http://2014.igem.org/wiki/index.php?title=Special:Contributions/Clormeau&feed=atom&limit=50&target=Clormeau&year=&month=2014.igem.org - User contributions [en]2024-03-28T20:11:38ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:ETH_Zurich/human/essayTeam:ETH Zurich/human/essay2014-10-18T03:42:39Z<p>Clormeau: </p>
<hr />
<div>{{:Team:ETH Zurich/tpl/head|Our insights}}<br />
<br />
<center><br />
{{:Team:ETH Zurich/tpl/scrollbutton|Reflection|red}}<br />
{{:Team:ETH Zurich/tpl/scrollbuttontworows|In|Our Project|blue}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton|Influence|green}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton|Adaptation|red}}<br />
</center><br />
<br />
{{:Team:ETH_Zurich/human/essay/answer}}<br />
<br />
{{:Team:ETH_Zurich/human/essay/project}}<br />
<br />
{{:Team:ETH_Zurich/human/essay/influence}}<br />
<br />
{{:Team:ETH_Zurich/human/essay/adaptation}}<br />
<br />
{{:Team:ETH Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/human/essay/answerTeam:ETH Zurich/human/essay/answer2014-10-18T03:42:17Z<p>Clormeau: </p>
<hr />
<div><html><article id='Reflection'></html><br />
== Our reflections ==<br />
This essay is the third of four pillars towards a better understanding of complexity. It brings elements from the survey, from the interviews, the outreach and from further reading together. Here, we reflect on how our project and science, in general, relate to these topics. <br />
<br />
<br />
Our human practice project was guided by the following questions:<br />
<br />
<br />
'''“How do people experience complexity? Which approaches do exist to approach complexity? How does complexity arise? Should people, scientists in particular, consider that subparts of a complex entity are mixed in a both ordered and unorganized way, and accept uncertainty? If yes, how can the uncertainty be taken into account? Or are simple parts strictly ordered, and complexity arises when these simple parts follow rules?”'''<br />
<br />
<br />
This questions splits up into two approaches. The first approach is needed to take into account uncertainty of intrinsic complexity of the parts we consider as of the environment. The second approach is necessary to understand the parts better in order to be able to predict results.<br />
<br />
<br />
On our way of answering the questions coming along with complexity we focused on four different components: Listening, discussing, sharing and thinking. <br />
<br />
<br />
The first component of listening was covered by a survey regarding complexity and its emergence. We listened to the public and learned about the existing ideas of complexity and how people relate to it. Something that we have observed is a trend of increasing complexity when going from non-living objects to living beings. A feature of living beings might be that they have emerging properties. This is what we experience as complex.<br />
<br />
<br />
70% of the participants of our survey have shown an interest to simplify and try to understand complexity instead of avoiding it. Another phenomenon observed was the deviation between languages. Depending on the language spoken, complexity was judged in a different way. This fact may indicate cultural variation. <br />
<br />
<br />
The survey has taught us how complexity is perceived in the public. From our survey we can conclude that in our sample population an interest in complexity exists. A point to consider is that often people are not forced to deal with complexity directly. A cell, a dog and a computer exist as items in our daily lives but most of us do not think about their complexity in relation to other items on a daily basis. Albeit we are surrounded by complexity, it is not easy for us to name and define it. <br />
<br />
<br />
Our second component involved interviews with experts from different backgrounds. This enabled us to broaden our horizons away from the complexity we are facing in our project to the complexity faced by people of other backgrounds. This exchange has enriched our project, as the professional fields of the interview partners as their approaches to complexity were very diverse. <br />
<br />
<br />
The knowledge gained from our survey, the interviews and the thoughts about them in combination we wanted to share. Sharing as our fourth pillar was done in lectures at a high school where we aimed at explaining the fundamentals of synthetic biology and how it can be a way of approaching complexity. A science slam is defined as a scientific presentation competition where scientists present their topics in a predefined timeslot and in a funny, accessible way for the open public. <br />
<br />
<br />
<br />
Our human practice has shown us the diversity of approaches of addressing complexity in our daily lives, in our professional fields, in science and when encountering complex situations. From the talk with the priest we learnt that in his opinion religion and believe help us to find a way away from complexity and towards God. Thus we can live a life in trust instead of confusion and despair. <br />
From Dr. Chikkadi and also from Mr. Veress the philosophy teacher we learned that in their point of view complexity arises from simple phenomena. <br />
<br />
From Dr. Garcia we got the following input on the perception of complexity. „Complexity is a property of a system and it can be measured. It can be shown whether a system is complex or not: for a complex system, the sum of its elements is higher than each one of them independently in superposition.“<br />
<br />
We learned that it is often useful to simplify the complexity to obtain a more accessible approach. In the process of simplification we should not forget the relationship to reality. <br />
<br />
