http://2014.igem.org/wiki/index.php?title=Special:Contributions/Angelicb&feed=atom&limit=50&target=Angelicb&year=&month=2014.igem.org - User contributions [en]2024-03-29T08:04:32ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:ETH_Zurich/expresults/qsTeam:ETH Zurich/expresults/qs2014-10-18T02:15:19Z<p>Angelicb: /* Quorum Sensing */</p>
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<div><br/><br />
== Quorum Sensing ==<br />
<br />
For our Mosia''coli'' project, we were looking for molecular systems that allow orthogonal [https://2014.igem.org/Team:ETH_Zurich/project/background#Quorum_Sensing cell-to-cell communication] in order to implement connected [https://2014.igem.org/Team:ETH_Zurich/modeling/xor#XOR_Logic_Gate XOR logic gates]. We decided to exploit the quorum sensing systems [https://2014.igem.org/Team:ETH_Zurich/data#Gene_Circuit LuxI/LuxR, LasI/LasR, and RhlI/RhlR] in order to achieve the required orthogonal cell-to-cell communication. We developed a [https://2014.igem.org/Team:ETH_Zurich/modeling/qs model] for these cellular information processing. Even though the corresponding inducer molecules are commercially available and the systems often used, in particular in iGEM projects (e.g. [http://parts.igem.org/Part:BBa_R0062 pLux (BBa_R0062)], '[http://parts.igem.org/Frequently_Used_Parts Top 10 Most used promoters]' with 246 uses), potential crosstalk activity between the different systems may be a severe problem (e. g. [https://2013.igem.org/Team:Tokyo_Tech/Project/Ninja_State_Switching Tokyo_Tech 2013], [https://2011.igem.org/Team:Peking_S/project/wire/matrix Peking University 2011]).<br />
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In order to address this challenge, we measured a) a given promoter with its corresponding regulator and a different inducer molecule, b) a given promoter with an unspecific regulator and a particular inducer, c) a given promoter with both regulator and inducer being unspecific, and always included the correct combination of inducer molecule, regulator and promoter as a positive control. This gives in total 27 possible combinations. The output was assessed via sfGFP and measured in terms of fluorescence on microtiter-plate scale.<br />
[[File:ETH Zurich Crosstalk.png|1500px|center|thumb|'''Figure 4''' Each quorum sensing system is based on three components: a signaling molecule, a regulatory protein and a promoter. These elements are here ordered into three layers. Cross-talk evaluation can be done by comparing all combinations of those three elements. After collecting the [https://2014.igem.org/Team:ETH_Zurich/expresults experimental data] of all possible pathways, we modeled their influence.]]<br />
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===Summary of experimental results regarding quorum sensing===<br />
The following matrices serve as an overview summarizing the most significant results of our experiments to characterize crosstalk on different levels. <br />
On the horizontal top row we see the three different inducer molecules (3OC12-HSL, 3OC6-HSL, C4-HSL). In the top left corner we see the quorum sensing promoter used for all the experiments summarized in this matrix. On the vertical axis we see the three regulators ( [https://2014.igem.org/Team:ETH_Zurich/data#Used_and_Characterized_Pre-Existing_Parts LuxR, LasR, RhlR]). <br />
These matrices are giving an overview of the experimental results conducted in relation with [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing quorum sensing] and crosstalk. The graph shown in each matrix on the very top left describes the situation where the correct autoinducer molecule has bound the corresponding regulator and this complex has then induced the correct promoter. <br />
The solid lines in the graphs show the [https://2014.igem.org/Team:ETH_Zurich/modeling/qs model data], whereas the data points indicated with standard deviation show experimental data in triplicates (mean values of triplicate micro titerplate measurements). <br />
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{{:Team:ETH_Zurich/expresults/qs/tab-plux}}<br />
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{{:Team:ETH_Zurich/expresults/qs/tab-plas}}<br />
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{{:Team:ETH_Zurich/expresults/qs/tab-prhl}}<br />
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===Conclusion of crosstalk experiments===<br />
<br />
As shown in the graphs in the matrices above, we found and quantitatively characterized all three levels of crosstalk. The three levels were the following:<br />
*A given promoter with its corresponding regulator and a different inducer molecule<br />
*A given promoter with an unspecific regulator and a particular inducer<br />
*A given promoter with both regulator and inducer being unspecific<br />
Unspecific inducers binding to the regulators as well as unspecific binding of the regulator to another promoter species was observed in almost all possible combinations. <br />
To conclude, we were not able to find an orthogonal quorum sensing pair out of the three systems investigated ([https://2014.igem.org/Team:ETH_Zurich/data#Used_and_Characterized_Pre-Existing_Parts LuxI/LuxR, LasI/LasR, or RhlI/RhlR]). However, implementing the influence of these crosstalks in our [https://2014.igem.org/Team:ETH_Zurich/modeling/whole whole cell model] does not show a dramatic influence on the functionality of our system.<br />
This due to inevitable crosstalk between the three components in the system (inducer, regulator, promoter).<br />
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{{:Team:ETH_Zurich/tpl/topbutton|red}}</div>Angelicbhttp://2014.igem.org/Team:ETH_Zurich/human/outreachTeam:ETH Zurich/human/outreach2014-10-18T00:58:09Z<p>Angelicb: /* Simplifying our wiki for the public */</p>
