles in numerous phsychic disorders and drug addiction. For many years now, the Dopamine hypothesis of Schizophrenia exists and evolves. It states that many symptoms of Schizophrenia correlate with a hyperactive disturbed dopaminergic signal transduction. [http://schizophreniabulletin.oxfordjournals.org/content/early/2009/03/26/schbul.sbp006.abstract Howes and Kapur (2009)] as well as [http://jop.sagepub.com/content/21/4/440.short Stone ''et al.'' (2007)] reviewed the hypothesis and its evolution.<br />
<br />
Concerning drug addiction, Dopamine and the brains reward system are of special interest in research [http://dx.doi.org/10.1016/j.brainresrev.2004.12.033 (Heidbreder, 2005)].<br />
<html><div style="clear:both;" /></html><br />
<br />
== Picric acid==<br />
<html><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=6954" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=6954" width="200"><br />
<h3>Picric acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=6954&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
Nitroaromatic compounds, such as Picrid acid (2,4,6-trinitrophenol), can be used as dyes, explosives, pesticides and energy sources for bacterial growth. The industrial use ([http://www.springerlink.com/content/q82535h870021540/fulltext.pdf Rajan, J 1996]) of picric acid contaminated ground water and is as nitritited aromatic compound a potential explosive ([http://search.barnesandnoble.com/Explosives-Engineering/Paul-W-Cooper/e/9780471186366 Cooper PW, 1997]; [http://onlinelibrary.wiley.com/doi/10.1002/1521-3773%2820010601%2940:11%3C2104::AID-ANIE2104%3E3.0.CO;2-%23/full Sohn H, 2001]). Picric Acid is still a problem by unfound warefare. Existing detection methods are time consuming and even more cost intensive, so that a high selective and sensitiv application by microbial screening would be desirable.<br />
<br />
<br />
<html><div style="clear:both;" /></html><br />
<br />
== Summary ==<br />
The VirA receptor is a great starting point for modulations, in order to address a wide variety of highly relevant compounds. By contructing a Vir-gene based sensing system we present a ''proof of concept'' for a general microbial sensing system adaptable for various other compounds. Since the basic sensing and reporting system was successfully established in ''E. coli'', the next essential steps are to improve the mutagenesis strategy for the receptor, and finally the screening for each candidate compound.<br />
<br />
Using [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Output-signal_amplification_by_Sensitivity_Tuner_implementation a set of sensitivity tuners], the reporter output may then be optimized. Furthermore, the speed of the systems are expected to require refinement.<br />
<br />
As a result, the MARSS has great potential to deliver biological tests for medical diagnosis, drug tests for ''e.g.'' doping conctrols, analysis of food for ''e.g.''allergy triggering compounds and soil contaminating substances such as picric acid.<br />
<br />
=Weblinks=<br />
<br />
*Boyle J, Davidson DF, Perry CG and Connell JMC, ''Comparison of Diagnostic Accuracy of Urinary Free Metanephrines, Vanillyl Mandelic Acid, and Catecholamines and Plasma Catecholamines for Diagnosis of Pheochromocytoma'' ,Journal of Clinical Endocrinology & Metabolism, Vol. 92, No. 12, pp. 4602-4608<br />
<br />
*Chandler JV and Blair SN (1980), '' The effect of amphetamines on selected physiological components related to athletic success''. Med. Sci. Sports Exercise, Vol. 12, No. 1, pp. 65-69<br />
<br />
*Cooper PW (1997), ''Explosives Engineering'', Edition 1, Wiley, John & Sons.<br />
<br />
* ''Eunice Kennedy Shriver'' National Institute of ''Child Health and Human Development'', Pheochromocytoma and Paragangliooma, 0-CH-0093<br />
<br />
*Heidbreder CA, Gardner EL, Xi ZX, Thanos PK, Mugnaini M, Hagan JJ, Ashby CR Jr. (2005), ''The role of central dopamine D3 receptors in drug addiction: a review of pharmacological evidence.'', Brain Research Reviews, Vol. 49, pp. 77 – 105 <br />
<br />
*Howes OD and Kapur S (2009), ''The Dopamine Hypothesis of Schizophrenia: Version III—The Final Common Pathway'', Schizophrenia Bulletin, Vol. 35, No. 3, pp.549-62<br />
<br />
*http://pubchem.ncbi.nlm.nih.gov/<br />
<br />
*Knoll E, Wisser H, Stamm D (1971), ''in Verfahren zur Bestimmung der 3-Methoxy-4-hydroxy-phenylessigsäure (Homovanillinsäure) im Harn durch in situ Remissionsmessung nach dünnschichtchromatographischer Trennung'', Z. klin. Chem. u. klin. Biochem., 1971<br />
<br />
*Lacayo NJ (2010), ''Neuroblastoma'', eMedicine from webMD<br />
<br />
*Rajan J,Valli K, Perkins RE, Sariaslani FS, Barns FM, Reysenbach A-L,Rehm S, Ehringer M and Pace NR (1996), ''Mineralization of 2,4,6-trinitrophenol (picric acid): characterization and phylogenetic identification of microbial strains'' Journal of Industrial Microbiology, 16, 319-324<br />
<br />
*Sohn H, Calhoun RM, Sailor MJ, Trogler WC (2001), ''Detection of TNT and Picric Acid on Surfaces and in Seawater by Using Photoluminescent Polysiloles'', Angewandte Chemie, Vol.40, pp.2104–2105<br />
<br />
*Sotnikova TD, Beaulieu J-M, Espinoza S, Masri B, Zhang X, Salahpour A, Barak LS, Caron MG, Gainetdinov RR (2010), ''The Dopamine Metabolite 3-Methoxytyramine Is a Neuromodulator'', PloSONE<br />
<br />
*Stone JM, Morrison PD, Pilowsky LS (2007), ''Review: Glutamate and dopamine dysregulation in schizophrenia — a synthesis and selective review'', Journal of Psychopharmacology, Vol. 21, No. 4, pp. 440-452</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/SafetyTeam:Bielefeld-Germany/Safety2010-10-27T15:30:00Z<p>Nkessler: /* Biosafety */</p>
<hr />
<div>{{Bielefeld_MainMenu_2010|<nowiki>http://igem-bielefeld.de/img/banner_safety.png</nowiki>}}<br />
<br />
= Biosafety and Biosecurity (English) =<br />
<br />
It is important to discriminate between Biosafety and Biosecurity in the context of working with genetically modified organisms and lab use. Biosecurity comprises the safety of the lab. The [http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_EPR_2006_6.pdf WHO] defines Biosecurity as a task of organisation and administration.The term Biosafety deals with the biological risks of the samples for the user, the enviroment and the lab. In this article, we are mostly summarizing the German law on Biosecurity and Biosafety regarding to the [http://www.bvl.bund.de/nn_495478/DE/06__Gentechnik/093__ZKBS/gentechnik__zkbs__node.html__nnn=true ZKBS (zentrale Kommission für Biologische Sicherheit)] and [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf their lists of unhazardous organisms] plus the comment on safety of the [http://www.dfg.de/download/pdf/dfg_im_profil/reden_stellungnahmen/2009/stellungnahme_synthetische_biologie.pdf DFG (Deutsche Forschungsgemeinschaft)].<br />
Furthermore we crosslinked the laws of the United States of America and the European law of Biosecurity and Biosafety in this article. We marked this statement in the text, so the reader can discrimante between the law systems. This kind of modulation makes sense, because our experimental work was done in Germany, shipped in europe (SDU Denmark) and used as a biobrick in United States of America.<br />
<br />
== Biosecurity ==<br />
[[Image:Safe labwork.jpg|200px|thumb|right|Picture 2: We are using a cleanbench to avoid any contamination of our samples. A proper use of the cleanbench can only be achieved by placing all the big things in the rear of the bench, by not disturbing the air circulation and by avoiding any kind of aerosoles. Use gloves for your own protection.]]<br />
<br />
<br />
In the context of "genetically engineered" it is quite important to keep unqualified personnel and trespassers away from your laboratory and your samples. In our case we secure our lab with a keypad. Every staff member needs a key to open the laboratory door. Nobody is able to open the door without the proper keys. Staff members receive a key, after beeing briefed by the safety officer on safety issues. You can only get a key for the safety stage you are working in. The labs with higher safety stages are cut off from the lower ones. Staff members need a continuative safety guide to receive a key for the higher security levels. Concludingly no unqualified personel can pass the door to the laboratory.<br />
The picture 1 shows the keypad in front of the lab.<br />
<br />
The laboratories, which are in use for our iGEM project are security stage 1 (S1). For the safety of the staff our labs are equiped with fire extinguisher, emergency showers and a heart defibrillator.<br />
<br />
== Biosafety ==<br />
[[Image:Keypad.JPG|200px|thumb|left| Picture 1: Security entrance to the lab. Every Member of the Team needs a key-token in order to get acces to the lab]]<br />
<br />
The term "biological safety" defines the effort to reduce or eliminate any potential risks caused by biotechnolgy or genetical engineering. The laboratories are scaled into four different saftey stages. The stage "S1" defines working with organsims and methods, which do no prooved harm neither to the enviroment nor mankind. The stages rise up to biosafety stage four, which defines working with prooved humanpathogenous organisms. The WHO defines for the United states the same safety stages, which are called risk groups [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf Manual of Biosecurity, WHO, page 1].<br />
<br />
The Biosafety is ruled by the "Zentrale Kommission für Biologische Sicherheit" (ZKBS) in Germany. The ZKBS reviewed and verified all commercial, research or privat laboratories about their safety issues. The iGEM Team Bielefeld is currently working with the organisms ''Agrobacterium tumefaciens'' and a derivate of ''E. coli'' K12 in a ZKBS reviewed S1-lab. The organisms strains are declared as riskfactor 1 by the german act of genetics (GenTSV, § 5; 15.06.2010). The act contains that the organisms (abstract):<br />
<br />
1.<br />
<br />
: - are no proved human-, phyto- or animalpathogen<br />
: - do not contain or release organism containing to a higher risk stage<br />
: - are prooved by experiments or long term evaluation or do not proliferate in the enviroment because of biological implanted boundaries<br />
<br />
<br />
2.<br />
Organisms, which confirm the paragraph 1, are defined by the $6 of the act of genetics (GenTSV):<br />
<br />
: a) as biological safe organisms<br />
: b) organims or strains, which are contaminated by organisms or strains of a higher security level<br />
: c) organisms defined by the risk level S1 (list of organisms used for genetically engineering research)<br />
<br />
<br />
The [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf list of organisms] categorize the strains ''E. coli'' K12 and ''Agrobacterium tumefaciens'' as biological safety risk 1 (S1). The strain [http://www.bvl.bund.de/cln_007/nn_520774/DE/06__Gentechnik/093__ZKBS/01__Allg__Stellungnahmen/02__bakterien/zkbs__bakterien__agrobacterium__tumefaciens,templateId=raw,property=publicationFile.pdf/zkbs_bakterien_agrobacterium_tumefaciens.pdf ''Agrobacterium tumefaciens''] is a known phytopathogen. Because of its frequent appearence in the earth (500 bacteria in 1g earth) it is generally regarded as safe.<br />
<br />
The WHO defines "security assesments" has the highest priority for the work with organisms. This includes a proper theoretically background of the organism. Further they defined rules for good molecular practice and proper lab security [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf WHO Manual Biosafety, page 9 following]. They note that most of the security guidelines are not quite neccessary for organism of risk stage 1. Although we play by the rules and work as carefully as possible (see, what we do to protect).<br />
<br />
= Biosafety und Biosecurity (German)=<br />
<br />
Es ist bei der Inbetriebnahme und Nutzung einer Gentechnischen Anlagen darauf zu achten, dass Vorkehrungen sowohl für den Betriebsschutz (Biosecurity) als auch für die biologische Sicherheit (Biosafety) getroffen werden. Biosecurity wird von der [http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_EPR_2006_6.pdf WHO] als Aufgabe der Adminstrative des Labors definiert.<br />
<br />
== Biosecurity ==<br />
Der Biosecurity wird Sorge getragen, in dem kein unbefugter Zugang zum Laborplatz hat. Die Türen sind nur per Schlüssel zu öffnen. Jeder Mitarbeiten muss eine Sicherheitsbelehrung beim Sicherheitsbeauftragten ablegen ehe er Zugang zu den Laboratorien bekommt. Die Labore sind je nach Sicherheitsgefährdung gekennzeichnet und verschlossen, so dass keine unterqualifizierte Person in ein höheres Sicherheitslevel eindringen kann. In dem vom iGEM genutzten Labor handelt es sich um die Sicherheitstufe S1. Die WHO definiert die gleichen Sicherheitsstufen, hier "risk group" genannt. [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf Manual of Biosecurity, WHO, page 1].<br />
<br />
== Biosafety ==<br />
[[Image:Bielefeld Sign.JPG|200px|thumb|left| Picture 3: The Sign says: No Trespassing, Security stage 1.]]<br />
<br />
Als Biologische Sicherheit (Biosafety) wird der Versuch zur Reduzierung bzw. Eleminierung potentieller Gefahren durch Biotechnologie definiert. In Deutschland werden die unterschiedlichen Organismen in vier Sicherheitstufen von S1 für keine bekannte Gefährdung bis S4 für erwiesene humanpathogene Gefährdung eingeteilt. Die zentrale Kommussion für Biologische Sicherheit (ZKBS) überprüft alle Laboratorien, ob privat, gewerblich oder für die Forschung, vor in Betriebnahme auf Sicherheitsmängel und Einhaltung der rechtlichen Grundlagen. Das iGEM Team Bielefeld nutzt die Stämme ''Agrobacterium tumefaciens'' und einen Ableger des ''E. coli'' K12 in einem ZKBS geprüften Labor der Sicherheitstufe S1 für gentechnische Arbeiten. Die Stämme sind nach dem Gentechnik Gesetz (GenTSV) § 5 Absatz 1 Satz 1 und AnhangI Nummer 1 GenTSV der Risikogruppe 1 zu zuordnen (Stand 15.06.2010). Das beinhaltet, dass die Organismen (Auszug):<br />
<br />
1.<br />
<br />
: - weder ein human-, pflanzen- noch tierpathogen sind<br />
: - keine Organismen höhere Risikogruppen abgeben<br />
: - sich durch experimentell erwiesene oder langfristig erprobte Anwendung auszeichnen oder eingebaute biologische Schranken […] die Überlebens- bzw. Vermehrungsfähigkeit in der Umwelt begrenzen<br />
<br />
2.<br />
Organismen, die die Punkte unter 1 erfüllen, sind gemäß §6 Abs. 1 in Verbindung mit Anhang II Teil A des GenTVS<br />
<br />
: a) Organismen, die als biologische Sicherheitsmaßnahme anerkannt sind (§6 Abs. 3 GenTSV)<br />
: b) Zellen oder Zelllinien, die nicht von außen mit Organismen höherer Risikogruppe (2-4) kontaminiert sind<br />
: c) Organismen der Risikogruppe 1 nach der Organismenliste (Spender- und Empfängerorganismen für gentechnische Arbeiten zu Forschungszwecken<br />
<br />
Die verwendeten Stämme ''E. coli'' K12 und ''Agrobacterium tumefaciens'' werden nach [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf dieser Organismenliste] als Risikogruppe 1 klassifiziert. [http://www.bvl.bund.de/cln_007/nn_520774/DE/06__Gentechnik/093__ZKBS/01__Allg__Stellungnahmen/02__bakterien/zkbs__bakterien__agrobacterium__tumefaciens,templateId=raw,property=publicationFile.pdf/zkbs_bakterien_agrobacterium_tumefaciens.pdf ''Agrobacterium tumefaciens''] ist zwar als phytopathogen vermerkt, ist jedoch ubiquitär, d.h. weltweit im Boden (ca. 500 Bakterien / 1 g Boden) verbreitet und wurde aus diesem Grund der Risikogruppe 1 zugeordnet. <br />
<br />
Zur Sicherheit des Personals und der Umwelt herrscht ein konstanter leichter Unterdruck in den Laboratorien und die Fenster sind geschlossen zu halten, so dass weder Organismen weder hinaus noch hineingelangen können. Zudem wird der anfallende Organismen-Müll in spezillen Säcken gesammelt, autoklaviert und entsorgt. Die Organismen werden abgetötet. Zudem werden benutzen Flächen vor und nach der Arbeit desinfiziert.<br />
<br />
= Risk Assesment =<br />
<br />
== What we do to protect ==<br />
<br />
:*Every single person in the lab is trained on their work<br />
:*Every working person in the lab is trained on safety issues<br />
:*We got a safety and a disinfection officer at our lab <br />
:*No unqualified personal has access to the lab<br />
:*No public access to the lab<br />
:*We reduced the risk of contamination of the environment or staff members by disinfection, autoclavation and using of protecting clothes<br />
:*In case of emergency there are telephones, fire extinguisher, defibrillator and alarm buttons around the lab<br />
:*We only used organism of the risk stage 1, which do not harm neither mankind nor the environment<br />
:*We modify a unharzadous organism with soecific, identified and characterized biobricks. So we can appraise the risk causing by the modified organisms<br />
:*Constantly low pressure, no open windows => no organism can get in or out<br />
:*Desinfection after work of all working places (everyday)<br />
:*Autoclavation of waste (solid/liquid)<br />
:*No pipetting with the mouth<br />
<br />
== Unsere Schutzmaßnahmen ==<br />
<br />
:*Jede Person, die sich im Labor aufhält und arbeitet ist in ihrer Arbeit ausgebildet und/oder unterwiesen<br />
:*Jede Person, die sich im Labor aufhält oder arbeitet hat eine Sicherheitsbelehrung unterlaufen<br />