In our outreach part we experienced how important it is to break the complexity of the own down to make it accessible for a broader public. On our way of spreading the word of synthetic biology we had many enriching encounters. We met many different people and encountered the phenomenon already described in our survey. The people we met all showed interest in trying to simplify complex problems and a will to understand what seems complex in first place. <br />
<br />
<br />
We did not find a universal answer to the question guiding our human practice project. What we found are many different approaches to address complexity arising in many different fields. This project helped us to improve our understanding of complexity as a whole and how we could profit from this profound, interdisciplinary knowledge. <br />
<br />
<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/human/essayTeam:ETH Zurich/human/essay2014-10-18T03:42:05Z<p>Clormeau: </p>
<hr />
<div>{{:Team:ETH Zurich/tpl/head|Our insights}}<br />
<br />
<center><br />
{{:Team:ETH Zurich/tpl/scrollbutton|Reflection|red}}<br />
{{:Team:ETH Zurich/tpl/scrollbuttontworows|In our|Project|blue}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton|Influence|green}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton|Adaptation|red}}<br />
</center><br />
<br />
{{:Team:ETH_Zurich/human/essay/answer}}<br />
<br />
{{:Team:ETH_Zurich/human/essay/project}}<br />
<br />
{{:Team:ETH_Zurich/human/essay/influence}}<br />
<br />
{{:Team:ETH_Zurich/human/essay/adaptation}}<br />
<br />
{{:Team:ETH Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/human/essay/influenceTeam:ETH Zurich/human/essay/influence2014-10-18T03:42:01Z<p>Clormeau: </p>
<hr />
<div><html><article id='Influence'></html><br />
== Influence of human practice on our scientific project ==<br />
<br/><br />
As soon as we agreed on a subject for our iGEM project, we started to observe patterns and complexity all among us. Our awareness of structures and compositions, as well as exchange and interaction between subunits, continuously increased. It quickly became a running gag among us to say: "Anyway, it is too complex". <br />
<br />
<br />
Here we aim at describing the influence human practice had on our scientific project. Our human practice part is intricately linked to our scientific project, since we investigated in a philosophical and sociological way the concepts we tried to reproduce biologically: The emergence of a complex pattern from simple rules. By analyzing complexity and pattern formation in depth, we learned new strategies to approach them. Our human practice project allowed us to consider our scientific project from different points of view. <br />
<br />
<br />
In our human practice project we interviewed experts of different fields, conducted a survey and sought to outreach our experience and knowledge. Thereby we learnt new strategies of how to approach complexity, but also ways to handle it. Furthermore, we found a source of motivation and reinforcement in our human practice project.<br />
<br />
<br />
In [https://2014.igem.org/Team:ETH_Zurich/human/interviews/expert2 town planning] it is crucial to not reduce a problem too much. Otherwise, the architectural intervention will be an isolated and foreign object in its surrounding. Such interventions have a high risk to fail, explained D. Übelhör. As urban systems, biological systems are highly complex. In order to understand them we tend to reduce them to single subunits. However, reductionist approaches run the risk of neglecting important information, especially when transferring behavior of subunits to the behavior of the whole. That is also what we learnt from D. Garcia, a member of the research team of [https://2014.igem.org/Team:ETH_Zurich/human/interviews/expert3 Systems Design]. He pointed out that in some situations reductionism is not only an unsuitable strategy but also unnecessary at the same time. Why should one struggle with details if they cannot explain the entity? <br />
<br />
<br />
Unexplainable noise is jointly responsible for complexity. There might be ways to reduce the noise and thus increase the predictability of systems, however, we have to accept that we do not know everything. K. Chikkadi who studies [https://2014.igem.org/Team:ETH_Zurich/human/interviews/expert5 micro- and nanosystems] expressed this fact in a scientific context, while J. Fuisz highlighted the [https://2014.igem.org/Team:ETH_Zurich/human/interviews/expert4 religious aspect] of acceptance. The trust in a higher power helps to overcome complexity. Of course, this cannot be transferred to a research project literally. Anyhow, to accept that there is no human omnipotence can protect from frustration. We cannot - but we also do not have to - solve all complexity. <br />
<br />
<br />
To not get lost while interpreting experimental data, we followed the advice of the [https://2014.igem.org/Team:ETH_Zurich/human/interviews/expert6 physicist and philosopher] E. Klein: we limited our ambitions in order to become efficient. Many times it is advisable to proceed in small steps rather than aiming at the full monty immediately. However, pedantic planning is always highly important especially when dealing with complexity, emphasized C. Veress, the [https://2014.igem.org/Team:ETH_Zurich/human/interviews/expert1 philosophy teacher]. Looking back on the past weeks and months we can only agree with that. Spending time with planning instead of rushing quite often saves time, money and work.<br />
<br />
<br />
While [https://2014.igem.org/Team:ETH_Zurich/human/outreach/ spreading the word about synthetic biology] we experienced the importance of a simple, comprehensible language. In fact, good communication and articulation is crucial for functional teamwork. Explaining concepts to high school students, interested visitors at the open house or during a science slam increased our awareness of the importance of communication and trained our skills at the same time.<br />