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<div>{{:Team:ETH Zurich/tpl/head|Outreach}}<br />
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<html><article></html><br />
'''During our project we seek the dialog with people through different platforms to spread the awareness of synthetic biology and our project.'''<br />
<html></article></html><br />
<br />
<html><article></html><br />
==School visits==<br />
One side of our human practice is to share knowledge with the public. For this reason one day mid-October Stefanie and Nadine had the chance to teach students at the age of 16-17 years at a high school in Bern, Switzerland. Through these lectures, we attempted to impart the basic principles of synthetic biology and encouraged the students to participate in a discussion of the concepts presented. In these discussions we tried to focus on risks to humans, health, the environment and the public. Furthermore former iGEM projects were quickly outlined and their possibilities and potential discussed. <br />
Thus, we increase the level of awareness of synthetic biology and simultaneously teach them how to approach complex problems.<br />
<br />
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<html></article></html><br />
<html><article></html><br />
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==Open house day at the department==<br />
<br />
On the open house day held by D-BSSE, ETHZ ([http://www.bsse.ethz.ch Department of Biosystems Science and Engineering]) and the [http://www.biozentrum.unibas.ch Biozentrum] the University of Basel the public could get an idea of the scientific laboratories and the work done there. Many labs opened their doors to the public and the scientists briefed them about their daily work. We as the ETHZ iGEM team 2014 also used this platform to talk about iGEM. The team was present with a poster showing the history of iGEM, explaining the projects of previous ETHZ iGEM teams and general information about synthetic biology. Additionally, we presented a collection of photos from previous years jamboree. The goal of this day was to inform the public about synthetic biology in general and specifically about the spirit and the projects of iGEM. Many people showed great interest and have been following the project developments during the course of the project through our wiki.<br />
<br />
[[File:20140510-openhouse-8265 960px.jpg|500px|center|thumb|'''Figure 1''' Max and Nadine at the open house day at D-BSSE]]<br />
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==Science Slams==<br />
<br />
A science slam is all about presenting scientific work to a diverse group of people in a short time, in an understandable and entertaining way. <br />
<br />
<br><br />
<br />
Elise from our team has participated in two science slams. She has given her best to explain about iGEM and our project. Her task is to simplify our complex project to make it comprehensible and catchy. The first one was in Brugg AG for the Open Door day of the Fachhochschule Nordwestschweiz. The audience mainly consisted of families. The second science slam was at the theater in Basel on October 10th: you can watch the video [https://www.youtube.com/watch?v=tSS2JVcBWiI here]. The audience mainly consisted of Bachelor and Master students, interested scientists and families. This last event was covered by the media : [http://www.telebasel.ch/de/tv-archiv/&id=366875892&search=&datefrom=&dateto=&group= TeleBasel] and BAZ.<br />
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[[File:ETH Zurich_scienceslam.jpg|center|450px|thumb|'''Figure 2''' Elise at the science slam in Basel]] <br />
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<html></article></html><br />
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==iGEM ETH Zurich 2014 and social media==<br />
<br />
Today’s society relies more and more on the internet. Since, a vast group of people today widely use Social media for communication, we decided to use [http://www.facebook.com/iGEM.ETH.Zurich?fref=ts Facebook] and Twitter [http://twitter.com/ETH_iGEM twitter] to share our results, news and updates. This enabled us to reach a large group of people almost instantaneously. Both accounts refer to our wiki where more background information can be found.<br />
We think sharing the news of our project on social media increases the attention given by people to synthetic biology. We observed that for many people, the curiosity increased after seeing a post or a picture with a short description of what we had done on Facebook. We also shared fascinating videos presented by other teams and links to their wikis. Thus, we were able to increase the overall awareness of synthetic biology and iGEM.<br />
<br />
<html></article></html><br />
<html><article></html><br />
<br />
==Discourse at the after work pint==<br />
<br />
D-BSSE hosts a weekly after work pint where scientists from different groups meet up to discuss work in a relaxed environment. In one of these weekly after work pint we spoke about our project and iGEM. We took advantage of the informal environment to discuss our ideas with scientists of the department. We received encouraging and supportive feedbacks and some very useful advice and suggestions. <br />
<br />
<br />
[[File:Afterworkpint.jpg|200px|center|thumb|'''Figure 3''' Advertisement for the after work pint]]<br />
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<html></article></html><br />
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==Interview with NZZ campus==<br />
<br />