:*Es gibt einen Sicherheits- und eine Desinfektionsfachkraft in unserem Labor<br />
:*Kein unqualifiziertes Personal gelangt ins Labor<br />
:*Kein unqualifiziertes Personal gelangt in eine höhere Sicherheitsstufe als ihr erlaubt<br />
:*Kein öffentlicher Zugang zum Labor möglich<br />
:*We reduzieren das Kontaminationsrisiko für Arbeiter und Umwelt durch Autoklavieren, Desinfizierren und das Nutzen von protektiver Arbeitskleidung auf ein Minimum<br />
:*Im Falle eines Notfalls gibt es ein Telefon zum Hilferuf, Feuerlöscherr, Defibrilatoren und Feueralarmschalter in Reichweite<br />
:*Wir benutzen ausschließlich Organismen der Sicherheitsstufe 1<br />
<br />
= Short Summary =<br />
: 1'''. Would any of your project ideas raise safety issues ?'''<br />
<br />
:The bacteria we are currently working with are defined as bio security stage 1 (S1). Our produced GMOs (genetically modified organisms) are therefore no proofed harm neither to mankind nor to the environment. Thus they are GRAS - generally regarded as safe.<br />
:We are working in a bio security laboratory of the stage 1 (S1). There is no possible access for the public. Every person working in the lab is trained and instructed by the safety rules for laboratories S1. <br />
<br />
: '''2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?''' <br />
:No. All GMOs are S1 biosafety level. We did a risk assesement and know what kind of modilation we did on the GMOs. There is no risk of hazard or biological safety issues to mankind.<br />
<br />
: ''' 3. Is there a local biosafety group, committee, or review board at your institution?'''<br />
:Yes. There is a safety and a desinfection officer at our institution.<br />
<br />
: '''4.Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?''' <br />
: Every team must be aware of the risk that might be based in the organisms they use and they modify. Maybe a general safety instruction, which can be downlaoded from the igem.org page will be helpful to secure the research done in this competition. Moreover a list of risk organisms, with easy acces on the igem.safety page, would be a great benefit. Additional the safety.page could be tidied up to ease the acces to the key facts. Hence a small safety summary would be usefull. Some kind of a checklist.<br />
Certainly, it has to be said that the safety of the project must be reviewed by the team performing the experiments.<br />
<br />
= Referenzen / References =<br />
<br />
<br />
Comment on synthetic biology of the DFG (German Research Association)/pdf, German<br />
<br />
[http://daccess-dds-ny.un.org/doc/UNDOC/GEN/G08/625/32/PDF/G0862532.pdf?OpenElement Comments on Safety UNOG Genova, 2008 / pdf, English]<br />
<br />
WHO; Laboratory Biosafety Manual (English)<br />
<br />
WHO; Laboratory biosecurity guidance (English, 2008)<br />
<br />
Stellungnahme der ZKBS zur Einstufung von ''Agrobacterium tumefaciens'' (Deutsch) / Statement of the ZKBS concerning the classification of ''Agrobacterium tumefaciens'' (German)]<br />
<br />
Kategorisierte Organismenliste (Deutsch) der ZKBS / List of categorized organisms (German) of the ZKBS<br />
<br />
Merkblatt zum Transportieren von GVOs / ZKBS<br />
<br />
ZKBS, 2010</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Project/OutlookTeam:Bielefeld-Germany/Project/Outlook2010-10-27T15:22:15Z<p>Nkessler: /* Weblinks */</p>
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= Outlook =<br />
== The techniques’ potential ==<br />
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<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1548942" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=1548942" width="200"><br />
<h3>Capsaicin</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1548942&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
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<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=17198" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=17198" width="200"><br />
<h3>Acetosyringone</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=17198&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
<br />
In our project we modulated the virA-gene from ’’A. tumefaciens’’, which is encoding a receptor for the plant-hormone acetosyringone, via error prone PCR. Slight mutations may alter the receptors conformation or its binding site. Both of these can cause shifts in specificity and sensitivity. This way we created a new receptor, which is capable of sensing capsaicin. The chemical formulas of capsaicin and acetosyringone are quite similar and there are other molecules of interest, that have some of [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Phenolic_Compounds the necessary properties], too. Thus, we suggest that it is possible to modulate VirA receptors in order to make them sense a variety of highly relevant compounds.<br />
<br />
In other words, there are other candidates our sensing system may be applied to. All of them have similiar molecular structures as the vir-systems natural inducor acetosyringone. In the following we will present some of the most relevant compounds other than capsaicin, that may be upcoming targets of our research.<br />
<br />
''All of the following images of the compounds molecular structures are linked to [http://pubchem.ncbi.nlm.nih.gov/ '''The PubChem Project'''].''<br />
<br />
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== Diagnosis of pheochromocytoma and neuroblastoma (child tumors) ==<br />
<html><br />
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<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=21100" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=21100" width="200"><br />
<h3>Metanephrine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=21100&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=23615482" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=23615482" width="200"><br />
<h3>Vanilmandelic Acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=23615482&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1738" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=1738" width="200"><br />
<h3>Homovanillic acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1738&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
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=== Pheochromocytoma ===<br />
Pheochromocytoma is a rare endocrine tumor originating in the medulla of adrenal glands, localized on top of the kidney. The adrenal glands produce several catecholamines, to which metanephrine and dopamine belong. These hormones regulate responses to stress, heart rate and blood pressure. In patients with pheochromocytoma these hormones are released excessively, potentially causing increased heart rate and blood pressure. Pheochromocytoma may become life threatening when not recognized and treated [http://pheopara.nichd.nih.gov/ (Pheochromocytoma and Paraganglioma website at the NIH)].<br />
<br />
These hormones can be quantified in urine and thus are important compounds of pheochromocytoma diagnostics. [http://jcem.endojournals.org/cgi/content/abstract/92/12/4602 Boyle et al. (2007)] compare different accuracies of diagnostic measures for the tumor and name urinary free metanephrine HPLC-measurement as the most sensitive and specific. Homovanillic acid and vanillyl mandelic acid are measured via HPLC aswell and are indicators for the same tumor.<br />
<br />
=== Neuroblastoma ===<br />
"Neuroblastoma is the most common extracranial solid tumor in infancy. It is an embryonal malignancy of the sympathetic nervous system arising from neuroblasts (pluripotent sympathetic cells). In the developing embryo, these cells invaginate, migrate along the neuraxis, and populate the sympathetic ganglia, adrenal medulla, and other sites. The pattern of distribution of these cells correlates with the sites of primary disease presentation." ([http://emedicine.medscape.com/article/988284-overview Lacayo, Davis 2010])<br />
<br />
Patients with high-risk disease still have very poor outcomes despite intensive therapy.<br />
<br />
"More than 90% of patients have elevated homovanillic acid (HVA) and/or vanillylmandelic acid (VMA) levels detectable in urine."<br />
<br />
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<br />
== 3-Methoxytyramine ==<br />
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<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=681" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=681" width="200"><br />
<h3>Dopamine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=681&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=11957621" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=11957621" width="200"><br />
<h3>3-Methoxytyramine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=11957621&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
<br />
[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0013452 Sotnikova et al. (2010)] suggest, that 3-Methoxytyramine (3-MT) plays an important role as a neuromodulator. 3-MT is a metabolite of Dopamine and is reported to be a potential indicator for dopamine-dependent diseases like the parkinson disease, schizophrenia and dyskinesia. 3-Methoxytyramin as metabolic product of the hormon dopamine can be detected in human urine ([http://edoc.hu-berlin.de/oa/degruyter/cclm.1971.9.6.478.pdf Knoll ''et al.'', 1971]).<br />
Dopamine is known as happiness hormone found in increased levels after taking stimulating substances such as cocaine or amphetamines. The latter is one of the worldwide most used substitutes in sportive competition as doping. Increased levels of 3-MT analyzed in urine taken from sportives can indicate to intake of stimulating substances ([http://journals.lww.com/acsm-msse/Abstract/1980/21000/The_effect_of_amphetamines_on_selected.13.aspx CHANDLER, JOE V. and STEVEN N BLAIR, 1980]).<br />
<br />
== Dopamine ==<br />
<br />
Even though being referred to as "happiness hormone", Dopamine and its receptors are said to play key roles in numerous phsychic disorders and drug addiction. For many years now, the Dopamine hypothesis of Schizophrenia exists and evolves. It states that many symptoms of Schizophrenia correlate with a hyperactive disturbed dopaminergic signal transduction. [http://schizophreniabulletin.oxfordjournals.org/content/early/2009/03/26/schbul.sbp006.abstract Howes and Kapur (2009)] as well as [http://jop.sagepub.com/content/21/4/440.short Stone ''et al.'' (2007)] reviewed the hypothesis and its evolution.<br />
<br />
Concerning drug addiction, Dopamine and the brains reward system are of special interest in research [http://dx.doi.org/10.1016/j.brainresrev.2004.12.033 (Heidbreder, 2005)].<br />
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<br />
== Picric acid==<br />
<html><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=6954" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=6954" width="200"><br />
<h3>Picric acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=6954&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
</html><br />
Picrid acid (2,4,6-trinitrophenol) still occurs as contaminat in ground water and is as nitritited aromatic compound a potential explosive ([http://search.barnesandnoble.com/Explosives-Engineering/Paul-W-Cooper/e/9780471186366 Cooper PW, 1997]), ([http://onlinelibrary.wiley.com/doi/10.1002/1521-3773%2820010601%2940:11%3C2104::AID-ANIE2104%3E3.0.CO;2-%23/full Sohn H, 2001]). Existing detection methods are time consuming and even more cost intensive, so that a high selective and sensitiv application by microbial screening would be desirable.<br />
<br />
<br />
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<br />
== Summary ==<br />
The VirA receptor is a great starting point for modulations, in order to address a wide variety of highly relevant compounds. By contructing a Vir-gene based sensing system we present a ''proof of concept'' for a general microbial sensing system adaptable for various other compounds. Since the basic sensing and reporting system was successfully established in ''E. coli'', the next essential steps are to improve the mutagenesis strategy for the receptor, and finally the screening for each candidate compound.<br />
<br />
Using [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Output-signal_amplification_by_Sensitivity_Tuner_implementation a set of sensitivity tuners], the reporter output may then be optimized. Furthermore, the speed of the systems are expected to require refinement.<br />
<br />
As a result, the MARSS has great potential to deliver biological tests for medical diagnosis, drug tests for ''e.g.'' doping conctrols, analysis of food for ''e.g.''allergy triggering compounds and soil contaminating substances such as picric acid.<br />
<br />
=Weblinks=<br />
<br />
*Boyle J, Davidson DF, Perry CG and Connell JMC, ''Comparison of Diagnostic Accuracy of Urinary Free Metanephrines, Vanillyl Mandelic Acid, and Catecholamines and Plasma Catecholamines for Diagnosis of Pheochromocytoma'' ,Journal of Clinical Endocrinology & Metabolism, Vol. 92, No. 12, pp. 4602-4608<br />
<br />
*Chandler JV and Blair SN (1980), '' The effect of amphetamines on selected physiological components related to athletic success''. Med. Sci. Sports Exercise, Vol. 12, No. 1, pp. 65-69<br />
<br />
*Cooper PW (1997), ''Explosives Engineering'', Edition 1, Wiley, John & Sons.<br />
<br />
* ''Eunice Kennedy Shriver'' National Institute of ''Child Health and Human Development'', Pheochromocytoma and Paragangliooma, 0-CH-0093<br />
<br />
*Howes OD and Kapur S (2009), ''The Dopamine Hypothesis of Schizophrenia: Version III—The Final Common Pathway'', Schizophrenia Bulletin, Vol. 35, No. 3, pp.549-62<br />
<br />
*http://pubchem.ncbi.nlm.nih.gov/<br />
<br />
*Knoll E, Wisser H, Stamm D (1971), ''in Verfahren zur Bestimmung der 3-Methoxy-4-hydroxy-phenylessigsäure (Homovanillinsäure) im Harn durch in situ Remissionsmessung nach dünnschichtchromatographischer Trennung'', Z. klin. Chem. u. klin. Biochem., 1971<br />
<br />
*Lacayo NJ (2010), ''Neuroblastoma'', eMedicine from webMD<br />
<br />
*Sohn H, Calhoun RM, Sailor MJ, Trogler WC (2001), ''Detection of TNT and Picric Acid on Surfaces and in Seawater by Using Photoluminescent Polysiloles'', Angewandte Chemie, Vol.40, pp.2104–2105<br />
<br />
*Sotnikova TD, Beaulieu J-M, Espinoza S, Masri B, Zhang X, Salahpour A, Barak LS, Caron MG, Gainetdinov RR (2010), ''The Dopamine Metabolite 3-Methoxytyramine Is a Neuromodulator'', PloSONE<br />
<br />
*Stone JM, Morrison PD, Pilowsky LS (2007), ''Review: Glutamate and dopamine dysregulation in schizophrenia — a synthesis and selective review'', Journal of Psychopharmacology, Vol. 21, No. 4, pp. 440-452</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/SafetyTeam:Bielefeld-Germany/Safety2010-10-27T14:53:09Z<p>Nkessler: /* Transportation of GMOs */</p>
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= Biosafety and Biosecurity (English) =<br />
<br />
It is important to discriminate between Biosafety and Biosecurity in the context of working with genetically modified organisms and lab use. Biosecurity comprises the safety of the lab. The [http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_EPR_2006_6.pdf WHO] defines Biosecurity as a task of organisation and administration.The term Biosafety deals with the biological risks of the samples for the user, the enviroment and the lab. In this article, we are mostly summarizing the German law on Biosecurity and Biosafety regarding to the [http://www.bvl.bund.de/nn_495478/DE/06__Gentechnik/093__ZKBS/gentechnik__zkbs__node.html__nnn=true ZKBS (zentrale Kommission für Biologische Sicherheit)] and [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf their lists of unhazardous organisms] plus the comment on safety of the [http://www.dfg.de/download/pdf/dfg_im_profil/reden_stellungnahmen/2009/stellungnahme_synthetische_biologie.pdf DFG (Deutsche Forschungsgemeinschaft)].<br />
Furthermore we crosslinked the laws of the United States of America and the European law of Biosecurity and Biosafety in this article. We marked this statement in the text, so the reader can discrimante between the law systems. This kind of modulation makes sense, because our experimental work was done in Germany, shipped in europe (SDU Denmark) and used as a biobrick in United States of America.<br />
<br />
== Biosecurity ==<br />
[[Image:Safe labwork.jpg|200px|thumb|right|clean bench in our lab.]]<br />
<br />
<br />
In the context of "genetically engineered" it is quite important to keep unqualified personnel and trespassers away from your laboratory and your samples. In our case we secure our lab with a keypad. Every staff member needs a key to open the laboratory door. Nobody is able to open the door without the proper keys. Staff members receive a key, after beeing briefed by the safety officer on safety issues. You can only get a key for the safety stage you are working in. The labs with higher safety stages are cut off from the lower ones. Staff members need a continuative safety guide to receive a key for the higher security levels. Concludingly no unqualified personel can pass the door to the laboratory.<br />
The picture 1 shows the keypad in front of the lab.<br />