<br />
<br />
Many participants of our [https://2014.igem.org/Team:ETH_Zurich/human/survey survey on emergence on complexity] encouraged us in our research project. They agreed on the importance of analyzing complexity and the emergence of patterns. We were very happy to get such a positive feedback and motivated to continue our studies.<br />
<br />
<br />
<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/human/essay/adaptationTeam:ETH Zurich/human/essay/adaptation2014-10-18T03:41:50Z<p>Clormeau: </p>
<hr />
<div><html><article id='Adaptation'></html><br />
<br />
== Going further ==<br />
<br />
In this part, we analyze the methods we used to answer our human practice question. We hope that this analysis will provide iGEMers material to think about their policy and practice project. <br />
<br/><br />
<br/><br />
;'''Survey'''<br />
:The internet as a media allowed us to reach more than 800 persons willing to participate in our survey. We thank each participant for its support! We are also grateful for all the people that provided us with additional comments; no matter whether these were suggestions for publications to read, complaints about the complexity of the survey or encouraging words. <br />
<br />
<br />
:We called for other iGEM team's solidarity for the survey. The idea of winning a badge for the wiki was appealing to many of them. The teams were motivated to participate as often as time allowed, since we introduced a two badge system (a colorful badge for 20 answers and a golden badge for 50 answers). In fact, two teams, namely the [https://2014.igem.org/Team:Hannover iGEM team Hannover] and the [https://2014.igem.org/Team:SDU-Denmark iGEM team SDU Denmark], handed in more than 50 filled in surveys. We are impressed and grateful!<br />
<br />
:We did not define a target population, as we were too ambitious and wanted to cover all subgroups of society. However, the data set we collected is strongly biased towards students. It could be interesting to particularly design a survey concerning this part of the population.<br />
<br />
:Our goal was to learn more about the people's understanding of complexity and emergence. Even though our survey included spaces for own answers, most people chose one of the preformed answers. It would be interesting to encourage people to express themselves, so as to receive more individual answers. One option could be to ask people on the streets to answer one question, e.g.: "what is complexity for you?". Audio or video records of the interviews could be used to supplement the survey study. It would be strongly dependent on the country of investigation but it could give some unexpected insights on the topic. Moreover, it would give an occasion to increase awareness of the public on synthetic biology.<br />
<br />
;'''Interviews'''<br />
:First, we would like to thank every person that accepted to answer our questions.<br />
<br />
:As one of our goals was to investigate how complexity is taken into account in different fields, interviews seemed to be the best way to get a broad overview of different fields. We achieved to get seven personal and professional points of view about complexity. It was highly interesting to explore in details different conceptions of complexity in scientific and non-scientific fields.<br />
<br />
:An interesting interview is not self-evident. It is advisable to practice the dialogue before the real interview so as to make full use of the meeting with the interviewee. Recording the interviews could provide raw material of interest.<br />
<br />
;'''Outreach'''<br />
:We managed to have diverse outreach projects targeting several population groups, from high school students to elder people.<br />
<br />
:If outreach is an interesting task in itself, it could be advisable to stick to a more strictly defined theme or topic to be more consistent. For instance, focusing on activities for a certain age group or on media outreach through television, audio and newsletter could give a coherent whole and give rise to multiple interpretations.<br />
<br />
;'''Literature Work'''<br />
:Defining a question to answer is a difficult starting point. One has to screen literature hoping to find interesting, promising hints. Being mentored in the first part could avoid a team to go into a dead end.<br />
<br />
:As many iGEM teams we were extremely busy with our project. Reading of scientific literature on the topic of human practice is probably one of the first things to be missed out. Doing a weekly journal club on human practice could broaden the horizon of all team members and allow them to discover interesting new points of view.<br />
<br />
<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/human/essay/adaptationTeam:ETH Zurich/human/essay/adaptation2014-10-18T03:40:39Z<p>Clormeau: </p>
<hr />
<div><html><article id='adaptation'></html><br />
<br />
== Going further ==<br />
<br />
In this part, we analyze the methods we used to answer our human practice question. We hope that this analysis will provide iGEMers material to think about their policy and practice project. <br />
<br/><br />
<br/><br />
;'''Survey'''<br />
:The internet as a media allowed us to reach more than 800 persons willing to participate in our survey. We thank each participant for its support! We are also grateful for all the people that provided us with additional comments; no matter whether these were suggestions for publications to read, complaints about the complexity of the survey or encouraging words. <br />
<br />
<br />