By the end of October an article about dealing with criticism related to the subject of study will appear in the [http://campus.nzz.ch NZZ campus]. The article will be based on an interview with one of our team members, Stefanie. In the interview Stefanie attempted to address the fears, ignorance or prejudices that are often associated with synthetic biology and microbiology and tried to emphasise on the importance of these fields. We believe that it is important to seek a constructive dialogue to increase awareness of biotechnology. <br />
<br />
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<html></article></html><br />
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<html><article></html><br />
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==Simplifying our wiki for the public==<br />
<br />
==Simplify: Explore synthetic biology at your own pace ==<br />
<br />
All the past teams did an incredible jobs to bring Synthetic Biology to the public, involving more and more curious people to have their first contact with the subject. While reaching for the general public and talking to people outside the academia we became aware of a fundamental disconnect. Several of the people we met were captivated by the idea of synthetic biology and after talking with us about our project they decided to independently explore past IGEM works and ideas. <br />
<br />
The main complain we registered was that they could understand the introduction pages and grasp the general idea of projects but often found challenging to move from the descriptive level to an understanding of the underlying mechanism without some external help. We think that at this stage as a community we are missing instruments that helps interested people to easily and independently explore and extend their knowledge. <br />
<br />
The Simplify buttons is our idea to create a constant and direct link between the popular science and the technical version of our work. Visitors from the general public can understand each piece of information in its descriptive version and then check how that specific element works more in detail. This should mitigate the divide between an inspiring introduction and a sudden high wall of complex biology or mathematical modeling.<br />
<br />
We are deeply interested in starting a discussion with people with deeper expertise in scientific writing and popularization in order to improve this approach and diffuse it among other team making easier for curious to explore the wide and inspiring collection of IGEM projects<br />
<br />
<br />
<br />
<html></article></html><br />
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{{:Team:ETH Zurich/tpl/foot}}</div>Angelicbhttp://2014.igem.org/Team:ETH_Zurich/human/outreachTeam:ETH Zurich/human/outreach2014-10-18T00:53:44Z<p>Angelicb: </p>
<hr />
<div>{{:Team:ETH Zurich/tpl/head|Outreach}}<br />
<br />
<br />
<html><article></html><br />
'''During our project we seek the dialog with people through different platforms to spread the awareness of synthetic biology and our project.'''<br />
<html></article></html><br />
<br />
<html><article></html><br />
==School visits==<br />
One side of our human practice is to share knowledge with the public. For this reason one day mid-October Stefanie and Nadine had the chance to teach students at the age of 16-17 years at a high school in Bern, Switzerland. Through these lectures, we attempted to impart the basic principles of synthetic biology and encouraged the students to participate in a discussion of the concepts presented. In these discussions we tried to focus on risks to humans, health, the environment and the public. Furthermore former iGEM projects were quickly outlined and their possibilities and potential discussed. <br />
Thus, we increase the level of awareness of synthetic biology and simultaneously teach them how to approach complex problems.<br />
<br />
<br />
<html></article></html><br />
<html><article></html><br />
<br />
==Open house day at the department==<br />
<br />
On the open house day held by D-BSSE, ETHZ ([http://www.bsse.ethz.ch Department of Biosystems Science and Engineering]) and the [http://www.biozentrum.unibas.ch Biozentrum] the University of Basel the public could get an idea of the scientific laboratories and the work done there. Many labs opened their doors to the public and the scientists briefed them about their daily work. We as the ETHZ iGEM team 2014 also used this platform to talk about iGEM. The team was present with a poster showing the history of iGEM, explaining the projects of previous ETHZ iGEM teams and general information about synthetic biology. Additionally, we presented a collection of photos from previous years jamboree. The goal of this day was to inform the public about synthetic biology in general and specifically about the spirit and the projects of iGEM. Many people showed great interest and have been following the project developments during the course of the project through our wiki.<br />
<br />
[[File:20140510-openhouse-8265 960px.jpg|500px|center|thumb|'''Figure 1''' Max and Nadine at the open house day at D-BSSE]]<br />
<br />
<br />
<html></article></html><br />
<html><article></html><br />
<br />
==Science Slams==<br />
<br />
A science slam is all about presenting scientific work to a diverse group of people in a short time, in an understandable and entertaining way. <br />
<br />
<br><br />
<br />
Elise from our team has participated in two science slams. She has given her best to explain about iGEM and our project. Her task is to simplify our complex project to make it comprehensible and catchy. The first one was in Brugg AG for the Open Door day of the Fachhochschule Nordwestschweiz. The audience mainly consisted of families. The second science slam was at the theater in Basel on October 10th: you can watch the video [https://www.youtube.com/watch?v=tSS2JVcBWiI here]. The audience mainly consisted of Bachelor and Master students, interested scientists and families. This last event was covered by the media : [http://www.telebasel.ch/de/tv-archiv/&id=366875892&search=&datefrom=&dateto=&group= TeleBasel] and BAZ.<br />