<br />
The laboratories, which are in use for our iGEM project are security stage 1 (S1). For the safety of the staff our labs are equiped with fire extinguisher, emergency showers and a heart defibrillator.<br />
<br />
== Biosafety ==<br />
[[Image:Keypad.JPG|200px|thumb|left| Picture 1: Security entrance to the lab. Every Member of the Team needs a key-token in order to get acces to the lab]]<br />
<br />
The term "biological safety" defines the effort to reduce or eliminate any potential risks caused by biotechnolgy or genetical engineering. The laboratories are scaled into four differnt saftey stages. The stage "S1" defines working with organsims and methods, which do no prooved harm neither to the enviroment nor mankind. The stages rise up to biosafety stage four, which defines working with prooved humanpathogenous organisms. The WHO defines for the United states the same safety stages, which are called risk groups [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf Manual of Biosecurity, WHO, page 1].<br />
<br />
The Biosafety is ruled by the "Zentrale Kommission für Biologische Sicherheit" (ZKBS) in Germany. The ZKBS reviewed and verified all commercial, research or privat laboratories about their safety issues. The iGEM Team Bielefeld is currently working with the organisms ''Agrobacterium tumefaciens'' and a derivate of ''E. coli'' K12 in a ZKBS reviewed S1-lab. The organisms strains are declared as riskfactor 1 by the german act of genetics (GenTSV, § 5; 15.06.2010). The act contains that the organisms (abstract):<br />
<br />
1.<br />
<br />
: - are no proved human-, phyto- or animalpathogen<br />
: - do not contain or release organism containing to a higher risk stage<br />
: - are prooved by experiments or long term evaluation or do not proliferate in the enviroment because of biological implanted boundaries<br />
<br />
<br />
2.<br />
Organisms, which confirm the paragraph 1, are defined by the $6 of the act of genetics (GenTSV):<br />
<br />
: a) as biological safe organisms<br />
: b) organims or strains, which are contaminated by organisms or strains of a higher security level<br />
: c) organisms defined by the risk level S1 (list of organisms used for genetically engineering research)<br />
<br />
<br />
The [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf list of organisms] categorize the strains ''E. coli'' K12 and ''Agrobacterium tumefaciens'' as biological safety risk 1 (S1). The strain [http://www.bvl.bund.de/cln_007/nn_520774/DE/06__Gentechnik/093__ZKBS/01__Allg__Stellungnahmen/02__bakterien/zkbs__bakterien__agrobacterium__tumefaciens,templateId=raw,property=publicationFile.pdf/zkbs_bakterien_agrobacterium_tumefaciens.pdf ''Agrobacterium tumefaciens''] is a known phytopathogen. Because of its frequent appearence in the earth (500 bacteria in 1g earth) it is generally regarded as safe.<br />
<br />
The WHO defines "security assesments" has the highest priority for the work with organisms. This includes a proper theoretically background of the organism. Further they defined rules for good molecular practice and proper lab security [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf WHO Manual Biosafety, page 9 following]. They note that most of the security guidelines are not quite neccessary for organism of risk stage 1. Although we play by the rules and work as carefully as possible (see, what we do to protect).<br />
<br />
= Transportation of GMOs =<br />
<br />
The transportation and shipping of genetically modified organisms (GMOs) has to be done under specific safety conditions in order to lower the risk GMOs released into the enviroment. Moreover it is important that no GMOs are sent to unqualified personnel or is used by unqualified personnel during the shipment.<br />
<br />
As a part of the European community, the German law for export of GMOs are similar to the rules of the European community law. Before you send a GMO, you must ensure that the recipient is allowed to work with the GMO. Moreover every release of the GMOs into the enviroment must be checked by gouvernmental law in the recipients country and the country of the dispatcher.<br />
<br />
The package must be signed as "shipped GMOs". Otherwise it will not be let cross any border in the European community.<br />
<br />
= What we do to protect=<br />
<br />
:*Every single person in the lab is trained on their work<br />
:*Every working person in the lab is trained on safety issues<br />
:*We got a safety and a disinfection officer at our lab <br />
:*No unqualified personal has access to the lab<br />
:*No public access to the lab<br />
:*We reduced the risk of contamination of the environment or staff members by disinfection, autoclavation and using of protecting clothes<br />
:*In case of emergency there are telephones, fire extinguisher, defibrillator and alarm buttons around the lab<br />
:*We only used organism of the risk stage 1, which do not harm neither mankind nor the environment<br />
:*We modify a unharzadous organism with soecific, identified and characterized biobricks. So we can appraise the risk causing by the modified organisms<br />
:*Constantly low pressure, no open windows => no organism can get in or out<br />
:*Desinfection after work of all working places (everyday)<br />
:*Autoclavation of waste (solid/liquid)<br />
:*No pipetting with the mouth<br />
<br />
<br />
= Biosafety und Biosecurity (German)=<br />
<br />
Es ist bei der Inbetriebnahme und Nutzung einer Gentechnischen Anlagen darauf zu achten, dass Vorkehrungen sowohl für den Betriebsschutz (Biosecurity) als auch für die biologische Sicherheit (Biosafety) getroffen werden. Biosecurity wird von der [http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_EPR_2006_6.pdf WHO] als Aufgabe der Adminstrative des Labors definiert.<br />
<br />
== Biosecurity ==<br />
Der Biosecurity wird Sorge getragen, in dem kein unbefugter Zugang zum Laborplatz hat. Die Türen sind nur per Schlüssel zu öffnen. Jeder Mitarbeiten muss eine Sicherheitsbelehrung beim Sicherheitsbeauftragten ablegen ehe er Zugang zu den Laboratorien bekommt. Die Labore sind je nach Sicherheitsgefährdung gekennzeichnet und verschlossen, so dass keine unterqualifizierte Person in ein höheres Sicherheitslevel eindringen kann. In dem vom iGEM genutzten Labor handelt es sich um die Sicherheitstufe S1. Die WHO definiert die gleichen Sicherheitsstufen, hier "risk group" genannt. [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf Manual of Biosecurity, WHO, page 1].<br />
<br />
== Biosafety ==<br />
[[Image:|200px|thumb|left| We are using a cleanbench to avoid any contamination of our samples. A proper use of the cleanbench can only be achieved by placing all the big things in the rear of the bench, by not disturbing the air circulation and by avoiding any kind of aerosoles. Use gloves for your own protection.]]<br />
<br />
Als Biologische Sicherheit (Biosafety) wird der Versuch zur Reduzierung bzw. Eleminierung potentieller Gefahren durch Biotechnologie definiert. In Deutschland werden die unterschiedlichen Organismen in vier Sicherheitstufen von S1 für keine bekannte Gefährdung bis S4 für erwiesene humanpathogene Gefährdung eingeteilt. Die zentrale Kommussion für Biologische Sicherheit (ZKBS) überprüft alle Laboratorien, ob privat, gewerblich oder für die Forschung, vor in Betriebnahme auf Sicherheitsmängel und Einhaltung der rechtlichen Grundlagen. Das iGEM Team Bielefeld nutzt die Stämme ''Agrobacterium tumefaciens'' und einen Ableger des ''E. coli'' K12 in einem ZKBS geprüften Labor der Sicherheitstufe S1 für gentechnische Arbeiten. Die Stämme sind nach dem Gentechnik Gesetz (GenTSV) § 5 Absatz 1 Satz 1 und AnhangI Nummer 1 GenTSV der Risikogruppe 1 zu zuordnen (Stand 15.06.2010). Das beinhaltet, dass die Organismen (Auszug):<br />
<br />
1.<br />
<br />
: - weder ein human-, pflanzen- noch tierpathogen sind<br />
: - keine Organismen höhere Risikogruppen abgeben<br />
: - sich durch experimentell erwiesene oder langfristig erprobte Anwendung auszeichnen oder eingebaute biologische Schranken […] die Überlebens- bzw. Vermehrungsfähigkeit in der Umwelt begrenzen<br />
<br />
2.<br />
Organismen, die die Punkte unter 1 erfüllen, sind gemäß §6 Abs. 1 in Verbindung mit Anhang II Teil A des GenTVS<br />
<br />
: a) Organismen, die als biologische Sicherheitsmaßnahme anerkannt sind (§6 Abs. 3 GenTSV)<br />
: b) Zellen oder Zelllinien, die nicht von außen mit Organismen höherer Risikogruppe (2-4) kontaminiert sind<br />
: c) Organismen der Risikogruppe 1 nach der Organismenliste (Spender- und Empfängerorganismen für gentechnische Arbeiten zu Forschungszwecken<br />
<br />
Die verwendeten Stämme ''E. coli'' K12 und ''Agrobacterium tumefaciens'' werden nach [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf dieser Organismenliste] als Risikogruppe 1 klassifiziert. [http://www.bvl.bund.de/cln_007/nn_520774/DE/06__Gentechnik/093__ZKBS/01__Allg__Stellungnahmen/02__bakterien/zkbs__bakterien__agrobacterium__tumefaciens,templateId=raw,property=publicationFile.pdf/zkbs_bakterien_agrobacterium_tumefaciens.pdf ''Agrobacterium tumefaciens''] ist zwar als phytopathogen vermerkt, ist jedoch ubiquitär, d.h. weltweit im Boden (ca. 500 Bakterien / 1 g Boden) verbreitet und wurde aus diesem Grund der Risikogruppe 1 zugeordnet. <br />
<br />
Zur Sicherheit des Personals und der Umwelt herrscht ein konstanter leichter Unterdruck in den Laboratorien und die Fenster sind geschlossen zu halten, so dass weder Organismen weder hinaus noch hineingelangen können. Zudem wird der anfallende Organismen-Müll in spezillen Säcken gesammelt, autoklaviert und entsorgt. Die Organismen werden abgetötet. Zudem werden benutzen Flächen vor und nach der Arbeit desinfiziert.<br />
<br />
= Transport von GVO =<br />
<br />
Der Transport von genetisch veränderten Organismen (GVOs) unterliegt innerhalb der Europäischen Union und außerhalb davon speziellen Sicherheitsbedingungen. Die deutschen Bestimmung sind denen der Europäischen Union ähnlich.<br />
<br />
= Unsere Schutzmaßnahmen=<br />
<br />
:*Jede Person, die sich im Labor aufhält und arbeitet ist in ihrer Arbeit ausgebildet und/oder unterwiesen<br />
:*Jede Person, die sich im Labor aufhält oder arbeitet hat eine Sicherheitsbelehrung unterlaufen<br />
:*Es gibt einen Sicherheits- und eine Desinfektionsfachkraft in unserem Labor<br />
:*Kein unqualifiziertes Personal gelangt ins Labor<br />
:*Kein unqualifiziertes Personal gelangt in eine höhere Sicherheitsstufe als ihr erlaubt<br />
:*Kein öffentlicher Zugang zum Labor möglich<br />
:*We rreduzieren das Kontaminationsrisiko für Arbeiter und Umwelt durch Autoklavieren, Desinfizierren und das Nutzen von protektiver Arbeitskleidung auf ein Minimum<br />
:*Im Falle eines Notfalls gibt es ein Telefon zum Hilferuf, Feuerlöscherr, Defibrilatoren und Feueralarmschalter in Reichweite<br />
:*Wir benutzen ausschließlich Organismen der Sicherheitsstufe 1<br />
<br />
<br />
<br />
= Short Summary =<br />
: 1'''. Would any of your project ideas raise safety issues ?'''<br />
<br />
:The bacteria we are currently working with are defined as bio security stage 1 (S1). Our produced GMOs (genetically modified organisms) are therefor no proofed harm neither to mankind nor to the environment. Thus they are GRAS - generally regarded as safe.<br />
:We are working in a bio security laboratory of the stage 1 (S1). There is no possible access for the public. Every person working in the lab is trained and instructed by the safety rules for laboratories S1. <br />
<br />
: '''2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?''' <br />
:No. All GMOs are S1 biosafety level. There is no risk of hazard or biological safety issues to mankind.<br />
<br />
: ''' 3. Is there a local biosafety group, committee, or review board at your institution?'''<br />
:Yes. There is a safety officer at our institution.<br />
<br />
: '''4.Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?''' <br />
: Every team must be aware of the risk that might be based in the organisms they use and they modify. Maybe a general safety instruction, which can be downlaoded from the igem.org page will be helpful to secure the research done in this competition. Moreover a list of risk organisms, with easy acces on the igem.safety page, would be a great benefit. Additional the safety.page could be tidied up to ease the acces to the key facts. Hence a small safety summary would be usefull. Some kind of a checklist.<br />
Certainly, it has to be said that the safety of the project must be reviewed by the team performing the experiments.<br />
<br />
= Referenzen / References =<br />
<br />
ZKBS, 2010<br />
<br />
Kategorisierte Organismenliste (Deutsch) der ZKBS / List of categorized organisms (German) of the ZKBS<br />
<br />
Stellungnahme der ZKBS zur Einstufung von ''Agrobacterium tumefaciens'' (Deutsch) / Statement of the ZKBS concerning the classification of ''Agrobacterium tumefaciens'' (German)]<br />
<br />
Comment on synthetic biology of the DFG (German Research Association)/pdf, German<br />
<br />
[http://daccess-dds-ny.un.org/doc/UNDOC/GEN/G08/625/32/PDF/G0862532.pdf?OpenElement COmments on Safety UNOG Genova, 2008 / pdf, English]<br />
<br />
WHO; Laboratory Biosafety Manual (English)<br />
<br />
WHO; Laboratory biosecurity guidance (English, 2008)</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/SafetyTeam:Bielefeld-Germany/Safety2010-10-27T14:48:10Z<p>Nkessler: /* Biosafety */</p>
<hr />
<div>{{Bielefeld_MainMenu_2010|<nowiki>http://igem-bielefeld.de/img/banner_safety.png</nowiki>}}<br />
<br />
= Biosafety and Biosecurity (English) =<br />
<br />
It is important to discriminate between Biosafety and Biosecurity in the context of working with genetically modified organisms and lab use. Biosecurity comprises the safety of the lab. The [http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_EPR_2006_6.pdf WHO] defines Biosecurity as a task of organisation and administration.The term Biosafety deals with the biological risks of the samples for the user, the enviroment and the lab. In this article, we are mostly summarizing the German law on Biosecurity and Biosafety regarding to the [http://www.bvl.bund.de/nn_495478/DE/06__Gentechnik/093__ZKBS/gentechnik__zkbs__node.html__nnn=true ZKBS (zentrale Kommission für Biologische Sicherheit)] and [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf their lists of unhazardous organisms] plus the comment on safety of the [http://www.dfg.de/download/pdf/dfg_im_profil/reden_stellungnahmen/2009/stellungnahme_synthetische_biologie.pdf DFG (Deutsche Forschungsgemeinschaft)].<br />
Furthermore we crosslinked the laws of the United States of America and the European law of Biosecurity and Biosafety in this article. We marked this statement in the text, so the reader can discrimante between the law systems. This kind of modulation makes sense, because our experimental work was done in Germany, shipped in europe (SDU Denmark) and used as a biobrick in United States of America.<br />
<br />
== Biosecurity ==<br />
[[Image:Safe labwork.jpg|200px|thumb|right|clean bench in our lab.]]<br />
<br />
<br />
In the context of "genetically engineered" it is quite important to keep unqualified personnel and trespassers away from your laboratory and your samples. In our case we secure our lab with a keypad. Every staff member needs a key to open the laboratory door. Nobody is able to open the door without the proper keys. Staff members receive a key, after beeing briefed by the safety officer on safety issues. You can only get a key for the safety stage you are working in. The labs with higher safety stages are cut off from the lower ones. Staff members need a continuative safety guide to receive a key for the higher security levels. Concludingly no unqualified personel can pass the door to the laboratory.<br />
The picture 1 shows the keypad in front of the lab.<br />
<br />
The laboratories, which are in use for our iGEM project are security stage 1 (S1). For the safety of the staff our labs are equiped with fire extinguisher, emergency showers and a heart defibrillator.<br />
<br />
== Biosafety ==<br />
[[Image:Keypad.JPG|200px|thumb|left| Picture 1: Security entrance to the lab. Every Member of the Team needs a key-token in order to get acces to the lab]]<br />
<br />
The term "biological safety" defines the effort to reduce or eliminate any potential risks caused by biotechnolgy or genetical engineering. The laboratories are scaled into four differnt saftey stages. The stage "S1" defines working with organsims and methods, which do no prooved harm neither to the enviroment nor mankind. The stages rise up to biosafety stage four, which defines working with prooved humanpathogenous organisms. The WHO defines for the United states the same safety stages, which are called risk groups [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf Manual of Biosecurity, WHO, page 1].<br />
<br />
The Biosafety is ruled by the "Zentrale Kommission für Biologische Sicherheit" (ZKBS) in Germany. The ZKBS reviewed and verified all commercial, research or privat laboratories about their safety issues. The iGEM Team Bielefeld is currently working with the organisms ''Agrobacterium tumefaciens'' and a derivate of ''E. coli'' K12 in a ZKBS reviewed S1-lab. The organisms strains are declared as riskfactor 1 by the german act of genetics (GenTSV, § 5; 15.06.2010). The act contains that the organisms (abstract):<br />