:We called for other iGEM team's solidarity for the survey. The idea of winning a badge for the wiki was appealing to many of them. The teams were motivated to participate as often as time allowed, since we introduced a two badge system (a colorful badge for 20 answers and a golden badge for 50 answers). In fact, two teams, namely the [https://2014.igem.org/Team:Hannover iGEM team Hannover] and the [https://2014.igem.org/Team:SDU-Denmark iGEM team SDU Denmark], handed in more than 50 filled in surveys. We are impressed and grateful!<br />
<br />
:We did not define a target population, as we were too ambitious and wanted to cover all subgroups of society. However, the data set we collected is strongly biased towards students. It could be interesting to particularly design a survey concerning this part of the population.<br />
<br />
:Our goal was to learn more about the people's understanding of complexity and emergence. Even though our survey included spaces for own answers, most people chose one of the preformed answers. It would be interesting to encourage people to express themselves, so as to receive more individual answers. One option could be to ask people on the streets to answer one question, e.g.: "what is complexity for you?". Audio or video records of the interviews could be used to supplement the survey study. It would be strongly dependent on the country of investigation but it could give some unexpected insights on the topic. Moreover, it would give an occasion to increase awareness of the public on synthetic biology.<br />
<br />
;'''Interviews'''<br />
:First, we would like to thank every person that accepted to answer our questions.<br />
<br />
:As one of our goals was to investigate how complexity is taken into account in different fields, interviews seemed to be the best way to get a broad overview of different fields. We achieved to get seven personal and professional points of view about complexity. It was highly interesting to explore in details different conceptions of complexity in scientific and non-scientific fields.<br />
<br />
:An interesting interview is not self-evident. It is advisable to practice the dialogue before the real interview so as to make full use of the meeting with the interviewee. Recording the interviews could provide raw material of interest.<br />
<br />
;'''Outreach'''<br />
:We managed to have diverse outreach projects targeting several population groups, from high school students to elder people.<br />
<br />
:If outreach is an interesting task in itself, it could be advisable to stick to a more strictly defined theme or topic to be more consistent. For instance, focusing on activities for a certain age group or on media outreach through television, audio and newsletter could give a coherent whole and give rise to multiple interpretations.<br />
<br />
;'''Literature Work'''<br />
:Defining a question to answer is a difficult starting point. One has to screen literature hoping to find interesting, promising hints. Being mentored in the first part could avoid a team to go into a dead end.<br />
<br />
:As many iGEM teams we were extremely busy with our project. Reading of scientific literature on the topic of human practice is probably one of the first things to be missed out. Doing a weekly journal club on human practice could broaden the horizon of all team members and allow them to discover interesting new points of view.<br />
<br />
<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/human/essay/influenceTeam:ETH Zurich/human/essay/influence2014-10-18T03:40:23Z<p>Clormeau: </p>
<hr />
<div><html><article id='influence'></html><br />
== Influence of human practice on our scientific project ==<br />
<br/><br />
As soon as we agreed on a subject for our iGEM project, we started to observe patterns and complexity all among us. Our awareness of structures and compositions, as well as exchange and interaction between subunits, continuously increased. It quickly became a running gag among us to say: "Anyway, it is too complex". <br />
<br />
<br />
Here we aim at describing the influence human practice had on our scientific project. Our human practice part is intricately linked to our scientific project, since we investigated in a philosophical and sociological way the concepts we tried to reproduce biologically: The emergence of a complex pattern from simple rules. By analyzing complexity and pattern formation in depth, we learned new strategies to approach them. Our human practice project allowed us to consider our scientific project from different points of view. <br />
<br />
<br />
In our human practice project we interviewed experts of different fields, conducted a survey and sought to outreach our experience and knowledge. Thereby we learnt new strategies of how to approach complexity, but also ways to handle it. Furthermore, we found a source of motivation and reinforcement in our human practice project.<br />
<br />
<br />
In [https://2014.igem.org/Team:ETH_Zurich/human/interviews/expert2 town planning] it is crucial to not reduce a problem too much. Otherwise, the architectural intervention will be an isolated and foreign object in its surrounding. Such interventions have a high risk to fail, explained D. Übelhör. As urban systems, biological systems are highly complex. In order to understand them we tend to reduce them to single subunits. However, reductionist approaches run the risk of neglecting important information, especially when transferring behavior of subunits to the behavior of the whole. That is also what we learnt from D. Garcia, a member of the research team of [https://2014.igem.org/Team:ETH_Zurich/human/interviews/expert3 Systems Design]. He pointed out that in some situations reductionism is not only an unsuitable strategy but also unnecessary at the same time. Why should one struggle with details if they cannot explain the entity? <br />