<br />
[[File:ETH Zurich_scienceslam.jpg|center|450px|thumb|'''Figure 2''' Elise at the science slam in Basel]] <br />
<br />
<html></article></html><br />
<html><article></html><br />
<br />
==iGEM ETH Zurich 2014 and social media==<br />
<br />
Today’s society relies more and more on the internet. Since, a vast group of people today widely use Social media for communication, we decided to use [http://www.facebook.com/iGEM.ETH.Zurich?fref=ts Facebook] and Twitter [http://twitter.com/ETH_iGEM twitter] to share our results, news and updates. This enabled us to reach a large group of people almost instantaneously. Both accounts refer to our wiki where more background information can be found.<br />
We think sharing the news of our project on social media increases the attention given by people to synthetic biology. We observed that for many people, the curiosity increased after seeing a post or a picture with a short description of what we had done on Facebook. We also shared fascinating videos presented by other teams and links to their wikis. Thus, we were able to increase the overall awareness of synthetic biology and iGEM.<br />
<br />
<html></article></html><br />
<html><article></html><br />
<br />
==Discourse at the after work pint==<br />
<br />
D-BSSE hosts a weekly after work pint where scientists from different groups meet up to discuss work in a relaxed environment. In one of these weekly after work pint we spoke about our project and iGEM. We took advantage of the informal environment to discuss our ideas with scientists of the department. We received encouraging and supportive feedbacks and some very useful advice and suggestions. <br />
<br />
<br />
[[File:Afterworkpint.jpg|200px|center|thumb|'''Figure 3''' Advertisement for the after work pint]]<br />
<br />
<br />
<html></article></html><br />
<html><article></html><br />
<br />
==Interview with NZZ campus==<br />
<br />
By the end of October an article about dealing with criticism related to the subject of study will appear in the [http://campus.nzz.ch NZZ campus]. The article will be based on an interview with one of our team members, Stefanie. In the interview Stefanie attempted to address the fears, ignorance or prejudices that are often associated with synthetic biology and microbiology and tried to emphasise on the importance of these fields. We believe that it is important to seek a constructive dialogue to increase awareness of biotechnology. <br />
<br />
<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
==Simplifying our wiki for the public==<br />
<br />
==Simplifying our wiki for the public==<br />
<br />
All the past teams did an incredible jobs to bring Synthetic Biology to the public, involving more and more curious people to have their first contact with the subject. While reaching for the general public and talking to people outside the academia we became aware of a fundamental disconnect. Several of the people we met were captivated by the idea of synthetic biology and after talking with us about our project they decided to independently explore past IGEM works and ideas. <br />
<br />
The main complain we registered was that they could understand the introduction pages and grasp the general idea of projects but often found challenging to move from the descriptive level to an understanding of the underlying mechanism without some external help. We think that at this stage as a community we are missing instruments that helps interested people to easily and independently explore and extend their knowledge. <br />
<br />
The Simplify buttons is our idea to create a constant and direct link between the popular science and the technical version of our work. Visitors from the general public can understand each piece of information in its descriptive version and then check how that specific element works more in detail. This should mitigate the divide between an inspiring introduction and a sudden high wall of complex biology or mathematical modeling.<br />
<br />
We are deeply interested in starting a discussion with people with deeper expertise in scientific writing and popularization in order to improve this approach and diffuse it among other team making easier for curious to explore the wide and inspiring collection of IGEM projects<br />
<br />
<br />
<br />
<html></article></html><br />
<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH Zurich/tpl/foot}}</div>Angelicbhttp://2014.igem.org/Team:ETH_Zurich/expresults/rrTeam:ETH Zurich/expresults/rr2014-10-17T23:47:45Z<p>Angelicb: /* Riboregulators */</p>
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<div>==Riboregulators==<br />
High basal expression from inactive inducible promoter (leakiness) is a common challenge in the implementation of robust biological control system. The problem is especially severe for systems that approximate digital Boolean logic or drive an amplified downstream response. In the first case an OFF state can be interpreted as ON resulting in incorrect computations. The second scenario leads to high levels of undesired final response. Many former iGEM teams have encountered unwanted basal expression of their genes of interest (''goi'') due to promoter leakiness ([https://2013.igem.org/Team:ETH_Zurich/Optimization ETH2013], [https://2012.igem.org/Team:Groningen/pigmentproduction Groningen2012], amongst others), resulting often in narrow optimal operation conditions for their biological devices. Since our system is based on Boolean Logic for its decisions and relies on downstream amplification to regenerate and propagate the signal as a first step we investigated strategies to improve tight gene control. Looking into the available literature we found riboregulators as a promising, highly generalizable approach to address the issue of leakiness<sup>[[Team:ETH_Zurich/project/references#refIsaacs|[32]]]</sup>. As a convenient proof of concept we coupled the system with [https://2014.igem.org/Team:ETH_Zurich/project/background#Quorum_Sensing quorum sensing modules] characterized the response and made the resulting parts available for the iGEM community in the [http://parts.igem.org/Main_Page Registry of Standard Biological Parts]. We suggest that this approach can be generalized to improve many of the inducible system contained in the registry<br />