<br />
1.<br />
<br />
: - are no proved human-, phyto- or animalpathogen<br />
: - do not contain or release organism containing to a higher risk stage<br />
: - are prooved by experiments or long term evaluation or do not proliferate in the enviroment because of biological implanted boundaries<br />
<br />
<br />
2.<br />
Organisms, which confirm the paragraph 1, are defined by the $6 of the act of genetics (GenTSV):<br />
<br />
: a) as biological safe organisms<br />
: b) organims or strains, which are contaminated by organisms or strains of a higher security level<br />
: c) organisms defined by the risk level S1 (list of organisms used for genetically engineering research)<br />
<br />
<br />
The [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf list of organisms] categorize the strains ''E. coli'' K12 and ''Agrobacterium tumefaciens'' as biological safety risk 1 (S1). The strain [http://www.bvl.bund.de/cln_007/nn_520774/DE/06__Gentechnik/093__ZKBS/01__Allg__Stellungnahmen/02__bakterien/zkbs__bakterien__agrobacterium__tumefaciens,templateId=raw,property=publicationFile.pdf/zkbs_bakterien_agrobacterium_tumefaciens.pdf ''Agrobacterium tumefaciens''] is a known phytopathogen. Because of its frequent appearence in the earth (500 bacteria in 1g earth) it is generally regarded as safe.<br />
<br />
The WHO defines "security assesments" has the highest priority for the work with organisms. This includes a proper theoretically background of the organism. Further they defined rules for good molecular practice and proper lab security [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf WHO Manual Biosafety, page 9 following]. They note that most of the security guidelines are not quite neccessary for organism of risk stage 1. Although we play by the rules and work as carefully as possible (see, what we do to protect).<br />
<br />
= Transportation of GMOs =<br />
<br />
The transportation and shipping of genetically modified organisms (GMOs) has to be done under specific safety conditions in order to lower the risk GMOs released into the enviroment. Moreover it is important that no GMOs is sent to unqualified personel or is used by unqualified personell during the shipment.<br />
<br />
As a part of the european community, the german law for export of GMOs are similar to the rules of the eurpean community law. Before you send a GMOs, you must ensure that the recipient is allowed to work with the GMO. Moreover every release of the GMOs into the enviroment must be checked by gouvermental law in the recipients country and the country of the dispatcher.<br />
<br />
The package must be signed as "shipped GMOs". Otherwise it will not be cross any boarder in the european community.<br />
<br />
<br />
= What we do to protect=<br />
<br />
:*Every single person in the lab is trained on their work<br />
:*Every working person in the lab is trained on safety issues<br />
:*We got a safety and a disinfection officer at our lab <br />
:*No unqualified personal has access to the lab<br />
:*No public access to the lab<br />
:*We reduced the risk of contamination of the environment or staff members by disinfection, autoclavation and using of protecting clothes<br />
:*In case of emergency there are telephones, fire extinguisher, defibrillator and alarm buttons around the lab<br />
:*We only used organism of the risk stage 1, which do not harm neither mankind nor the environment<br />
:*We modify a unharzadous organism with soecific, identified and characterized biobricks. So we can appraise the risk causing by the modified organisms<br />
:*Constantly low pressure, no open windows => no organism can get in or out<br />
:*Desinfection after work of all working places (everyday)<br />
:*Autoclavation of waste (solid/liquid)<br />
:*No pipetting with the mouth<br />
<br />
<br />
= Biosafety und Biosecurity (German)=<br />
<br />
Es ist bei der Inbetriebnahme und Nutzung einer Gentechnischen Anlagen darauf zu achten, dass Vorkehrungen sowohl für den Betriebsschutz (Biosecurity) als auch für die biologische Sicherheit (Biosafety) getroffen werden. Biosecurity wird von der [http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_EPR_2006_6.pdf WHO] als Aufgabe der Adminstrative des Labors definiert.<br />
<br />
== Biosecurity ==<br />
Der Biosecurity wird Sorge getragen, in dem kein unbefugter Zugang zum Laborplatz hat. Die Türen sind nur per Schlüssel zu öffnen. Jeder Mitarbeiten muss eine Sicherheitsbelehrung beim Sicherheitsbeauftragten ablegen ehe er Zugang zu den Laboratorien bekommt. Die Labore sind je nach Sicherheitsgefährdung gekennzeichnet und verschlossen, so dass keine unterqualifizierte Person in ein höheres Sicherheitslevel eindringen kann. In dem vom iGEM genutzten Labor handelt es sich um die Sicherheitstufe S1. Die WHO definiert die gleichen Sicherheitsstufen, hier "risk group" genannt. [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf Manual of Biosecurity, WHO, page 1].<br />
<br />
== Biosafety ==<br />
[[Image:|200px|thumb|left| We are using a cleanbench to avoid any contamination of our samples. A proper use of the cleanbench can only be achieved by placing all the big things in the rear of the bench, by not disturbing the air circulation and by avoiding any kind of aerosoles. Use gloves for your own protection.]]<br />
<br />
Als Biologische Sicherheit (Biosafety) wird der Versuch zur Reduzierung bzw. Eleminierung potentieller Gefahren durch Biotechnologie definiert. In Deutschland werden die unterschiedlichen Organismen in vier Sicherheitstufen von S1 für keine bekannte Gefährdung bis S4 für erwiesene humanpathogene Gefährdung eingeteilt. Die zentrale Kommussion für Biologische Sicherheit (ZKBS) überprüft alle Laboratorien, ob privat, gewerblich oder für die Forschung, vor in Betriebnahme auf Sicherheitsmängel und Einhaltung der rechtlichen Grundlagen. Das iGEM Team Bielefeld nutzt die Stämme ''Agrobacterium tumefaciens'' und einen Ableger des ''E. coli'' K12 in einem ZKBS geprüften Labor der Sicherheitstufe S1 für gentechnische Arbeiten. Die Stämme sind nach dem Gentechnik Gesetz (GenTSV) § 5 Absatz 1 Satz 1 und AnhangI Nummer 1 GenTSV der Risikogruppe 1 zu zuordnen (Stand 15.06.2010). Das beinhaltet, dass die Organismen (Auszug):<br />
<br />
1.<br />
<br />
: - weder ein human-, pflanzen- noch tierpathogen sind<br />
: - keine Organismen höhere Risikogruppen abgeben<br />
: - sich durch experimentell erwiesene oder langfristig erprobte Anwendung auszeichnen oder eingebaute biologische Schranken […] die Überlebens- bzw. Vermehrungsfähigkeit in der Umwelt begrenzen<br />
<br />
2.<br />
Organismen, die die Punkte unter 1 erfüllen, sind gemäß §6 Abs. 1 in Verbindung mit Anhang II Teil A des GenTVS<br />
<br />
: a) Organismen, die als biologische Sicherheitsmaßnahme anerkannt sind (§6 Abs. 3 GenTSV)<br />
: b) Zellen oder Zelllinien, die nicht von außen mit Organismen höherer Risikogruppe (2-4) kontaminiert sind<br />
: c) Organismen der Risikogruppe 1 nach der Organismenliste (Spender- und Empfängerorganismen für gentechnische Arbeiten zu Forschungszwecken<br />
<br />
Die verwendeten Stämme ''E. coli'' K12 und ''Agrobacterium tumefaciens'' werden nach [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf dieser Organismenliste] als Risikogruppe 1 klassifiziert. [http://www.bvl.bund.de/cln_007/nn_520774/DE/06__Gentechnik/093__ZKBS/01__Allg__Stellungnahmen/02__bakterien/zkbs__bakterien__agrobacterium__tumefaciens,templateId=raw,property=publicationFile.pdf/zkbs_bakterien_agrobacterium_tumefaciens.pdf ''Agrobacterium tumefaciens''] ist zwar als phytopathogen vermerkt, ist jedoch ubiquitär, d.h. weltweit im Boden (ca. 500 Bakterien / 1 g Boden) verbreitet und wurde aus diesem Grund der Risikogruppe 1 zugeordnet. <br />
<br />
Zur Sicherheit des Personals und der Umwelt herrscht ein konstanter leichter Unterdruck in den Laboratorien und die Fenster sind geschlossen zu halten, so dass weder Organismen weder hinaus noch hineingelangen können. Zudem wird der anfallende Organismen-Müll in spezillen Säcken gesammelt, autoklaviert und entsorgt. Die Organismen werden abgetötet. Zudem werden benutzen Flächen vor und nach der Arbeit desinfiziert.<br />
<br />
= Transport von GVO =<br />
<br />
Der Transport von genetisch veränderten Organismen (GVOs) unterliegt innerhalb der Europäischen Union und außerhalb davon speziellen Sicherheitsbedingungen. Die deutschen Bestimmung sind denen der Europäischen Union ähnlich.<br />
<br />
= Unsere Schutzmaßnahmen=<br />
<br />
:*Jede Person, die sich im Labor aufhält und arbeitet ist in ihrer Arbeit ausgebildet und/oder unterwiesen<br />
:*Jede Person, die sich im Labor aufhält oder arbeitet hat eine Sicherheitsbelehrung unterlaufen<br />
:*Es gibt einen Sicherheits- und eine Desinfektionsfachkraft in unserem Labor<br />
:*Kein unqualifiziertes Personal gelangt ins Labor<br />
:*Kein unqualifiziertes Personal gelangt in eine höhere Sicherheitsstufe als ihr erlaubt<br />
:*Kein öffentlicher Zugang zum Labor möglich<br />
:*We rreduzieren das Kontaminationsrisiko für Arbeiter und Umwelt durch Autoklavieren, Desinfizierren und das Nutzen von protektiver Arbeitskleidung auf ein Minimum<br />
:*Im Falle eines Notfalls gibt es ein Telefon zum Hilferuf, Feuerlöscherr, Defibrilatoren und Feueralarmschalter in Reichweite<br />
:*Wir benutzen ausschließlich Organismen der Sicherheitsstufe 1<br />
<br />
<br />
<br />
= Short Summary =<br />
: 1'''. Would any of your project ideas raise safety issues ?'''<br />
<br />
:The bacteria we are currently working with are defined as bio security stage 1 (S1). Our produced GMOs (genetically modified organisms) are therefor no proofed harm neither to mankind nor to the environment. Thus they are GRAS - generally regarded as safe.<br />
:We are working in a bio security laboratory of the stage 1 (S1). There is no possible access for the public. Every person working in the lab is trained and instructed by the safety rules for laboratories S1. <br />
<br />
: '''2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?''' <br />
:No. All GMOs are S1 biosafety level. There is no risk of hazard or biological safety issues to mankind.<br />
<br />
: ''' 3. Is there a local biosafety group, committee, or review board at your institution?'''<br />
:Yes. There is a safety officer at our institution.<br />
<br />
: '''4.Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?''' <br />
: Every team must be aware of the risk that might be based in the organisms they use and they modify. Maybe a general safety instruction, which can be downlaoded from the igem.org page will be helpful to secure the research done in this competition. Moreover a list of risk organisms, with easy acces on the igem.safety page, would be a great benefit. Additional the safety.page could be tidied up to ease the acces to the key facts. Hence a small safety summary would be usefull. Some kind of a checklist.<br />
Certainly, it has to be said that the safety of the project must be reviewed by the team performing the experiments.<br />
<br />
= Referenzen / References =<br />
<br />
ZKBS, 2010<br />
<br />
Kategorisierte Organismenliste (Deutsch) der ZKBS / List of categorized organisms (German) of the ZKBS<br />
<br />
Stellungnahme der ZKBS zur Einstufung von ''Agrobacterium tumefaciens'' (Deutsch) / Statement of the ZKBS concerning the classification of ''Agrobacterium tumefaciens'' (German)]<br />
<br />
Comment on synthetic biology of the DFG (German Research Association)/pdf, German<br />
<br />
[http://daccess-dds-ny.un.org/doc/UNDOC/GEN/G08/625/32/PDF/G0862532.pdf?OpenElement COmments on Safety UNOG Genova, 2008 / pdf, English]<br />
<br />
WHO; Laboratory Biosafety Manual (English)<br />
<br />
WHO; Laboratory biosecurity guidance (English, 2008)</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/SafetyTeam:Bielefeld-Germany/Safety2010-10-27T14:43:47Z<p>Nkessler: /* Biosecurity */</p>
<hr />
<div>{{Bielefeld_MainMenu_2010|<nowiki>http://igem-bielefeld.de/img/banner_safety.png</nowiki>}}<br />
<br />
= Biosafety and Biosecurity (English) =<br />
<br />
It is important to discriminate between Biosafety and Biosecurity in the context of working with genetically modified organisms and lab use. Biosecurity comprises the safety of the lab. The [http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_EPR_2006_6.pdf WHO] defines Biosecurity as a task of organisation and administration.The term Biosafety deals with the biological risks of the samples for the user, the enviroment and the lab. In this article, we are mostly summarizing the German law on Biosecurity and Biosafety regarding to the [http://www.bvl.bund.de/nn_495478/DE/06__Gentechnik/093__ZKBS/gentechnik__zkbs__node.html__nnn=true ZKBS (zentrale Kommission für Biologische Sicherheit)] and [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf their lists of unhazardous organisms] plus the comment on safety of the [http://www.dfg.de/download/pdf/dfg_im_profil/reden_stellungnahmen/2009/stellungnahme_synthetische_biologie.pdf DFG (Deutsche Forschungsgemeinschaft)].<br />
Furthermore we crosslinked the laws of the United States of America and the European law of Biosecurity and Biosafety in this article. We marked this statement in the text, so the reader can discrimante between the law systems. This kind of modulation makes sense, because our experimental work was done in Germany, shipped in europe (SDU Denmark) and used as a biobrick in United States of America.<br />
<br />
== Biosecurity ==<br />
[[Image:Safe labwork.jpg|200px|thumb|right|clean bench in our lab.]]<br />
<br />
<br />
In the context of "genetically engineered" it is quite important to keep unqualified personnel and trespassers away from your laboratory and your samples. In our case we secure our lab with a keypad. Every staff member needs a key to open the laboratory door. Nobody is able to open the door without the proper keys. Staff members receive a key, after beeing briefed by the safety officer on safety issues. You can only get a key for the safety stage you are working in. The labs with higher safety stages are cut off from the lower ones. Staff members need a continuative safety guide to receive a key for the higher security levels. Concludingly no unqualified personel can pass the door to the laboratory.<br />
The picture 1 shows the keypad in front of the lab.<br />
<br />
The laboratories, which are in use for our iGEM project are security stage 1 (S1). For the safety of the staff our labs are equiped with fire extinguisher, emergency showers and a heart defibrillator.<br />
<br />
== Biosafety ==<br />
[[Image:Keypad.JPG|200px|thumb|left| Picture 1: Security entrance to the lab. Every Member of the Team needs a key-token in order to get acces to the lab]]<br />
<br />
The term "biological safety" defines the effort to reduce or elimnate any potential risks caused by biotechnolgy or genetical engineering. The laboratories are scaled into four differnt saftey stages. The stage "S1" defines working with organsims and methods, which do no prooved harm neither to the enviroment or mankind. The stages rises up to Biosafety stage four, which defines working with prooved humanpathogenous organisms. The WHO defines for the United states the same safety stages, which are called risk groups [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf Manual of Biosecurity, WHO, page 1].<br />
<br />
The Biosafety is ruled by the "Zentrale Kommission für Biologische Sicherheit" (ZKBS) in Germany. The ZKBS reviewd and verified all commercial, research or privat laboraties about their safety issues. The iGEM TEAM Bielefeld is currently working with the organisms ''Agrobacterium tumefaciens'' and a derivate of ''E. coli'' K12 in a ZKBS reviewed S1-lab. The organisms strains are declared as riskfactor 1 by the german act of genetics (GenTSV, § 5; 15.06.2010). The act contains that the organisms (abstract):<br />
<br />
1.<br />
<br />
: - are no proved human-,phyto or animalpathogen<br />
: - do not contain or release organism containing to a higher risk stage<br />
: - are prooved by experiments or long term evaluation or do not proliferate in the enviroment because of biological implanted boundaries<br />
<br />
<br />
2.<br />
Organisms, which confirm the paragraph 1, are defined by the $6 of the act of genetics (GenTSV):<br />
<br />
: a) as biological safe organisms<br />
: b) organims or strains, which are contaminated by organisms or strains of a higher security level<br />
: c) organisms definied by the risk level S1 (list of organisms used for genetically engineering research)<br />
<br />
<br />
The [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf list of organisms] categorize the strains ''E. coli'' K12 and ''Agrobacterium tumefaciens'' as biological safety risk 1 (S1). The strain [http://www.bvl.bund.de/cln_007/nn_520774/DE/06__Gentechnik/093__ZKBS/01__Allg__Stellungnahmen/02__bakterien/zkbs__bakterien__agrobacterium__tumefaciens,templateId=raw,property=publicationFile.pdf/zkbs_bakterien_agrobacterium_tumefaciens.pdf ''Agrobacterium tumefaciens''] is a known phytopathogen. Because of its frequent appearence in the earth (500 bacteria in 1g earth) it is generally regarded as safe.<br />