<br />
<br />
Unexplainable noise is jointly responsible for complexity. There might be ways to reduce the noise and thus increase the predictability of systems, however, we have to accept that we do not know everything. K. Chikkadi who studies [https://2014.igem.org/Team:ETH_Zurich/human/interviews/expert5 micro- and nanosystems] expressed this fact in a scientific context, while J. Fuisz highlighted the [https://2014.igem.org/Team:ETH_Zurich/human/interviews/expert4 religious aspect] of acceptance. The trust in a higher power helps to overcome complexity. Of course, this cannot be transferred to a research project literally. Anyhow, to accept that there is no human omnipotence can protect from frustration. We cannot - but we also do not have to - solve all complexity. <br />
<br />
<br />
To not get lost while interpreting experimental data, we followed the advice of the [https://2014.igem.org/Team:ETH_Zurich/human/interviews/expert6 physicist and philosopher] E. Klein: we limited our ambitions in order to become efficient. Many times it is advisable to proceed in small steps rather than aiming at the full monty immediately. However, pedantic planning is always highly important especially when dealing with complexity, emphasized C. Veress, the [https://2014.igem.org/Team:ETH_Zurich/human/interviews/expert1 philosophy teacher]. Looking back on the past weeks and months we can only agree with that. Spending time with planning instead of rushing quite often saves time, money and work.<br />
<br />
<br />
While [https://2014.igem.org/Team:ETH_Zurich/human/outreach/ spreading the word about synthetic biology] we experienced the importance of a simple, comprehensible language. In fact, good communication and articulation is crucial for functional teamwork. Explaining concepts to high school students, interested visitors at the open house or during a science slam increased our awareness of the importance of communication and trained our skills at the same time.<br />
<br />
<br />
Many participants of our [https://2014.igem.org/Team:ETH_Zurich/human/survey survey on emergence on complexity] encouraged us in our research project. They agreed on the importance of analyzing complexity and the emergence of patterns. We were very happy to get such a positive feedback and motivated to continue our studies.<br />
<br />
<br />
<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/human/essay/projectTeam:ETH Zurich/human/essay/project2014-10-18T03:40:00Z<p>Clormeau: </p>
<hr />
<div><html><article id='In'></html><br />
== Complexity in our project ==<br />
;'''Wet lab'''<br />
<br />
:At the very beginning the work in the wet lab seemed straightforward. The way we planed our time was ambitious, but soon after the start we faced first problems. In different experiments we could observe cross-talk between the different quorum sensing molecules. In a next step we focused on quantifying the [https://2014.igem.org/Team:ETH_Zurich/expresults crosstalk]. We found interactions between the subparts (AHLs, AHL binding molecules and promoters) on different levels. The various ways of crosstalk are characteristics of a complex system with emerging features. The whole is more than the sum of its parts; living beings, even if not multicellular have various naturally emergent properties. <br />
<br />
:In fact the observation of emergence was one of the central topics of our project. The techniques of rapid prototyping and 3D-printing followed by PDMS molding allowed us to use custom-designed [https://2014.igem.org/Team:ETH_Zurich/lab/chip millifluidic chips]. The chip with the [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate bead] grid on it enabled us to observe the phenomenon of guided [https://2014.igem.org/Team:ETH_Zurich/project/background/emergence#Emergence.2C_Complexity_and_Simplicity emergence]. In this case we do not have a simplification but a contextualisation. The design of our experiments aimed at the consideration of different interactive factors rather than a complete reduction of complexity. Since the knowledge of biological systems is limited such an approach seems to be nearby.<br />
<br />
:During our project we experienced that many times biological systems are not behaving as expected. While in some cases mistakes of the person planning or conducting the experiment were discovered, we could many times not localize a causation for the unexpected behavior. This taught us about the influence of parameters that have a priori not been taken into account or that are beyond our control. A cell is an open system that interacts with its environment; it is possible to reduce the number of factors influencing the system, but is not possible to eradicate them all.<br />
<br />
<br />
;'''Modeling'''<br />
: A model is always a simplistic representation of reality. Using standard descriptions, like chemical reactions and mass action, we analytically derived each formula we wanted to fit. This derivation was only possible thanks to some assumptions. As this [https://2014.igem.org/Team:ETH_Zurich/modeling/int page] shows, every assumption was carefully made and thought about. The process of simplification was put into question because our human practice project tends to investigate why simplification is powerful but not enough to understand the surrounding world. In our case, a reductionist approach was indispensable. Thus we tried to legitimate every assumption in a biological way. <br />
<br />