<br />
The riboregulator systems include two parts: 1) a ''cis''-repressed RBS in front of the ''goi'', and 2) a co-expressed ''trans''-activating RNA. <br />
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<br />
To characterize this system for reliable cell-to-cell communication, we combined the [https://2014.igem.org/Team:ETH_Zurich/data#Our_Favorite_New_Characterized_Parts riboregulator part] with promoters of the quorum-input-sensing systems used by our team ([https://2014.igem.org/Team:ETH_Zurich/data#Used_and_Characterized_Pre-Existing_Parts LuxI/LuxR, LasI/LasR, and RhlI/RhlR]). In our final [https://2014.igem.org/Team:ETH_Zurich/data#Gene_Circuit_and_Parts gene circuit], the riboregulators were intended for tight control of [https://2014.igem.org/Team:ETH_Zurich/project/background#Integrases integrases]. However, to have an easily quantifiable and proportional output, we initially used GFP as our riboregulated ''goi'' and measured the output fluorescence with a [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Tecan_Infinite_M200_Pro.E2.84.A2 Tecan plate reader]. This approach is described below in the figure 1.<br />
<br />
<br />
[[File:ETH Zurich2014 Riboregulator expresults.png|center|800px|thumb| '''Figure.1''' '''Characterization of the riboregulator system.''' First, a defined amount of the incoming signal molecule 3OC6-HSL binds to LuxR. After dimerization, the complex binds to its corresponding promoter pLux. Both, the ''trans''-activator(taR12y) and the ''cis''-repressor (crR12y) together with superfolder green fluorecent protein (sfGFP), are under control of pLux. Upon induction with LuxR/3OC6-HSL, both are transcribed simultaneously and the taR12y (key symbol) opens up the crR12y sequence (lock) in front of the sfGFP mRNA. This enables translation and sfGFP production (left-hand side). Low transcription (leakiness) gives only small concentrations of mRNA, as a result 'lock' (''cis''-repressor) and 'key' (''trans''-activator) structures do not encounter each other and the translation is not enabled (right-hand side).]]<br />
The first set of experiments was conducted with cells transformed with two different pPaB plasmids. These plasmids contained either the pBR322 origin (pMB1) or the p15A origin, yielding a stable two-plasmid system with about 15-20 copies per plasmid and cell (medium-copy). Furthermore the published riboregulator sequence includes two forbidden restriction sites (EcoRI and XbaI), since the sites removal could affect the folding or functionality of the part, the original sequences were used as a control. After characterizing the original part the restriction sites were removed by two different approaches: a) multiple site-directed mutagenesis, b) blunting and ligation (Klenow and T4 DNA polymerase). The new Biobrick compatible riboregulator was tested to confirm that no severe loss-of-function occurred due to the site-removal.<br />
In a third step, the construct was transferred into the [http://parts.igem.org/Part:pSB1C3 pSB1C3] backbone (pMB1 origin, high copy number of 100-300) to be in line with the [http://parts.igem.org/Help:Standards/Assembly BioBrick Standard]. Below, you can find the corresponding graphs in figure. 2 and figure. 3, respectively. Future characterization of these constructs should allow us to explore the leakiness dependence on plasmid amount and if the tight expression is robust for different copy numbers. <br />
<br />
We have characterized promoters response of the three quorum-sensing systems. These are [https://2014.igem.org/Team:ETH_Zurich/data#Used_and_Characterized_Pre-Existing_Parts pLux, pLas, and pRhl]. We first measured the transfer functions with a non-regulated RBS in front of sfGFP, i. e. the amount of fluorescence in dependence of the inducer concentration present and compared the response with the corresponding riboregulated constructs with and without the forbidden restriction sites. All experiments were carried out in 96-well [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Tecan_Infinite_M200_Pro.E2.84.A2 microtiter plate format] for 10 h. This kinetic data-sets were also used for parameter-fitting in our [https://2014.igem.org/Team:ETH_Zurich/modeling/qs#Retrieving_degradation_rates modeling approach].<br />
<br />
We found an about 60-fold reduced basal GFP expression and an increased signal-to-noise ratio of about 6-fold when using our riboregulator construct, as compared to a non-regulated [http://parts.igem.org/Part:BBa_B0034 reference RBS (B0034)]. These results are summarized in figure 2. The BioBrick conform module was confirmed to show no loss-of-funtion due to EcoRI and XbaI site removal. Interestingly the sensitivity towards the inducer was reduced (see figure 3). Is interesting to speculate if small changes in the sequences flanking the riboregulator could be used as a way to switch the response curve.<br />
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{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 with regulator.png|center|600px]] <br />