<br />
The WHO defines "security assesments" has the highest priority for the work with organisms. This includes a proper theoretically background of the organism. Further they defined rules for good molecular practice and proper lab security [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf WHO Manual Biosafety, page 9 following]. They note that most of the security guidelines are not quite neccessary for organism of risk stage 1. Although we play by the rules and work as carefully as possible (see, what we do to protect).<br />
<br />
= Transportation of GMOs =<br />
<br />
The transportation and shipping of genetically modified organisms (GMOs) has to be done under specific safety conditions in order to lower the risk GMOs released into the enviroment. Moreover it is important that no GMOs is sent to unqualified personel or is used by unqualified personell during the shipment.<br />
<br />
As a part of the european community, the german law for export of GMOs are similar to the rules of the eurpean community law. Before you send a GMOs, you must ensure that the recipient is allowed to work with the GMO. Moreover every release of the GMOs into the enviroment must be checked by gouvermental law in the recipients country and the country of the dispatcher.<br />
<br />
The package must be signed as "shipped GMOs". Otherwise it will not be cross any boarder in the european community.<br />
<br />
<br />
= What we do to protect=<br />
<br />
:*Every single person in the lab is trained on their work<br />
:*Every working person in the lab is trained on safety issues<br />
:*We got a safety and a disinfection officer at our lab <br />
:*No unqualified personal has access to the lab<br />
:*No public access to the lab<br />
:*We reduced the risk of contamination of the environment or staff members by disinfection, autoclavation and using of protecting clothes<br />
:*In case of emergency there are telephones, fire extinguisher, defibrillator and alarm buttons around the lab<br />
:*We only used organism of the risk stage 1, which do not harm neither mankind nor the environment<br />
:*We modify a unharzadous organism with soecific, identified and characterized biobricks. So we can appraise the risk causing by the modified organisms<br />
:*Constantly low pressure, no open windows => no organism can get in or out<br />
:*Desinfection after work of all working places (everyday)<br />
:*Autoclavation of waste (solid/liquid)<br />
:*No pipetting with the mouth<br />
<br />
<br />
= Biosafety und Biosecurity (German)=<br />
<br />
Es ist bei der Inbetriebnahme und Nutzung einer Gentechnischen Anlagen darauf zu achten, dass Vorkehrungen sowohl für den Betriebsschutz (Biosecurity) als auch für die biologische Sicherheit (Biosafety) getroffen werden. Biosecurity wird von der [http://www.who.int/csr/resources/publications/biosafety/WHO_CDS_EPR_2006_6.pdf WHO] als Aufgabe der Adminstrative des Labors definiert.<br />
<br />
== Biosecurity ==<br />
Der Biosecurity wird Sorge getragen, in dem kein unbefugter Zugang zum Laborplatz hat. Die Türen sind nur per Schlüssel zu öffnen. Jeder Mitarbeiten muss eine Sicherheitsbelehrung beim Sicherheitsbeauftragten ablegen ehe er Zugang zu den Laboratorien bekommt. Die Labore sind je nach Sicherheitsgefährdung gekennzeichnet und verschlossen, so dass keine unterqualifizierte Person in ein höheres Sicherheitslevel eindringen kann. In dem vom iGEM genutzten Labor handelt es sich um die Sicherheitstufe S1. Die WHO definiert die gleichen Sicherheitsstufen, hier "risk group" genannt. [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf Manual of Biosecurity, WHO, page 1].<br />
<br />
== Biosafety ==<br />
[[Image:|200px|thumb|left| We are using a cleanbench to avoid any contamination of our samples. A proper use of the cleanbench can only be achieved by placing all the big things in the rear of the bench, by not disturbing the air circulation and by avoiding any kind of aerosoles. Use gloves for your own protection.]]<br />
<br />
Als Biologische Sicherheit (Biosafety) wird der Versuch zur Reduzierung bzw. Eleminierung potentieller Gefahren durch Biotechnologie definiert. In Deutschland werden die unterschiedlichen Organismen in vier Sicherheitstufen von S1 für keine bekannte Gefährdung bis S4 für erwiesene humanpathogene Gefährdung eingeteilt. Die zentrale Kommussion für Biologische Sicherheit (ZKBS) überprüft alle Laboratorien, ob privat, gewerblich oder für die Forschung, vor in Betriebnahme auf Sicherheitsmängel und Einhaltung der rechtlichen Grundlagen. Das iGEM Team Bielefeld nutzt die Stämme ''Agrobacterium tumefaciens'' und einen Ableger des ''E. coli'' K12 in einem ZKBS geprüften Labor der Sicherheitstufe S1 für gentechnische Arbeiten. Die Stämme sind nach dem Gentechnik Gesetz (GenTSV) § 5 Absatz 1 Satz 1 und AnhangI Nummer 1 GenTSV der Risikogruppe 1 zu zuordnen (Stand 15.06.2010). Das beinhaltet, dass die Organismen (Auszug):<br />
<br />
1.<br />
<br />
: - weder ein human-, pflanzen- noch tierpathogen sind<br />
: - keine Organismen höhere Risikogruppen abgeben<br />
: - sich durch experimentell erwiesene oder langfristig erprobte Anwendung auszeichnen oder eingebaute biologische Schranken […] die Überlebens- bzw. Vermehrungsfähigkeit in der Umwelt begrenzen<br />
<br />
2.<br />
Organismen, die die Punkte unter 1 erfüllen, sind gemäß §6 Abs. 1 in Verbindung mit Anhang II Teil A des GenTVS<br />
<br />
: a) Organismen, die als biologische Sicherheitsmaßnahme anerkannt sind (§6 Abs. 3 GenTSV)<br />
: b) Zellen oder Zelllinien, die nicht von außen mit Organismen höherer Risikogruppe (2-4) kontaminiert sind<br />
: c) Organismen der Risikogruppe 1 nach der Organismenliste (Spender- und Empfängerorganismen für gentechnische Arbeiten zu Forschungszwecken<br />
<br />
Die verwendeten Stämme ''E. coli'' K12 und ''Agrobacterium tumefaciens'' werden nach [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf dieser Organismenliste] als Risikogruppe 1 klassifiziert. [http://www.bvl.bund.de/cln_007/nn_520774/DE/06__Gentechnik/093__ZKBS/01__Allg__Stellungnahmen/02__bakterien/zkbs__bakterien__agrobacterium__tumefaciens,templateId=raw,property=publicationFile.pdf/zkbs_bakterien_agrobacterium_tumefaciens.pdf ''Agrobacterium tumefaciens''] ist zwar als phytopathogen vermerkt, ist jedoch ubiquitär, d.h. weltweit im Boden (ca. 500 Bakterien / 1 g Boden) verbreitet und wurde aus diesem Grund der Risikogruppe 1 zugeordnet. <br />
<br />
Zur Sicherheit des Personals und der Umwelt herrscht ein konstanter leichter Unterdruck in den Laboratorien und die Fenster sind geschlossen zu halten, so dass weder Organismen weder hinaus noch hineingelangen können. Zudem wird der anfallende Organismen-Müll in spezillen Säcken gesammelt, autoklaviert und entsorgt. Die Organismen werden abgetötet. Zudem werden benutzen Flächen vor und nach der Arbeit desinfiziert.<br />
<br />
= Transport von GVO =<br />
<br />
Der Transport von genetisch veränderten Organismen (GVOs) unterliegt innerhalb der Europäischen Union und außerhalb davon speziellen Sicherheitsbedingungen. Die deutschen Bestimmung sind denen der Europäischen Union ähnlich.<br />
<br />
= Unsere Schutzmaßnahmen=<br />
<br />
:*Jede Person, die sich im Labor aufhält und arbeitet ist in ihrer Arbeit ausgebildet und/oder unterwiesen<br />
:*Jede Person, die sich im Labor aufhält oder arbeitet hat eine Sicherheitsbelehrung unterlaufen<br />
:*Es gibt einen Sicherheits- und eine Desinfektionsfachkraft in unserem Labor<br />
:*Kein unqualifiziertes Personal gelangt ins Labor<br />
:*Kein unqualifiziertes Personal gelangt in eine höhere Sicherheitsstufe als ihr erlaubt<br />
:*Kein öffentlicher Zugang zum Labor möglich<br />
:*We rreduzieren das Kontaminationsrisiko für Arbeiter und Umwelt durch Autoklavieren, Desinfizierren und das Nutzen von protektiver Arbeitskleidung auf ein Minimum<br />
:*Im Falle eines Notfalls gibt es ein Telefon zum Hilferuf, Feuerlöscherr, Defibrilatoren und Feueralarmschalter in Reichweite<br />
:*Wir benutzen ausschließlich Organismen der Sicherheitsstufe 1<br />
<br />
<br />
<br />
= Short Summary =<br />
: 1'''. Would any of your project ideas raise safety issues ?'''<br />
<br />
:The bacteria we are currently working with are defined as bio security stage 1 (S1). Our produced GMOs (genetically modified organisms) are therefor no proofed harm neither to mankind nor to the environment. Thus they are GRAS - generally regarded as safe.<br />
:We are working in a bio security laboratory of the stage 1 (S1). There is no possible access for the public. Every person working in the lab is trained and instructed by the safety rules for laboratories S1. <br />
<br />
: '''2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?''' <br />
:No. All GMOs are S1 biosafety level. There is no risk of hazard or biological safety issues to mankind.<br />
<br />
: ''' 3. Is there a local biosafety group, committee, or review board at your institution?'''<br />
:Yes. There is a safety officer at our institution.<br />
<br />
: '''4.Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?''' <br />
: Every team must be aware of the risk that might be based in the organisms they use and they modify. Maybe a general safety instruction, which can be downlaoded from the igem.org page will be helpful to secure the research done in this competition. Moreover a list of risk organisms, with easy acces on the igem.safety page, would be a great benefit. Additional the safety.page could be tidied up to ease the acces to the key facts. Hence a small safety summary would be usefull. Some kind of a checklist.<br />
Certainly, it has to be said that the safety of the project must be reviewed by the team performing the experiments.<br />
<br />
= Referenzen / References =<br />
<br />
ZKBS, 2010<br />
<br />
Kategorisierte Organismenliste (Deutsch) der ZKBS / List of categorized organisms (German) of the ZKBS<br />
<br />
Stellungnahme der ZKBS zur Einstufung von ''Agrobacterium tumefaciens'' (Deutsch) / Statement of the ZKBS concerning the classification of ''Agrobacterium tumefaciens'' (German)]<br />
<br />
Comment on synthetic biology of the DFG (German Research Association)/pdf, German<br />
<br />
[http://daccess-dds-ny.un.org/doc/UNDOC/GEN/G08/625/32/PDF/G0862532.pdf?OpenElement COmments on Safety UNOG Genova, 2008 / pdf, English]<br />
<br />
WHO; Laboratory Biosafety Manual (English)<br />
<br />
WHO; Laboratory biosecurity guidance (English, 2008)</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/SafetyTeam:Bielefeld-Germany/Safety2010-10-27T14:39:39Z<p>Nkessler: /* Biosafety and Biosecurity (English) */</p>
<hr />
<div>{{Bielefeld_MainMenu_2010|<nowiki>http://igem-bielefeld.de/img/banner_safety.png</nowiki>}}<br />
<br />
= Biosafety and Biosecurity (English) =<br />
<br />
It is important to discriminate between Biosafety and Biosecurity in the context of working with genetically modified organisms and lab use. Biosecurity comprises the safety of the lab. The term Biosafety deals with the biological risks of the samples for the user, the enviroment and the lab. In this article, we are mostly summarizing the German law on Biosecurity and Biosafety regarding to the [http://www.bvl.bund.de/nn_495478/DE/06__Gentechnik/093__ZKBS/gentechnik__zkbs__node.html__nnn=true ZKBS (zentrale Kommission für Biologische Sicherheit)] and [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf their lists of unhazardous organisms] plus the comment on safety of the [http://www.dfg.de/download/pdf/dfg_im_profil/reden_stellungnahmen/2009/stellungnahme_synthetische_biologie.pdf DFG (Deutsche Forschungsgemeinschaft)].<br />
Furthermore we crosslinked the laws of the United States of America and the European law of Biosecurity and Biosafety in this article. We marked this statement in the text, so the reader can discrimante between the law systems. This kind of modulation makes sense, because our experimental work was done in Germany, shipped in europe (SDU Denmark) and used as a biobrick in United States of America.<br />
<br />
== Biosecurity ==<br />
[[Image:Safe labwork.jpg|200px|thumb|right|clean bench in our lab.]]<br />
In the context of "genetically engineered" it is quite important to keep unqualified and trespasser away from your laboratory and your samples. In our case we secure our lab with a keypad. Every staff member needs a key to open the laboratory door. Nobody is able to open the door without the proper keys. Staff members receive a key, after beeing briefed by the safety officer on safety issues. You can only get a key for the safety stage you are working in. The labs with higher safety stages are cut off from the lower ones. Staff members need a continuative safety guide to receive a key for the higher security levels. Concludingly no unqualified personel can pass the door to the laboratory.<br />
The picture 1 shows the keypad in front of the lab.<br />
<br />
The laboratories, which are in use for our iGEM project are security stage 1 (S1). For the safety of the staff our labs are equiped with fire extinguisher, emergency showers and a heart defibrillator.<br />
<br />
== Biosafety ==<br />
[[Image:Keypad.JPG|200px|thumb|left| Picture 1: Security entrance to the lab. Every Member of the Team needs a key-token in order to get acces to the lab]]<br />
<br />
The term "biological safety" defines the effort to reduce or elimnate any potential risks caused by biotechnolgy or genetical engineering. The laboratories are scaled into four differnt saftey stages. The stage "S1" defines working with organsims and methods, which do no prooved harm neither to the enviroment or mankind. The stages rises up to Biosafety stage four, which defines working with prooved humanpathogenous organisms. The WHO defines for the United states the same safety stages, which are called risk groups [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf Manual of Biosecurity, WHO, page 1].<br />
<br />
The Biosafety is ruled by the "Zentrale Kommission für Biologische Sicherheit" (ZKBS) in Germany. The ZKBS reviewd and verified all commercial, research or privat laboraties about their safety issues. The iGEM TEAM Bielefeld is currently working with the organisms ''Agrobacterium tumefaciens'' and a derivate of ''E. coli'' K12 in a ZKBS reviewed S1-lab. The organisms strains are declared as riskfactor 1 by the german act of genetics (GenTSV, § 5; 15.06.2010). The act contains that the organisms (abstract):<br />
<br />
1.<br />
<br />
: - are no proved human-,phyto or animalpathogen<br />
: - do not contain or release organism containing to a higher risk stage<br />
: - are prooved by experiments or long term evaluation or do not proliferate in the enviroment because of biological implanted boundaries<br />
<br />
<br />
2.<br />
Organisms, which confirm the paragraph 1, are defined by the $6 of the act of genetics (GenTSV):<br />
<br />
: a) as biological safe organisms<br />
: b) organims or strains, which are contaminated by organisms or strains of a higher security level<br />
: c) organisms definied by the risk level S1 (list of organisms used for genetically engineering research)<br />
<br />
<br />
The [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf list of organisms] categorize the strains ''E. coli'' K12 and ''Agrobacterium tumefaciens'' as biological safety risk 1 (S1). The strain [http://www.bvl.bund.de/cln_007/nn_520774/DE/06__Gentechnik/093__ZKBS/01__Allg__Stellungnahmen/02__bakterien/zkbs__bakterien__agrobacterium__tumefaciens,templateId=raw,property=publicationFile.pdf/zkbs_bakterien_agrobacterium_tumefaciens.pdf ''Agrobacterium tumefaciens''] is a known phytopathogen. Because of its frequent appearence in the earth (500 bacteria in 1g earth) it is generally regarded as safe.<br />
<br />
The WHO defines "security assesments" has the highest priority for the work with organisms. This includes a proper theoretically background of the organism. Further they defined rules for good molecular practice and proper lab security [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf WHO Manual Biosafety, page 9 following]. They note that most of the security guidelines are not quite neccessary for organism of risk stage 1. Although we play by the rules and work as carefully as possible (see, what we do to protect).<br />
<br />
= Transportation of GMOs =<br />
<br />
The transportation and shipping of genetically modified organisms (GMOs) has to be done under specific safety conditions in order to lower the risk GMOs released into the enviroment. Moreover it is important that no GMOs is sent to unqualified personel or is used by unqualified personell during the shipment.<br />
<br />
As a part of the european community, the german law for export of GMOs are similar to the rules of the eurpean community law. Before you send a GMOs, you must ensure that the recipient is allowed to work with the GMO. Moreover every release of the GMOs into the enviroment must be checked by gouvermental law in the recipients country and the country of the dispatcher.<br />
<br />
The package must be signed as "shipped GMOs". Otherwise it will not be cross any boarder in the european community.<br />
<br />
<br />