: Our modeling part focuses on parameter fitting (see our [https://2014.igem.org/Team:ETH_Zurich/modeling/parameters parameter page]). We used a classical deterministic model and tried to fit it to the experiments. Matching the reality level with the description level is a complicated task, as there is no optimal match. Differences and similarities between experiments and simulations give insights on where emergent phenomena could happen.<br />
<br />
: The fact that randomness is a property of complex systems motivated us to derive a stochastic model. Some biological events, like binding, are typically stochastic phenomena.<br />
<br />
: We opted for an engineered representation (see the [https://2014.igem.org/Team:ETH_Zurich/project/infopro information processing page]), thus to divide our systems into simpler submodules seemed obvious. We took the fact into account that the different levels of description can give several insights on how the systems work. The decomposition into interacting submodules (see the [https://2014.igem.org/Team:ETH_Zurich/modeling/overview modeling overview page]) was crucial to explain the complex problem.<br />
<br />
<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/human/essay/answerTeam:ETH Zurich/human/essay/answer2014-10-18T03:39:17Z<p>Clormeau: </p>
<hr />
<div><html><article id='Reflections'></html><br />
== Our reflections ==<br />
This essay is the third of four pillars towards a better understanding of complexity. It brings elements from the survey, from the interviews, the outreach and from further reading together. Here, we reflect on how our project and science, in general, relate to these topics. <br />
<br />
<br />
Our human practice project was guided by the following questions:<br />
<br />
<br />
'''“How do people experience complexity? Which approaches do exist to approach complexity? How does complexity arise? Should people, scientists in particular, consider that subparts of a complex entity are mixed in a both ordered and unorganized way, and accept uncertainty? If yes, how can the uncertainty be taken into account? Or are simple parts strictly ordered, and complexity arises when these simple parts follow rules?”'''<br />
<br />
<br />
This questions splits up into two approaches. The first approach is needed to take into account uncertainty of intrinsic complexity of the parts we consider as of the environment. The second approach is necessary to understand the parts better in order to be able to predict results.<br />
<br />
<br />
On our way of answering the questions coming along with complexity we focused on four different components: Listening, discussing, sharing and thinking. <br />
<br />
<br />
The first component of listening was covered by a survey regarding complexity and its emergence. We listened to the public and learned about the existing ideas of complexity and how people relate to it. Something that we have observed is a trend of increasing complexity when going from non-living objects to living beings. A feature of living beings might be that they have emerging properties. This is what we experience as complex.<br />
<br />
<br />
70% of the participants of our survey have shown an interest to simplify and try to understand complexity instead of avoiding it. Another phenomenon observed was the deviation between languages. Depending on the language spoken, complexity was judged in a different way. This fact may indicate cultural variation. <br />
<br />
<br />
The survey has taught us how complexity is perceived in the public. From our survey we can conclude that in our sample population an interest in complexity exists. A point to consider is that often people are not forced to deal with complexity directly. A cell, a dog and a computer exist as items in our daily lives but most of us do not think about their complexity in relation to other items on a daily basis. Albeit we are surrounded by complexity, it is not easy for us to name and define it. <br />
<br />
<br />
Our second component involved interviews with experts from different backgrounds. This enabled us to broaden our horizons away from the complexity we are facing in our project to the complexity faced by people of other backgrounds. This exchange has enriched our project, as the professional fields of the interview partners as their approaches to complexity were very diverse. <br />
<br />
<br />
The knowledge gained from our survey, the interviews and the thoughts about them in combination we wanted to share. Sharing as our fourth pillar was done in lectures at a high school where we aimed at explaining the fundamentals of synthetic biology and how it can be a way of approaching complexity. A science slam is defined as a scientific presentation competition where scientists present their topics in a predefined timeslot and in a funny, accessible way for the open public. <br />
<br />
<br />
<br />
Our human practice has shown us the diversity of approaches of addressing complexity in our daily lives, in our professional fields, in science and when encountering complex situations. From the talk with the priest we learnt that in his opinion religion and believe help us to find a way away from complexity and towards God. Thus we can live a life in trust instead of confusion and despair. <br />
From Dr. Chikkadi and also from Mr. Veress the philosophy teacher we learned that in their point of view complexity arises from simple phenomena. <br />
<br />
From Dr. Garcia we got the following input on the perception of complexity. „Complexity is a property of a system and it can be measured. It can be shown whether a system is complex or not: for a complex system, the sum of its elements is higher than each one of them independently in superposition.“<br />
<br />
We learned that it is often useful to simplify the complexity to obtain a more accessible approach. In the process of simplification we should not forget the relationship to reality. <br />