|[[File:ETH Zurich 2014 without sites.png|center|600px]]<br />
|-<br />
|'''Figure. 2''' '''Improved signal-to-noise ratio and decreased basal GFP expression (leakiness) due to the use of a riboregulator in combination with a quorum-sensing module.''' The fluorescence per OD<sub>600</sub> is shown for the LuxR-system with a complete riboregulator over an inducer-range of 10<sup>-13</sup> M to 10<sup>-5</sup> M (dashed, light blue). An incomplete riboregulator without the ''trans''-activator shows the expected reduced sensitivity towards the inducer (dark blue). As a reference, a system with a [http://parts.igem.org/Part:BBa_B0034 non-regulated RBS (BBa_B0034)] is shown (light blue). Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction &plusmn; standard deviation. For the full data set and kinetics please [https://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [https://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.<br />
|'''Figure. 3''' '''Confirmation of the improved signal-to-noise ratio and decreased basal GFP expression (leakiness) due to the use of a riboregulator without (w/o) EcoRI and XbaI restriction sites in combination with a quorum-sensing module.''' The fluorescence per OD<sub>600</sub> is shown for the LuxR-system with an unchanged riboregulator (dashed, light blue) and a regulator with a changed sequence due to EcoRI and XbaI restriction site removal (dashed, dark blue). The inducer range covers 10<sup>-13</sup> M to 10<sup>-5</sup> M. As a reference, a system with a [http://parts.igem.org/Part:BBa_B0034 non-regulated RBS (BBa_B0034)] is shown (light blue). Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction &plusmn; standard deviation. For the full data set and kinetics please [https://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [https://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.<br />
|}</div>Angelicbhttp://2014.igem.org/Team:ETH_Zurich/expresults/rrTeam:ETH Zurich/expresults/rr2014-10-17T23:10:36Z<p>Angelicb: /* Riboregulators */</p>
<hr />
<div>==Riboregulators==<br />
<br />
As many former iGEM teams have encountered unwanted basal expression of their genes of interest (''goi'') due to promoter leakiness ([https://2013.igem.org/Team:ETH_Zurich/Optimization ETH2013], [https://2012.igem.org/Team:Groningen/pigmentproduction Groningen2012], amongst others), we investigated this severe obstacle to tightly controllable systems. We looked into the available literature and found with riboregulators a promising approach to challenge the issue of leakiness<sup>[[Team:ETH_Zurich/project/references#refIsaacs|[32]]]</sup>. In addition, we chose this approach, to study and characterize such regulators combined with [https://2014.igem.org/Team:ETH_Zurich/project/background#Quorum_Sensing quorum sensing modules] and make the resulting parts available for the iGEM community in the [http://parts.igem.org/Main_Page Registry of Standard Biological Parts].<br />
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<br />
The riboregulator systems include two parts: 1) a ''cis''-repressed RBS in front of the ''goi'', and 2) a co-expressed ''trans''-activating RNA. <br />
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<br />
To characterize this system for reliable cell-to-cell communication, we combined the [https://2014.igem.org/Team:ETH_Zurich/data#Our_Favorite_New_Characterized_Parts riboregulator part] with promoters of the quorum-input-sensing systems used by our team ([https://2014.igem.org/Team:ETH_Zurich/data#Used_and_Characterized_Pre-Existing_Parts LuxI/LuxR, LasI/LasR, and RhlI/RhlR]). In our final [https://2014.igem.org/Team:ETH_Zurich/data#Gene_Circuit_and_Parts gene circuit], the riboregulators were intended for the control of [https://2014.igem.org/Team:ETH_Zurich/project/background#Integrases integrases]. However, to have an easily quantifiable output, we used GFP as our riboregulated ''goi'' and measured the output fluorescence with a [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Tecan_Infinite_M200_Pro.E2.84.A2 Tecan plate reader]. This approach is described below in the figure 1.<br />
<br />
<br />
[[File:ETH Zurich2014 Riboregulator expresults.png|center|800px|thumb| '''Figure.1''' '''Characterization of the riboregulator system.''' First, a defined amount of the incoming signal molecule 3OC6-HSL binds to LuxR. After dimerization, the complex binds to its corresponding promoter pLux. Both, the ''trans''-activator(taR12y) and the ''cis''-repressor (crR12y) together with superfolder green fluorecent protein (sfGFP), are under control of pLux. Upon induction with LuxR/3OC6-HSL, both are transcribed simultaneously and the taR12y (key symbol) opens up the crR12y sequence (lock) in front of the sfGFP mRNA. This enables translation and sfGFP production (left-hand side). Low transcription (leakiness) gives only small concentrations of mRNA, as a result 'lock' (''cis''-repressor) and 'key' (''trans''-activator) structures do not encounter each other and the translation is not enabled (right-hand side).]]<br />
The first set of experiments was conducted with cells transformed with two different pPaB plasmids. These plasmids contained either the pBR322 origin (pMB1) or the p15A origin, yielding a stable two-plasmid system with about 15-20 copies per plasmid and cell (medium-copy). Furthermore the published riboregulator sequence includes two forbidden restriction sites (EcoRI and XbaI), since the sites removal could affect the folding or functionality of the part, the original sequences were used as a control. After characterizing the original part the restriction sites were removed by two different approaches: a) multiple site-directed mutagenesis, b) blunting and ligation (Klenow and T4 DNA polymerase). The new Biobrick compatible riboregulator was tested to confirm that no severe loss-of-function occurred due to the site-removal.<br />