= What we do to protect=<br />
<br />
:*Every single person in the lab is trained on their work<br />
:*Every working person in the lab is trained on safety issues<br />
:*We got a safety and a disinfection officer at our lab <br />
:*No unqualified personal has access to the lab<br />
:*No public access to the lab<br />
:*We reduced the risk of contamination of the environment or staff members by disinfection, autoclavation and using of protecting clothes<br />
:*In case of emergency there are telephones, fire extinguisher, defibrillator and alarm buttons around the lab<br />
:*We only used organism of the risk stage 1, which do not harm neither mankind nor the environment<br />
:*We modify a unharzadous organism with soecific, identified and characterized biobricks. So we can appraise the risk causing by the modified organisms<br />
:*Constantly low pressure, no open windows => no organism can get in or out<br />
:*Desinfection after work of all working places (everyday)<br />
:*Autoclavation of waste (solid/liquid)<br />
:*No pipetting with the mouth<br />
<br />
<br />
= Biosafety und Biosecurity (German)=<br />
<br />
Es ist bei der Inbetriebnahme und Nutzung einer Gentechnischen Anlagen darauf zu achten, dass Vorkehrungen sowohl für den Betriebsschutz (Biosecurity) als auch für die biologische Sicherheit (Biosafety) getroffen werden.<br />
<br />
== Biosecurity ==<br />
Der Biosecurity wird Sorge getragen, in dem kein unbefugter Zugang zum Laborplatz hat. Die Türen sind nur per Schlüssel zu öffnen. Jeder Mitarbeiten muss eine Sicherheitsbelehrung beim Sicherheitsbeauftragten ablegen ehe er Zugang zu den Laboratorien bekommt. Die Labore sind je nach Sicherheitsgefährdung gekennzeichnet und verschlossen, so dass keine unterqualifizierte Person in ein höheres Sicherheitslevel eindringen kann. In dem vom iGEM genutzten Labor handelt es sich um die Sicherheitstufe S1. Die WHO definiert die gleichen Sicherheitsstufen, hier "risk group" genannt. [http://www.who.int/csr/resources/publications/biosafety/Biosafety7.pdf Manual of Biosecurity, WHO, page 1].<br />
<br />
== Biosafety ==<br />
[[Image:|200px|thumb|left| We are using a cleanbench to avoid any contamination of our samples. A proper use of the cleanbench can only be achieved by placing all the big things in the rear of the bench, by not disturbing the air circulation and by avoiding any kind of aerosoles. Use gloves for your own protection.]]<br />
<br />
Als Biologische Sicherheit (Biosafety) wird der Versuch zur Reduzierung bzw. Eleminierung potentieller Gefahren durch Biotechnologie definiert. In Deutschland werden die unterschiedlichen Organismen in vier Sicherheitstufen von S1 für keine bekannte Gefährdung bis S4 für erwiesene humanpathogene Gefährdung eingeteilt. Die zentrale Kommussion für Biologische Sicherheit (ZKBS) überprüft alle Laboratorien, ob privat, gewerblich oder für die Forschung, vor in Betriebnahme auf Sicherheitsmängel und Einhaltung der rechtlichen Grundlagen. Das iGEM Team Bielefeld nutzt die Stämme ''Agrobacterium tumefaciens'' und einen Ableger des ''E. coli'' K12 in einem ZKBS geprüften Labor der Sicherheitstufe S1 für gentechnische Arbeiten. Die Stämme sind nach dem Gentechnik Gesetz (GenTSV) § 5 Absatz 1 Satz 1 und AnhangI Nummer 1 GenTSV der Risikogruppe 1 zu zuordnen (Stand 15.06.2010). Das beinhaltet, dass die Organismen (Auszug):<br />
<br />
1.<br />
<br />
: - weder ein human-, pflanzen- noch tierpathogen sind<br />
: - keine Organismen höhere Risikogruppen abgeben<br />
: - sich durch experimentell erwiesene oder langfristig erprobte Anwendung auszeichnen oder eingebaute biologische Schranken […] die Überlebens- bzw. Vermehrungsfähigkeit in der Umwelt begrenzen<br />
<br />
2.<br />
Organismen, die die Punkte unter 1 erfüllen, sind gemäß §6 Abs. 1 in Verbindung mit Anhang II Teil A des GenTVS<br />
<br />
: a) Organismen, die als biologische Sicherheitsmaßnahme anerkannt sind (§6 Abs. 3 GenTSV)<br />
: b) Zellen oder Zelllinien, die nicht von außen mit Organismen höherer Risikogruppe (2-4) kontaminiert sind<br />
: c) Organismen der Risikogruppe 1 nach der Organismenliste (Spender- und Empfängerorganismen für gentechnische Arbeiten zu Forschungszwecken<br />
<br />
Die verwendeten Stämme ''E. coli'' K12 und ''Agrobacterium tumefaciens'' werden nach [http://www.bvl.bund.de/cln_027/nn_491872/DE/06__Gentechnik/00__doks__downloads/06__Register__Datenbanken/organismenliste,templateId=raw,property=publicationFile.pdf/organismenliste.pdf dieser Organismenliste] als Risikogruppe 1 klassifiziert. [http://www.bvl.bund.de/cln_007/nn_520774/DE/06__Gentechnik/093__ZKBS/01__Allg__Stellungnahmen/02__bakterien/zkbs__bakterien__agrobacterium__tumefaciens,templateId=raw,property=publicationFile.pdf/zkbs_bakterien_agrobacterium_tumefaciens.pdf ''Agrobacterium tumefaciens''] ist zwar als phytopathogen vermerkt, ist jedoch ubiquitär, d.h. weltweit im Boden (ca. 500 Bakterien / 1 g Boden) verbreitet und wurde aus diesem Grund der Risikogruppe 1 zugeordnet. <br />
<br />
Zur Sicherheit des Personals und der Umwelt herrscht ein konstanter leichter Unterdruck in den Laboratorien und die Fenster sind geschlossen zu halten, so dass weder Organismen weder hinaus noch hineingelangen können. Zudem wird der anfallende Organismen-Müll in spezillen Säcken gesammelt, autoklaviert und entsorgt. Die Organismen werden abgetötet. Zudem werden benutzen Flächen vor und nach der Arbeit desinfiziert.<br />
<br />
= Transport von GVO =<br />
<br />
Der Transport von genetisch veränderten Organismen (GVOs) unterliegt innerhalb der Europäischen Union und außerhalb davon speziellen Sicherheitsbedingungen. Die deutschen Bestimmung sind denen der Europäischen Union ähnlich.<br />
<br />
= Unsere Schutzmaßnahmen=<br />
<br />
:*Jede Person, die sich im Labor aufhält und arbeitet ist in ihrer Arbeit ausgebildet und/oder unterwiesen<br />
:*Jede Person, die sich im Labor aufhält oder arbeitet hat eine Sicherheitsbelehrung unterlaufen<br />
:*Es gibt einen Sicherheits- und eine Desinfektionsfachkraft in unserem Labor<br />
:*Kein unqualifiziertes Personal gelangt ins Labor<br />
:*Kein unqualifiziertes Personal gelangt in eine höhere Sicherheitsstufe als ihr erlaubt<br />
:*Kein öffentlicher Zugang zum Labor möglich<br />
:*We rreduzieren das Kontaminationsrisiko für Arbeiter und Umwelt durch Autoklavieren, Desinfizierren und das Nutzen von protektiver Arbeitskleidung auf ein Minimum<br />
:*Im Falle eines Notfalls gibt es ein Telefon zum Hilferuf, Feuerlöscherr, Defibrilatoren und Feueralarmschalter in Reichweite<br />
:*Wir benutzen ausschließlich Organismen der Sicherheitsstufe 1<br />
<br />
<br />
<br />
= Short Summary =<br />
: 1'''. Would any of your project ideas raise safety issues ?'''<br />
<br />
:The bacteria we are currently working with are defined as bio security stage 1 (S1). Our produced GMOs (genetically modified organisms) are therefor no proofed harm neither to mankind nor to the environment. Thus they are GRAS - generally regarded as safe.<br />
:We are working in a bio security laboratory of the stage 1 (S1). There is no possible access for the public. Every person working in the lab is trained and instructed by the safety rules for laboratories S1. <br />
<br />
: '''2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?''' <br />
:No. All GMOs are S1 biosafety level. There is no risk of hazard or biological safety issues to mankind.<br />
<br />
: ''' 3. Is there a local biosafety group, committee, or review board at your institution?'''<br />
:Yes. There is a safety officer at our institution.<br />
<br />
: '''4.Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?''' <br />
: Every team must be aware of the risk that might be based in the organisms they use and they modify. Maybe a general safety instruction, which can be downlaoded from the igem.org page will be helpful to secure the research done in this competition. Moreover a list of risk organisms, with easy acces on the igem.safety page, would be a great benefit. Additional the safety.page could be tidied up to ease the acces to the key facts. Hence a small safety summary would be usefull. Some kind of a checklist.<br />
Certainly, it has to be said that the safety of the project must be reviewed by the team performing the experiments.<br />
<br />
= Referenzen / References =<br />
<br />
ZKBS, 2010<br />
<br />
Kategorisierte Organismenliste (Deutsch) der ZKBS / List of categorized organisms (German) of the ZKBS<br />
<br />
Stellungnahme der ZKBS zur Einstufung von ''Agrobacterium tumefaciens'' (Deutsch) / Statement of the ZKBS concerning the classification of ''Agrobacterium tumefaciens'' (German)]<br />
<br />
Comment on synthetic biology of the DFG (German Research Association)/pdf, German<br />
<br />
[http://daccess-dds-ny.un.org/doc/UNDOC/GEN/G08/625/32/PDF/G0862532.pdf?OpenElement COmments on Safety UNOG Genova, 2008 / pdf, English]<br />
<br />
WHO; Laboratory Biosafety Manual (English)<br />
<br />
WHO; Laboratory biosecurity guidance (English, 2008)</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Public_Relations/Public_DiscussionTeam:Bielefeld-Germany/Public Relations/Public Discussion2010-10-27T14:30:00Z<p>Nkessler: /* Public Discussion */</p>
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= Public Discussion =<br />
<br />
Public Relation is one of the keynotes of the iGEM competition. The goal is to access science to the public. This is especially important in the German speaking area, where the broad public has above-average concerns about biotechnology. Terms like "'''''synthetic''''' biology", "'''''mutation'''''" or "'''''directed evolution'''''" have unusal bad connotations in the German culture.<br />
<br />
== Public articles ==<br />
<br />
<br />
In order to get more people in touch with the competition, we created the first german Wikipedia article. We appreciate all the slight corrections done by all the unknown wikipedia reviewers.<br />
<br />
*We created the first german iGEM wikipedia article<br />
<br />
[http://de.wikipedia.org/wiki/IGEM iGEM Wiki Article]<br />
<br />
== Public Events ==<br />
<br />
'''Science Café, Ratscafé KaffeeKunst, Bielefeld 10/26/2010:'''<br />
<br />
Access scientific issues to the public is one of our main keynotes during the project. Hence we did a public discussion concerning synthetic biology. For this porpuse we presented our project after a brief introduction of synthetic biology given by [http://de.wikipedia.org/wiki/Alfred_P%C3%BChler Prof. Alfred Pühler], one of the German pioneers of genetics. After the talks there was an open discussion hosted by the chief of editorial of the local radio broadcaster "radio bielefeld". The event took place in an ambient café in the center of Bielefeld.<br />
<br />
[[Image:Bielefeld sc3.JPG|200px|left|thumb|The audience]]<br />
<br />
Additionally to the talks, we arranged an info point with laboratory stuff like pipettes, tipes, gloves and shaking flaks. We presented a poster about our project, in order to adress more information to the auditorium. People should get an inside look on our work and the practical lab work. We observed that most of the people preferably asked their question in a personal discussion with the iGEM Team members at the info point. We tried to feed information and positive examples of synthetic biology to the people, in order to rearrange their image of synthetic biology and genetics. This is quite important, because:<br />
<br />
<u>Quote of Michael Spector </u> ([https://2010.igem.org/Security iGEM security page])<br />
<br />
''"It is a debate about words, about metaphors - it is ideology, it is not science... It is law, it is morality, it is patent stuff. Science in not a company. It is not a country. It is not even an idea. It is a process; some times it works and some times it does not. The idea we should not allow science to do it's job, because we are afraid, is really deadening and it is preventing millions of people from prospering."''<br />
<br />
Michael Spector describes the impressions people often think about in the context of genetics and biology. Moreover he mentioned a very important attribute of science. Science is not political! Unfortunately science gets political involved in the moment, it discovers improvements of everyday life or solves problems of mankind. Therefore the political aspect has to be discussed. So, frequently asked questions out of the auditorium concerning all biological, political and safety topics. Most people still fear hidden risks, especially in genetics: <br />
<br />
<br />
<br />
<br />
:*Can I control everything i am able to do in the lab?<br />
:*Is it possible for the bacteria to get out of the lab?<br />
:*What is the differnce between synthetic biology and genetics?<br />
:*How do I work on a molecular level? What kind of tools are used for cutting and pasting DNA?<br />
:*Do I really need a test on spiciness? <br />
:*Can science be more than just an interim solution? Can we rule out the source of most problems through scientific improvement? Do starving people in foreign countries really get the improvements of green biotechnology or is there only a company profit?<br />
<br />
<br />
People recognized that we are aware of possible safety and ethic issues. We pointed out that we modificated an already as security stage 1 categorized organism. So we are aware of the hazard potential of final molecular products. People get that we do not build a risky bacteria by choice. Further they noted that building DNA devices out of BioBricks is a lot of experimental work.<br />
Some people tried to lead the conversation on political topics. The connection between the potential of genetics and synthethic biology and politics is quite obvious. The research might invent something that might help other people all around the world. Therefore it has to be discussed in a global context, how these inventions can be used by anybody. <br />
It was really exciting for us to get to know the publics opinion on safety, ethics and biological issues. We discussed a long time after the talks with different persons of different social groups. <br />
<br />
<br />
<br />
<br />
[[Image:Sc.jpg|200px|right|thumb|Briefing]]<br />
[[Image:Bielefeld sc1.JPG|200px|left|thumb|Controversy]] [[Image:A1.JPG|200px|center|thumb|Prof. Alfred Pühler]]<br />
<br />
<br />
''weblinks'' und References:<br />
<br />
[http://www.bielefeld.de/de/kf/veranstaltungen/kalender/detailanzeigen.html?id=2010-01-12-11.05.45.727686 Science café advertisment]<br />
<br />
[http://www.bio-owl.de/php/start.php Advertisment on Bio OWL (14.10.2010)]<br />
<br />
[https://2010.igem.org/Security iGEM Security]<br />
<br />
[http://de.wikipedia.org/wiki/Alfred_P%C3%BChler Wikipedia Prof. Alfred Pühler (German article)]<br />
<br />
<br />
<br />
'''Waffles:'''<br />
<br />
We kill two birds with one stone by feeding the students of the bielefeld university with waffles and knoweledge. For this porpuse we build up an infopoint with a poster and sold waffles. A lot of students took their time to study our project, eat a waffle and asked questions concerning the iGEM competition and our project. It was a lot of fun for us and more important it was quite delicious. <br />
<br />
[[Image:Bielefeld Waffeln.JPG|200px|right|thumb|Feeding Bielefeld]]<br />
[[Image:Bielefeld Waffeln2.JPG|200px|left|thumb|Timo is discussing our project]] [[Image:Bielefeld Waffel2.JPG|200px|center|thumb|Simon convincing students about our projects with the help of our poster]]</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Public_Relations/Public_DiscussionTeam:Bielefeld-Germany/Public Relations/Public Discussion2010-10-27T14:19:47Z<p>Nkessler: /* Public Events */</p>
<hr />
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<html><br />
<head><br />
<style type="text/css"><br />
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margin-left:90px;<br />
margin-right:90px;<br />
}<br />
</style><br />
</head><br />
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<li><a href="/Team:Bielefeld-Germany/Public_Relations">Public Relations</a></li><br />
<li><a href="/Team:Bielefeld-Germany/Public_Relations/Fotos">Fotos</a></li><br />
<li><a href="/Team:Bielefeld-Germany/Public_Relations/Press">Press</a></li><br />
<li><a href="/Team:Bielefeld-Germany/Public_Relations/Public_Discussion">Public Discussion</a></li><br />
</div><br />
</body><br />
</html><br />
= Public Discussion =<br />
<br />
Public Relation is one of the keynotes of the iGEM competition. The goal is to access science to the public. <br />
<br />
== Public articles ==<br />
<br />
<br />
In order to get more people in touch with the competition, we created the first german Wikipedia article. We appreciate all the slight corrections done by all the unknown wikipedia reviewers.<br />
<br />
*We created the first german iGEM wikipedia article<br />
<br />
[http://de.wikipedia.org/wiki/IGEM iGEM Wiki Article]<br />
<br />
== Public Events ==<br />
<br />
'''Science Café, Ratscafé KaffeeKunst, Bielefeld 10/26/2010:'''<br />
<br />