<br />
In our outreach part we experienced how important it is to break the complexity of the own down to make it accessible for a broader public. On our way of spreading the word of synthetic biology we had many enriching encounters. We met many different people and encountered the phenomenon already described in our survey. The people we met all showed interest in trying to simplify complex problems and a will to understand what seems complex in first place. <br />
<br />
<br />
We did not find a universal answer to the question guiding our human practice project. What we found are many different approaches to address complexity arising in many different fields. This project helped us to improve our understanding of complexity as a whole and how we could profit from this profound, interdisciplinary knowledge. <br />
<br />
<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/lab/beadTeam:ETH Zurich/lab/bead2014-10-18T03:28:59Z<p>Clormeau: /* Loading the Chip */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Beads}}<br />
<br />
<html><article></html><br />
==Overview==<br />
<br />
{|<br />
[[File:ETH2014_BeadLogo.jpg|left|300px|thumb|'''Figure 1''' Na<sup>2+</sup> alginate is extruded through a needle device]]<br />
Alginates are present as structural components in both the cell walls of brown algae and the capsules of soil bacteria. However, commercially available alginate is mainly extracted from algae. The polysaccharides find a broad application in various fields: in textile printing, in food industry or in research. The chemical features of alginates allow immobilization of macromolecules and cells, thus, the compound is commonly used in biotechnology, biomedicine and pharmacy<sup>[[Team:ETH_Zurich/project/references#refEmergence|[24]]]</sup>. In our project we encapsulated bacteria in alginate beads so as to ensure local separation of the different units (here strains) and directional communication between them. These prerequisites are required for controlled pattern formation.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
<br />
==Properties==<br />
<br />
{|<br />
[[File:ETH2014_BeadColonies.jpg|right|300px|thumb| '''Figure 2''' The green spots are ''E. coli'' colonies inside an alginate bead]]<br />
Na<sup>2+</sup> alginate is a viscous liquid, however, upon addition multivalent ions such as Ca<sup>2+</sup> cross-linking of the polysaccharides occurs. Thus gelling of alginate can be achieved by the addition of a Ca<sup>2+</sup>. For encapsulation the cells or macromolecules are added to Na<sup>2+</sup> alginate and subsequently immobilized during the gelling process. In fact, the encapsulation process is mild and compatible with most living cells. The high porosity of the ionically cross-linked polysaccharide lattice allows diffusion of nutrients and other substances into and out of the bead. This property of Ca<sup>2+</sup> alginate allows cultivation of bacteria inside beads and does not prevent communication via small molecules between colonies of different beads. Substances such as phosphate or EDTA are sequestrating Ca<sup>2+</sup> and thus destabilizing the alginate gel<sup>[[Team:ETH_Zurich/project/references#refEmergence|[25]]]</sup>. This fact should be considered when choosing the cultivation medium. Beads are permeable for our signal molecules HSL, which allows bead to bead communication.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
==Production==<br />
{|<br />
[[File:ETH2014_BeadsProduction.jpg|left|250px|thumb| '''Figure 3''' Bead production. Alginate droplets are gelling in the CaCl<sub>2</sub> solution in the beaker placed under the needle device]]<br />
Bacteria are resuspended and diluted in NaCl-solution (0.9 % in H<sub>2</sub>O) so as to achieve the desired cell density. Here, we aimed at a comparably high concentration of 10<sup>7</sup> bacteria per bead (3 mm diameter). The resuspension was added to alginate (2.5%) to reach a alginate concentration of 2%. The viscous solution is filled into a syringe containing an appropriate needle device and extruded at a constant, slow velocity. Ideally, the droplets should fall form a height of approximately 30 cm into a 100 mM CaCl<sub>2</sub> solution. In the CaCl<sub>2</sub> solution gelling of the alginate droplets will occur instantaneously. To avoid excessive salt stress for the bacteria the beads should be transferred to a 10 mM CaCl<sub>2</sub> solution. <br />
<br><br />
<br><br />
A more detailed protocol can be found [https://2014.igem.org/Team:ETH_Zurich/lab/protocols here.]<br />
|}<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
==Loading the Chip==<br />
{|<br />
[[File:ETH_Zurich_2014_Beadloding_with_mobile_phone.jpg|right|350px|thumb| '''Figure 4''' Loading of the chip. The cell phone light facilitates to locate the beads loaded in the PDMS chip]]<br />
The beads are better visible in liquid when light shines from the side. So it is easier to load the [https://2014.igem.org/Team:ETH_Zurich/lab/chip the chip] with help of a cell phone light. An inoculation loop with a diameter of around 2 mm can be used to conveniently transfer the beads.<br />
|}<br />
<html></article></html><br />
<br />
<br />
<br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/lab/beadTeam:ETH Zurich/lab/bead2014-10-18T03:28:52Z<p>Clormeau: /* Production */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Beads}}<br />
<br />
<html><article></html><br />
==Overview==<br />
<br />
{|<br />
[[File:ETH2014_BeadLogo.jpg|left|300px|thumb|'''Figure 1''' Na<sup>2+</sup> alginate is extruded through a needle device]]<br />