In a third step, the construct was transferred into the [http://parts.igem.org/Part:pSB1C3 pSB1C3] backbone (pMB1 origin, high copy number of 100-300) to be in line with the [http://parts.igem.org/Help:Standards/Assembly BioBrick Standard]. Below, you can find the corresponding graphs in figure. 2 and figure. 3, respectively. Future characterization of these constructs should allow us to explore the leakiness dependence on plasmid amount and if the tight expression is robust for different copy numbers. <br />
<br />
We have characterized promoters response of the three quorum-sensing systems. These are [https://2014.igem.org/Team:ETH_Zurich/data#Used_and_Characterized_Pre-Existing_Parts pLux, pLas, and pRhl]. We first measured the transfer functions with a non-regulated RBS in front of sfGFP, i. e. the amount of fluorescence in dependence of the inducer concentration present and compared the response with the corresponding riboregulated constructs with and without the forbidden restriction sites. All experiments were carried out in 96-well [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Tecan_Infinite_M200_Pro.E2.84.A2 microtiter plate format] for 10 h. This kinetic data-sets were also used for parameter-fitting in our [https://2014.igem.org/Team:ETH_Zurich/modeling/qs#Retrieving_degradation_rates modeling approach].<br />
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We found an about 60-fold reduced basal GFP expression and an increased signal-to-noise ratio of about 6-fold when using our riboregulator construct, as compared to a non-regulated [http://parts.igem.org/Part:BBa_B0034 reference RBS (B0034)]. These results are summarized in figure 2. The BioBrick conform module was confirmed to show no loss-of-funtion due to EcoRI and XbaI site removal. Interestingly the sensitivity towards the inducer was reduced (see figure 3). Is interesting to speculate if small changes in the sequences flanking the riboregulator could be used as a way to switch the response curve.<br />
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<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 with regulator.png|center|600px]] <br />
|[[File:ETH Zurich 2014 without sites.png|center|600px]]<br />
|-<br />
|'''Figure. 2''' '''Improved signal-to-noise ratio and decreased basal GFP expression (leakiness) due to the use of a riboregulator in combination with a quorum-sensing module.''' The fluorescence per OD<sub>600</sub> is shown for the LuxR-system with a complete riboregulator over an inducer-range of 10<sup>-13</sup> M to 10<sup>-5</sup> M (dashed, light blue). An incomplete riboregulator without the ''trans''-activator shows the expected reduced sensitivity towards the inducer (dark blue). As a reference, a system with a [http://parts.igem.org/Part:BBa_B0034 non-regulated RBS (BBa_B0034)] is shown (light blue). Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction &plusmn; standard deviation. For the full data set and kinetics please [https://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [https://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.<br />
|'''Figure. 3''' '''Confirmation of the improved signal-to-noise ratio and decreased basal GFP expression (leakiness) due to the use of a riboregulator without (w/o) EcoRI and XbaI restriction sites in combination with a quorum-sensing module.''' The fluorescence per OD<sub>600</sub> is shown for the LuxR-system with an unchanged riboregulator (dashed, light blue) and a regulator with a changed sequence due to EcoRI and XbaI restriction site removal (dashed, dark blue). The inducer range covers 10<sup>-13</sup> M to 10<sup>-5</sup> M. As a reference, a system with a [http://parts.igem.org/Part:BBa_B0034 non-regulated RBS (BBa_B0034)] is shown (light blue). Data points are mean values of triplicate measurements in 96-well microtiter plates 200 min after induction &plusmn; standard deviation. For the full data set and kinetics please [https://2014.igem.org/Team:ETH_Zurich/contact contact] us or visit the [https://2014.igem.org/Team:ETH_Zurich/data/raw raw data] page.<br />
|}</div>Angelicbhttp://2014.igem.org/Team:ETH_Zurich/project/background/modelingTeam:ETH Zurich/project/background/modeling2014-10-17T19:18:13Z<p>Angelicb: /* Cellular Automata */</p>
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<div>== Pattern Formation ==<br />
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==='''Cellular Automata''' ===<br />
Pattern formation was formalized by Neuman in the concept of cellular automata. Following a simple pre-programmed logic rule, the state of a cell is determined by the states of three parent cells (from the previous computation round). Those logic rules are discrete computations: they can be either implementing an AND logic gate or another rule. Wolfram <sup>[[Team:ETH_Zurich/project/references#refWolfram|[11]]]</sup> elaborated a whole theory on these cellular automata.<br />
<br />
[[File:ETHZurich_CAWolfram.jpg|300px|center|thumb|Here are three different cellular automata with their logic rule.]]<br />
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According to cellular automata theory, emergent patterns offer a large panel of properties: striking examples are the rule 30 which gives an apparently random pattern and the rule 110 which has been proven to be Turing complete. With cellular automata, you cannot predict how the final pattern will look like even if you know the rule that governs its property. Thus, the intricated computations of steps poses the problem of complexity.<br />
<br/> <br />