Access scientific issues to the public is one of our main keynotes during the project. Hence we did a public discussion concerning synthetic biology. For this porpuse we presented our project after a brief introduction of synthetic biology given by [http://de.wikipedia.org/wiki/Alfred_P%C3%BChler Prof. Alfred Pühler], one of the German pioneers of genetics. After the talks there was an open discussion hosted by the chief of editorial of the local radio broadcaster "radio bielefeld". The event took place in an ambient café in the center of Bielefeld.<br />
<br />
[[Image:Bielefeld sc3.JPG|200px|left|thumb|The audience]]<br />
<br />
Additionally to the talks, we arranged an info point with laboratory stuff like pipettes, tipes, gloves and shaking flaks. We presented a poster about our project, in order to adress more information to the auditorium. People should get an inside look on our work and the practical lab work. We observed that most of the people preferably asked their question in a personal discussion with the iGEM Team members at the info point. We tried to feed information and positive examples of synthetic biology to the people, in order to rearrange their image of synthetic biology and genetics. This is quite important, because:<br />
<br />
<u>Quote of Michael Spector </u> ([https://2010.igem.org/Security iGEM security page])<br />
<br />
''"It is a debate about words, about metaphors - it is ideology, it is not science... It is law, it is morality, it is patent stuff. Science in not a company. It is not a country. It is not even an idea. It is a process; some times it works and some times it does not. The idea we should not allow science to do it's job, because we are afraid, is really deadening and it is preventing millions of people from prospering."''<br />
<br />
Michael Spector describes the impressions people often think about in the context of genetics and biology. Moreover he mentioned a very important attribute of science. Science is not political! Unfortunately science gets political involved in the moment, it discovers improvements of everyday life or solves problems of mankind. Therefore the political aspect has to be discussed. So, frequently asked questions out of the auditorium concerning all biological, political and safety topics. Most people still fear hidden risks, especially in genetics: <br />
<br />
<br />
<br />
<br />
:*Can I control everything i am able to do in the lab?<br />
:*Is it possible for the bacteria to get out of the lab?<br />
:*What is the differnce between synthetic biology and genetics?<br />
:*How do I work on a molecular level? What kind of tools are used for cutting and pasting DNA?<br />
:*Do I really need a test on spiciness? <br />
:*Can science be more than just an interim solution? Can we rule out the source of most problems through scientific improvement? Do starving people in foreign countries really get the improvements of green biotechnology or is there only a company profit?<br />
<br />
<br />
People recognized that we are aware of possible safety and ethic issues. We pointed out that we modificated an already as security stage 1 categorized organism. So we are aware of the hazard potential of final molecular products. People get that we do not build a risky bacteria by choice. Further they noted that building DNA devices out of BioBricks is a lot of experimental work.<br />
Some people tried to lead the conversation on political topics. The connection between the potential of genetics and synthethic biology and politics is quite obvious. The research might invent something that might help other people all around the world. Therefore it has to be discussed in a global context, how these inventions can be used by anybody. <br />
It was really exciting for us to get to know the publics opinion on safety, ethics and biological issues. We discussed a long time after the talks with different persons of different social groups. <br />
<br />
<br />
<br />
<br />
[[Image:Sc.jpg|200px|right|thumb|Briefing]]<br />
[[Image:Bielefeld sc1.JPG|200px|left|thumb|Controversy]] [[Image:A1.JPG|200px|center|thumb|Prof. Alfred Pühler]]<br />
<br />
<br />
''weblinks'' und References:<br />
<br />
[http://www.bielefeld.de/de/kf/veranstaltungen/kalender/detailanzeigen.html?id=2010-01-12-11.05.45.727686 Science café advertisment]<br />
<br />
[http://www.bio-owl.de/php/start.php Advertisment on Bio OWL (14.10.2010)]<br />
<br />
[https://2010.igem.org/Security iGEM Security]<br />
<br />
[http://de.wikipedia.org/wiki/Alfred_P%C3%BChler Wikipedia Prof. Alfred Pühler (German article)]<br />
<br />
<br />
<br />
'''Waffles:'''<br />
<br />
We kill two birds with one stone by feeding the students of the bielefeld university with waffles and knoweledge. For this porpuse we build up an infopoint with a poster and sold waffles. A lot of students took their time to study our project, eat a waffle and asked questions concerning the iGEM competition and our project. It was a lot of fun for us and more important it was quite delicious. <br />
<br />
[[Image:Bielefeld Waffeln.JPG|200px|right|thumb|Feeding Bielefeld]]<br />
[[Image:Bielefeld Waffeln2.JPG|200px|left|thumb|Timo is discussing our project]] [[Image:Bielefeld Waffel2.JPG|200px|center|thumb|Simon convincing students about our projects with the help of our poster]]</div>Nkesslerhttp://2010.igem.org/Template:Bielefeld_NewsTemplate:Bielefeld News2010-10-27T14:09:41Z<p>Nkessler: </p>
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<div class="calendar_day">26</div><br />
<div class="calendar_month">October</div><br />
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<div class="calendar_title">Public Discussion</div><br />
<div class="calendar_text">We had the great opportunity to present ourselves and our project in a café in Bielefeld to the general public. There, about 100 interested visitors listened to Prof. Dr. Alfred Pühler's introduction to synthetic biology and to our talk about iGEM and our MARSS project. The feedback was awesome! Prof. Dr. Pühler and we answered loads of questions of the curious audience concerning both technical and ethical aspects of our research.</div><br />
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<div class="calendar_day">19</div><br />
<div class="calendar_month">October</div><br />
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<div class="calendar_title">Waffles!</div><br />
<div class="calendar_text">As the deadline is pretty close and time is rare, we decided to bake some waffles. Besides making about 1000 delicious waffles, we took the chance to present our iGEM-project to the local students and folks of our university. Combining food and public relations was very successful... and tasty!</div><br />
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<div class="calendar_day">15</div><br />
<div class="calendar_month">October</div><br />
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<div class="calendar_title">Public Talk on October 26th</div><br />
<div class="calendar_text">On October 26th the iGEM Team Bielefeld will give a public talk about the comoetition and the project. After a brief introduction in Synthetic Biology by Prof. Dr. A. Pühler we are going to explain our iGEM approach. The Event is hosted by Bielefeld Marketing.<br /><br /><a href="http://www.bio-owl.de/pdf/Science Cafe Bielelfeld _ Bakterien 101014.pdf" target="_blank">Donwload the Flyer here.</a></div><br />
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<div class="calendar_day">11</div><br />
<div class="calendar_month">October</div><br />
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<div class="calendar_title">Radio interview on WDR5 - part 2</div><br />
<div class="calendar_text">And the show is going on. There is another interview about iGEM and our team available at the science broadcast of WDR5, called "Leonardo".<br /><br /><a href="http://www.wdr5.de/sendungen/leonardo/s/d/11.10.2010-16.05/b/leo-2-go-lange-winkelheide-unterwegs-101011.html" target="_blank">Listen to the german podcast here.</a></div><br />
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<div class="calendar_day">8</div><br />
<div class="calendar_month">October</div><br />
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<div class="calendar_title">TV team number 1</div><br />
<div class="calendar_text">Today we had a TV team in our lab - they were shooting some scenes and interviews with us. The coverage about our team will be in the science broadcasting "nano" on 3sat (german TV station) during the Jamboree in november. </div><br />
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<div class="calendar_day">24-26</div><br />
<div class="calendar_month">September</div><br />
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<div class="calendar_title">Meet the iGEM team from Odense<br />(SDU Denmark)</div><br />
<div class="calendar_text">We visited the iGEM team from the SDU Denmark in Odense this weekend. We had a lot of fun (there will be some photos soon) and agreed upon broadening our cooperation for the remaining iGEM competition (e.g. test each others BioBricks).</div><br />
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<div class="calendar_day">13</div><br />
<div class="calendar_month">September</div><br />
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<div class="calendar_title">Radio interview on WDR5 - part 1</div><br />
<div class="calendar_text">A short radio coverage in the science broadcast of WDR5 "Leonardo" about our iGEM team is broadcasted today. <a href="http://www.wdr5.de/sendungen/leonardo/s/d/13.09.2010-16.05.html" target="_blank">Link's here</a> and <a href="http://gffstream-3.vo.llnwd.net/c1/m/1284390840/radio/leonardo/wdr5_leonardo_20100913.mp3" target="_blank"/>here's the podcast</a></div><br />
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<div class="calendar_day">30</div><br />
<div class="calendar_month">August</div><br />
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<div class="calendar_title">Interview with our team on biotechnologie.tv</div><br />
<div class="calendar_text">Here is a short interview with our team in today's broadcast of biotechnologie.tv: <a href="Here is a short interview with our team in today's broadcast of biotechnologie.tv: http://www.youtube.com/watch?v=I2SA-bTERR4" target="_blank">http://www.youtube.com/watch?v=I2SA-bTERR4</a></div><br />
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<div class="calendar_day">4</div><br />
<div class="calendar_month">August</div><br />
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<div class="calendar_title">Leading article in Westfalen-Blatt</div><br />
<div class="calendar_text">A two-sided leading article about our team and synthetic biology was published in today's edition of the newspaper Westfalen-Blatt. </div><br />
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<div class="calendar_day">2</div><br />
<div class="calendar_month">August</div><br />
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<div class="calendar_title">Team up with the iGEM crew of Odense</div><br />
<div class="calendar_text">Today we attended a video conference with the iGEM-Team of the SDU. We plan to cooperate with them by sharing knowledge and - more important - we plan to get to know the people personally and to have some fun together. </div><br />
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= Outlook =<br />
== The techniques’ potential ==<br />
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<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1548942" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=1548942" width="200"><br />
<h3>Capsaicin</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1548942&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
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<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=17198" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=17198" width="200"><br />
<h3>Acetosyringone</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=17198&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
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<br />
In our project we modulated the virA-gene from ’’A. tumefaciens’’, which is encoding a receptor for the plant-hormone acetosyringone, via error prone PCR. Slight mutations may alter the receptors conformation or its binding site. Both of these can cause shifts in specificity and sensitivity. This way we created a new receptor, which is capable of sensing capsaicin. The chemical formulas of capsaicin and acetosyringone are quite similar and there are other molecules of interest, that have some of [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Phenolic_Compounds the necessary properties], too. Thus, we suggest that it is possible to modulate VirA receptors in order to make them sense a variety of highly relevant compounds.<br />
<br />
In other words, there are other candidates our sensing system may be applied to. All of them have similiar molecular structures as the vir-systems natural inducor acetosyringone. In the following we will present some of the most relevant compounds other than capsaicin, that may be upcoming targets of our research.<br />
<br />
''All of the following images of the compounds molecular structures are linked to [http://pubchem.ncbi.nlm.nih.gov/ '''The PubChem Project'''].''<br />
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== Diagnosis of pheochromocytoma and neuroblastoma (child tumors) ==<br />
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<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=21100" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=21100" width="200"><br />
<h3>Metanephrine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=21100&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=23615482" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=23615482" width="200"><br />
<h3>Vanilmandelic Acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=23615482&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
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<br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1738" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=1738" width="200"><br />
<h3>Homovanillic acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1738&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
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=== Pheochromocytoma ===<br />
Pheochromocytoma is a rare endocrine tumor originating in the medulla of adrenal glands, localized on top of the kidney. The adrenal glands produce several catecholamines, to which metanephrine and dopamine belong. These hormones regulate responses to stress, heart rate and blood pressure. In patients with pheochromocytoma these hormones are released excessively, potentially causing increased heart rate and blood pressure. Pheochromocytoma may become life threatening when not recognized and treated [http://pheopara.nichd.nih.gov/ (Pheochromocytoma and Paraganglioma website at the NIH)].<br />
<br />
These hormones can be quantified in urine and thus are important compounds of pheochromocytoma diagnostics. [http://jcem.endojournals.org/cgi/content/abstract/92/12/4602 Boyle et al. (2007)] compare different accuracies of diagnostic measures for the tumor and name urinary free metanephrine HPLC-measurement as the most sensitive and specific. Homovanillic acid and vanillyl mandelic acid are measured via HPLC aswell and are indicators for the same tumor.<br />
<br />
=== Neuroblastoma ===<br />
"Neuroblastoma is the most common extracranial solid tumor in infancy. It is an embryonal malignancy of the sympathetic nervous system arising from neuroblasts (pluripotent sympathetic cells). In the developing embryo, these cells invaginate, migrate along the neuraxis, and populate the sympathetic ganglia, adrenal medulla, and other sites. The pattern of distribution of these cells correlates with the sites of primary disease presentation." ([http://emedicine.medscape.com/article/988284-overview Lacayo, Davis 2010])<br />
<br />
Patients with high-risk disease still have very poor outcomes despite intensive therapy.<br />
<br />
"More than 90% of patients have elevated homovanillic acid (HVA) and/or vanillylmandelic acid (VMA) levels detectable in urine."<br />
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== 3-Methoxytyramine ==<br />
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<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=681" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=681" width="200"><br />
<h3>Dopamine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=681&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
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<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=11957621" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=11957621" width="200"><br />
<h3>3-Methoxytyramine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=11957621&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
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<br />
[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0013452 Sotnikova et al. (2010)] suggest, that 3-Methoxytyramine (3-MT) plays an important role as a neuromodulator. 3-MT is a metabolite of Dopamine and is reported to be a potential indicator for dopamine-dependent diseases like the parkinson disease, schizophrenia and dyskinesia. 3-Methoxytyramin as metabolic product of the hormon dopamine can be detected in human urine ([http://edoc.hu-berlin.de/oa/degruyter/cclm.1971.9.6.478.pdf Knoll ''et al.'', 1971]).<br />
Dopamine is known as happiness hormone found in increased levels after taking stimulating substances such as cocaine or amphetamines. The latter is one of the worldwide most used substitutes in sportive competition as doping. Increased levels of 3-MT analyzed in urine taken from sportives can indicate to intake of stimulating substances ([http://journals.lww.com/acsm-msse/Abstract/1980/21000/The_effect_of_amphetamines_on_selected.13.aspx CHANDLER, JOE V. and STEVEN N BLAIR, 1980]).<br />
<br />
== Dopamine ==<br />
<br />
Even though being referred to as "happiness hormone", Dopamine and its receptors are said to play key roles in numerous phsychic disorders and drug addiction. For many years now, the Dopamine hypothesis of Schizophrenia exists and evolves. It states that many symptoms of Schizophrenia correlate with a hyperactive disturbed dopaminergic signal transduction. [http://schizophreniabulletin.oxfordjournals.org/content/early/2009/03/26/schbul.sbp006.abstract Howes and Kapur (2009)] as well as [http://jop.sagepub.com/content/21/4/440.short Stone ''et al.'' (2007)] reviewed the hypothesis and its evolution.<br />
<br />
Concerning drug addiction, Dopamine and the brains reward system are of special interest in research [http://dx.doi.org/10.1016/j.brainresrev.2004.12.033 (Heidbreder, 2005)].<br />
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<br />
== Picric acid==<br />
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<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=6954" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=6954" width="200"><br />
<h3>Picric acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=6954&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
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Picrid acid (2,4,6-trinitrophenol) still occurs as contaminat in ground water and is as nitritited aromatic compound a potential explosive ([http://search.barnesandnoble.com/Explosives-Engineering/Paul-W-Cooper/e/9780471186366 Cooper PW, 1997]), ([http://onlinelibrary.wiley.com/doi/10.1002/1521-3773%2820010601%2940:11%3C2104::AID-ANIE2104%3E3.0.CO;2-%23/full Sohn H, 2001]). Existing detection methods are time consuming and even more cost intensive, so that a high selective and sensitiv application by microbial screening would be desirable.<br />
<br />
<br />
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<br />
== Summary ==<br />
The VirA receptor is a great starting point for modulations, in order to address a wide variety of highly relevant compounds. By contructing a Vir-gene based sensing system we present a ''proof of concept'' for a general microbial sensing system adaptable for various other compounds. Since the basic sensing and reporting system was successfully established in ''E. coli'', the next essential steps are to improve the mutagenesis strategy for the receptor, and finally the screening for each candidate compound.<br />
<br />
Using [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Output-signal_amplification_by_Sensitivity_Tuner_implementation a set of sensitivity tuners], the reporter output may then be optimized. Furthermore, the speed of the systems are expected to require refinement.<br />
<br />
As a result, the MARSS has great potential to deliver biological tests for medical diagnosis, drug tests for ''e.g.'' doping conctrols, analysis of food for ''e.g.''allergy triggering compounds and soil contaminating substances such as picric acid.<br />
<br />
=Weblinks=<br />
<br />
*Cooper PW, 1997, ''Explosives Engineering'', Edition 1, Wiley, John & Sons.<br />
<br />
* ''Eunice Kennedy Shriver'' National Institute of ''Child Health and Human Development'', Pheochromocytoma and Paragangliooma, 0-CH-0093<br />
<br />
*Boyle J, Davidson DF, Perry CG and Connell JMC, ''Comparison of Diagnostic Accuracy of Urinary Free Metanephrines, Vanillyl Mandelic Acid, and Catecholamines and Plasma Catecholamines for Diagnosis of Pheochromocytoma'' ,Journal of Clinical Endocrinology & Metabolism, Vol. 92, No. 12 4602-4608<br />
<br />
*http://pubchem.ncbi.nlm.nih.gov/<br />
<br />
*KNOLL E, WISSER H, STAMM D, ''in Verfahren zur Bestimmung der 3-Methoxy-4-hydroxy-phenylessigsäure (Homovanillinsäure) im Harn durch in situ Remissionsmessung nach dünnschichtchromatographischer Trennung'', Z. klin. Chem. u. klin. Biochem., 1971.<br />
<br />
*Lacayo NJ, ''Neuroblastoma'', eMedicine from webMD, 2010<br />
<br />
*Sohn H, Calhoun RM, Sailor MJ, Trogler WC, 2001, ''Detection of TNT and Picric Acid on Surfaces and in Seawater by Using Photoluminescent Polysiloles'', Angewandte Chemie, Vol.40 pp.2104–2105<br />
<br />
*Sotnikova TD,Beaulieu J-M, Espinoza S, Masri B, Zhang X., Salahpour A, Barak LS, Caron MG, Gainetdinov RR, ''The Dopamine Metabolite 3-Methoxytyramine Is a Neuromodulator'', PloSONE, 2010<br />
<br />
*CHANDLER, JOE V. and STEVEN N. BLAIR.'' The effect of amphetamines on selected physiological components related to athletic success''. Med. Sci. Sports Exercise, Vol. 12, No. 1, pp. 65-69, 1980.</div>Nkesslerhttp://2010.igem.org/Team:Bielefeld-Germany/Project/OutlookTeam:Bielefeld-Germany/Project/Outlook2010-10-27T13:19:57Z<p>Nkessler: /* Picric acid */</p>
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= Outlook =<br />
== The techniques’ potential ==<br />
<html><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1548942" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=1548942" width="200"><br />
<h3>Capsaicin</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1548942&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=17198" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=17198" width="200"><br />
<h3>Acetosyringone</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=17198&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
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<br />
In our project we modulated the virA-gene from ’’A. tumefaciens’’, which is encoding a receptor for the plant-hormone acetosyringone, via error prone PCR. Slight mutations may alter the receptors conformation or its binding site. Both of these can cause shifts in specificity and sensitivity. This way we created a new receptor, which is capable of sensing capsaicin. The chemical formulas of capsaicin and acetosyringone are quite similar and there are other molecules of interest, that have some of [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Phenolic_Compounds the necessary properties], too. Thus, we suggest that it is possible to modulate VirA receptors in order to make them sense a variety of highly relevant compounds.<br />
<br />
In other words, there are other candidates our sensing system may be applied to. All of them have similiar molecular structures as the vir-systems natural inducor acetosyringone. In the following we will present some of the most relevant compounds other than capsaicin, that may be upcoming targets of our research.<br />
<br />
''All of the following images of the compounds molecular structures are linked to [http://pubchem.ncbi.nlm.nih.gov/ '''The PubChem Project'''].''<br />
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== Diagnosis of pheochromocytoma and neuroblastoma (child tumors) ==<br />
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<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=21100" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=21100" width="200"><br />
<h3>Metanephrine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=21100&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=23615482" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=23615482" width="200"><br />
<h3>Vanilmandelic Acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=23615482&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
<br />
<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=1738" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=1738" width="200"><br />
<h3>Homovanillic acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=1738&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
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=== Pheochromocytoma ===<br />
Pheochromocytoma is a rare endocrine tumor originating in the medulla of adrenal glands, localized on top of the kidney. The adrenal glands produce several catecholamines, to which metanephrine and dopamine belong. These hormones regulate responses to stress, heart rate and blood pressure. In patients with pheochromocytoma these hormones are released excessively, potentially causing increased heart rate and blood pressure. Pheochromocytoma may become life threatening when not recognized and treated [http://pheopara.nichd.nih.gov/ (Pheochromocytoma and Paraganglioma website at the NIH)].<br />
<br />
These hormones can be quantified in urine and thus are important compounds of pheochromocytoma diagnostics. [http://jcem.endojournals.org/cgi/content/abstract/92/12/4602 Boyle et al. (2007)] compare different accuracies of diagnostic measures for the tumor and name urinary free metanephrine HPLC-measurement as the most sensitive and specific. Homovanillic acid and vanillyl mandelic acid are measured via HPLC aswell and are indicators for the same tumor.<br />
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=== Neuroblastoma ===<br />
"Neuroblastoma is the most common extracranial solid tumor in infancy. It is an embryonal malignancy of the sympathetic nervous system arising from neuroblasts (pluripotent sympathetic cells). In the developing embryo, these cells invaginate, migrate along the neuraxis, and populate the sympathetic ganglia, adrenal medulla, and other sites. The pattern of distribution of these cells correlates with the sites of primary disease presentation." ([http://emedicine.medscape.com/article/988284-overview Lacayo, Davis 2010])<br />
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Patients with high-risk disease still have very poor outcomes despite intensive therapy.<br />
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"More than 90% of patients have elevated homovanillic acid (HVA) and/or vanillylmandelic acid (VMA) levels detectable in urine."<br />
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== 3-Methoxytyramine ==<br />
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<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=11957621" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=11957621" width="200"><br />
<h3>3-Methoxytyramine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=11957621&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
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<br />
[http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0013452 Sotnikova et al. (2010)] suggest, that 3-Methoxytyramine (3-MT) plays an important role as a neuromodulator. 3-MT is a metabolite of Dopamine and is reported to be a potential indicator for dopamine-dependent diseases like the parkinson disease, schizophrenia and dyskinesia. 3-Methoxytyramin as metabolic product of the hormon dopamine can be detected in human urine ([http://edoc.hu-berlin.de/oa/degruyter/cclm.1971.9.6.478.pdf Knoll ''et al.'', 1971]).<br />
Dopamine is known as happiness hormone found in increased levels after taking stimulating substances such as cocaine or amphetamines. The latter is one of the worldwide most used substitutes in sportive competition as doping. Increased levels of 3-MT analyzed in urine taken from sportives can indicate to intake of stimulating substances ([http://journals.lww.com/acsm-msse/Abstract/1980/21000/The_effect_of_amphetamines_on_selected.13.aspx CHANDLER, JOE V. and STEVEN N BLAIR, 1980]).<br />
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== Dopamine ==<br />
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<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=681" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=681" width="200"><br />
<h3>Dopamine</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=681&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
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<br />
Even though being referred to as "happiness hormone", Dopamine and its receptors are said to play key roles in numerous phsychic disorders and drug addiction. For many years now, the Dopamine hypothesis of Schizophrenia exists and evolves. It states that many symptoms of Schizophrenia correlate with a hyperactive disturbed dopaminergic signal transduction. [http://schizophreniabulletin.oxfordjournals.org/content/early/2009/03/26/schbul.sbp006.abstract Howes and Kapur (2009)] as well as [http://jop.sagepub.com/content/21/4/440.short Stone ''et al.'' (2007)] reviewed the hypothesis and its evolution.<br />
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Concerning drug addiction, Dopamine and the brains reward system are of special interest in research [http://dx.doi.org/10.1016/j.brainresrev.2004.12.033 (Heidbreder, 2005)].<br />
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== Picric acid==<br />
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<div class="molFormulas"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/imgsrv.fcgi?t=l&cid=6954" width="200"><br />
<img src="http://pubchem.ncbi.nlm.nih.gov/image/img3d.cgi?cid=6954" width="200"><br />
<h3>Picric acid</h3><br />
<a href="http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=6954&loc=ec_rcs" target="_blank">see pubchem datasheet</a><br />
</div><br />
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Picrid acid (2,4,6-trinitrophenol) still occurs as contaminat in ground water and is as nitritited aromatic compound a potential explosive ([http://search.barnesandnoble.com/Explosives-Engineering/Paul-W-Cooper/e/9780471186366 Cooper PW, 1997]), ([http://onlinelibrary.wiley.com/doi/10.1002/1521-3773%2820010601%2940:11%3C2104::AID-ANIE2104%3E3.0.CO;2-%23/full Sohn H, 2001]). Existing detection methods are time consuming and even more cost intensive, so that a high selective and sensitiv application by microbial screening would be desirable.<br />
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<br />
== Summary ==<br />
The VirA receptor is a great starting point for modulations, in order to address a wide variety of highly relevant compounds. By contructing a Vir-gene based sensing system we present a ''proof of concept'' for a general microbial sensing system adaptable for various other compounds. Since the basic sensing and reporting system was successfully established in ''E. coli'', the next essential steps are to improve the mutagenesis strategy for the receptor, and finally the screening for each candidate compound.<br />
<br />
Using [https://2010.igem.org/Team:Bielefeld-Germany/Project/Theory#Output-signal_amplification_by_Sensitivity_Tuner_implementation a set of sensitivity tuners], the reporter output may then be optimized. Furthermore, the speed of the systems are expected to require refinement.<br />
<br />
As a result, the MARSS has great potential to deliver biological tests for medical diagnosis, drug tests for ''e.g.'' doping conctrols, analysis of food for ''e.g.''allergy triggering compounds and soil contaminating substances such as picric acid.<br />
<br />
=Weblinks=<br />
<br />
*Cooper PW, 1997, ''Explosives Engineering'', Edition 1, Wiley, John & Sons.<br />
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* ''Eunice Kennedy Shriver'' National Institute of ''Child Health and Human Development'', Pheochromocytoma and Paragangliooma, 0-CH-0093<br />
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*Boyle J, Davidson DF, Perry CG and Connell JMC, ''Comparison of Diagnostic Accuracy of Urinary Free Metanephrines, Vanillyl Mandelic Acid, and Catecholamines and Plasma Catecholamines for Diagnosis of Pheochromocytoma'' ,Journal of Clinical Endocrinology & Metabolism, Vol. 92, No. 12 4602-4608<br />
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*http://pubchem.ncbi.nlm.nih.gov/<br />
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*KNOLL E, WISSER H, STAMM D, ''in Verfahren zur Bestimmung der 3-Methoxy-4-hydroxy-phenylessigsäure (Homovanillinsäure) im Harn durch in situ Remissionsmessung nach dünnschichtchromatographischer Trennung'', Z. klin. Chem. u. klin. Biochem., 1971.<br />
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*Lacayo NJ, ''Neuroblastoma'', eMedicine from webMD, 2010<br />
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*Sohn H, Calhoun RM, Sailor MJ, Trogler WC, 2001, ''Detection of TNT and Picric Acid on Surfaces and in Seawater by Using Photoluminescent Polysiloles'', Angewandte Chemie, Vol.40 pp.2104–2105<br />
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*Sotnikova TD,Beaulieu J-M, Espinoza S, Masri B, Zhang X., Salahpour A, Barak LS, Caron MG, Gainetdinov RR, ''The Dopamine Metabolite 3-Methoxytyramine Is a Neuromodulator'', PloSONE, 2010<br />
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*CHANDLER, JOE V. and STEVEN N. BLAIR.'' The effect of amphetamines on selected physiological components related to athletic success''. Med. Sci. Sports Exercise, Vol. 12, No. 1, pp. 65-69, 1980.</div>Nkessler