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We chose to implement the XOR gate with two inputs, corresponding to rule 6. It can be considered as a first step towards the rule 110, which is an XOR with 3 inputs and is known to be Turing complete.<br />
<br />
[[File:ETH_Zurich_XOR_Logic_Gate.png|200px|center|thumb|Truth table of an XOR logic gate.]]</div>Angelicbhttp://2014.igem.org/Team:ETH_Zurich/project/overviewTeam:ETH Zurich/project/overview2014-10-17T18:44:24Z<p>Angelicb: /* Implementation in E. coli */</p>
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== Implementation in ''E. coli'' ==<br />
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Here you can see our circuit in action. More details on how it works are <html> <a class="circled scrolly" href="#implementation"> just below</a></html>. For a comprehensive inventory of all parts used, you can check our [https://2014.igem.org/Team:ETH_Zurich/data Data page].<br />
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<h3 style='text-align:center; font-weight:800;'>See how the information is processed to get the right output : </h3><br />
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<img class='animation' id='luxred' src='https://static.igem.org/mediawiki/2014/7/73/ETH_Zurich_Animation_luxred.gif'><br />
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<img class='animation' id='noneblue' src='https://static.igem.org/mediawiki/2014/6/6e/ETH_Zurich_Animation_noneblue.gif'><br />
<img class='animation' id='luxblue' src='https://static.igem.org/mediawiki/2014/c/c4/ETH_Zurich_Animation_luxblue.gif'><br />
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{{:Team:ETH Zurich/tpl/foot}}</div>Angelicbhttp://2014.igem.org/Team:ETH_Zurich/project/overviewTeam:ETH Zurich/project/overview2014-10-17T18:40:53Z<p>Angelicb: /* Implementation in E. coli */</p>
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== Implementation in ''E. coli'' ==<br />
<center><br />
Here you can see our circuit in action. More details on how it works are <html> <a class="circled scrolly" href="#implementation"> just below</a></html>. For a detailed inventory of all parts used, you can check our [https://2014.igem.org/Team:ETH_Zurich/data Data page].<br />
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<h3 style='text-align:center; font-weight:800;'>See how the information is processed to get the right output : </h3><br />
<center><br />
<img class='animation' id='luxred' src='https://static.igem.org/mediawiki/2014/7/73/ETH_Zurich_Animation_luxred.gif'><br />
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<img class='animation' id='noneblue' src='https://static.igem.org/mediawiki/2014/6/6e/ETH_Zurich_Animation_noneblue.gif'><br />
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{{:Team:ETH Zurich/tpl/foot}}</div>Angelicbhttp://2014.igem.org/Team:ETH_Zurich/project/overview/summarysimpleTeam:ETH Zurich/project/overview/summarysimple2014-10-17T18:37:40Z<p>Angelicb: /* Mosaicoli : from simplicity to complexity with biologic gates */</p>
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== Mosai''coli'' : from simplicity to complexity with bio''logic'' gates ==<br />
</center><br />
<br />
[[File:ETH Zurich snails.jpg|center|400px|thumb|A Textile Cone Snail ([http://en.wikipedia.org/wiki/Conus_textile ''Conus textile''])<br/>Location: Cod Hole, Great Barrier Reef, Australia<br/>[http://commons.wikimedia.org/wiki/File:Textile_cone.JPG Photographer: Richard Ling (CC-BY-SA 3.0)]]]<br />
<br />
<br />
What if these surprising patterns on sea snail shells would come from a simple rule, followed by all cells on the shell?<br />
<br />
For example <html><a href="</html>#cont<html>" class="circled scrolly" style="text-decoration:none;">in the picture below</a></html>, you can see that the same kind of pattern appears, if every cell decides on its state (white or green) depending on the state of the 2 cells above it. This rule is called an XOR gate: If one of the two cells above is ON, the cell below them is ON, if both cells above are ON or if both cells above are OFF, the cell below is OFF.<br />
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<br />
As a matter of fact, many of the complex patterns you can see in nature come from simple rules. It is the case for hurricanes, flocks of birds, neural networks etc. We call this phenomenon emergence. Emergent phenomena are not predictable from the initial conditions and this is why they surprise us. If you are interested in emergence of complexity in general, you can read more about the [[Team:ETH_Zurich/project/background|background]] of our project. <br />
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<br/><br />
<br />
We are driven by our fascination for the emergence of complexity and try to make these snail shells patterns, called Sierpinski triangles, emerge on our own grid of bacteria. To achieve this goal we encode the rule that leads to emergence of these patterns in the DNA of these bacteria. This approach, defined Synthetic Biology, is the application of engineering principles to the fundamental components of biology. Here we are using [[Team:ETH_Zurich/project/infopro|information processing principles]] in order to control the path of the information through all cells.<br />
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<br/><br />
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This way, Mosaicoli not only investigates how a complex pattern can emerge from simple rules, but also develops new tools for controlling communication between cells and implementing reliable biological circuits. For a more detailed description of our goals, go to the [[Team:ETH_Zurich/project/goals| Goals]] section. For an outlook on the possible applications of our project, jump directly to [[Team:ETH_Zurich/project/applications|Applications and outlook]].</div>Angelicb