Alginates are present as structural components in both the cell walls of brown algae and the capsules of soil bacteria. However, commercially available alginate is mainly extracted from algae. The polysaccharides find a broad application in various fields: in textile printing, in food industry or in research. The chemical features of alginates allow immobilization of macromolecules and cells, thus, the compound is commonly used in biotechnology, biomedicine and pharmacy<sup>[[Team:ETH_Zurich/project/references#refEmergence|[24]]]</sup>. In our project we encapsulated bacteria in alginate beads so as to ensure local separation of the different units (here strains) and directional communication between them. These prerequisites are required for controlled pattern formation.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
<br />
==Properties==<br />
<br />
{|<br />
[[File:ETH2014_BeadColonies.jpg|right|300px|thumb| '''Figure 2''' The green spots are ''E. coli'' colonies inside an alginate bead]]<br />
Na<sup>2+</sup> alginate is a viscous liquid, however, upon addition multivalent ions such as Ca<sup>2+</sup> cross-linking of the polysaccharides occurs. Thus gelling of alginate can be achieved by the addition of a Ca<sup>2+</sup>. For encapsulation the cells or macromolecules are added to Na<sup>2+</sup> alginate and subsequently immobilized during the gelling process. In fact, the encapsulation process is mild and compatible with most living cells. The high porosity of the ionically cross-linked polysaccharide lattice allows diffusion of nutrients and other substances into and out of the bead. This property of Ca<sup>2+</sup> alginate allows cultivation of bacteria inside beads and does not prevent communication via small molecules between colonies of different beads. Substances such as phosphate or EDTA are sequestrating Ca<sup>2+</sup> and thus destabilizing the alginate gel<sup>[[Team:ETH_Zurich/project/references#refEmergence|[25]]]</sup>. This fact should be considered when choosing the cultivation medium. Beads are permeable for our signal molecules HSL, which allows bead to bead communication.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
==Production==<br />
{|<br />
[[File:ETH2014_BeadsProduction.jpg|left|250px|thumb| '''Figure 3''' Bead production. Alginate droplets are gelling in the CaCl<sub>2</sub> solution in the beaker placed under the needle device]]<br />
Bacteria are resuspended and diluted in NaCl-solution (0.9 % in H<sub>2</sub>O) so as to achieve the desired cell density. Here, we aimed at a comparably high concentration of 10<sup>7</sup> bacteria per bead (3 mm diameter). The resuspension was added to alginate (2.5%) to reach a alginate concentration of 2%. The viscous solution is filled into a syringe containing an appropriate needle device and extruded at a constant, slow velocity. Ideally, the droplets should fall form a height of approximately 30 cm into a 100 mM CaCl<sub>2</sub> solution. In the CaCl<sub>2</sub> solution gelling of the alginate droplets will occur instantaneously. To avoid excessive salt stress for the bacteria the beads should be transferred to a 10 mM CaCl<sub>2</sub> solution. <br />
<br><br />
<br><br />
A more detailed protocol can be found [https://2014.igem.org/Team:ETH_Zurich/lab/protocols here.]<br />
|}<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
===Loading the Chip===<br />
{|<br />
[[File:ETH_Zurich_2014_Beadloding_with_mobile_phone.jpg|right|350px|thumb| '''Figure 4''' Loading of the chip. The cell phone light facilitates to locate the beads loaded in the PDMS chip]]<br />
The beads are better visible in liquid when light shines from the side. So it is easier to load the [https://2014.igem.org/Team:ETH_Zurich/lab/chip the chip] with help of a cell phone light. An inoculation loop with a diameter of around 2 mm can be used to conveniently transfer the beads.<br />
|}<br />
<html></article></html><br />
<br />
<br />
<br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/lab/beadTeam:ETH Zurich/lab/bead2014-10-18T03:28:43Z<p>Clormeau: /* Properties */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Beads}}<br />
<br />
<html><article></html><br />
==Overview==<br />
<br />
{|<br />
[[File:ETH2014_BeadLogo.jpg|left|300px|thumb|'''Figure 1''' Na<sup>2+</sup> alginate is extruded through a needle device]]<br />
Alginates are present as structural components in both the cell walls of brown algae and the capsules of soil bacteria. However, commercially available alginate is mainly extracted from algae. The polysaccharides find a broad application in various fields: in textile printing, in food industry or in research. The chemical features of alginates allow immobilization of macromolecules and cells, thus, the compound is commonly used in biotechnology, biomedicine and pharmacy<sup>[[Team:ETH_Zurich/project/references#refEmergence|[24]]]</sup>. In our project we encapsulated bacteria in alginate beads so as to ensure local separation of the different units (here strains) and directional communication between them. These prerequisites are required for controlled pattern formation.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
<br />
==Properties==<br />
<br />
{|<br />
[[File:ETH2014_BeadColonies.jpg|right|300px|thumb| '''Figure 2''' The green spots are ''E. coli'' colonies inside an alginate bead]]<br />
Na<sup>2+</sup> alginate is a viscous liquid, however, upon addition multivalent ions such as Ca<sup>2+</sup> cross-linking of the polysaccharides occurs. Thus gelling of alginate can be achieved by the addition of a Ca<sup>2+</sup>. For encapsulation the cells or macromolecules are added to Na<sup>2+</sup> alginate and subsequently immobilized during the gelling process. In fact, the encapsulation process is mild and compatible with most living cells. The high porosity of the ionically cross-linked polysaccharide lattice allows diffusion of nutrients and other substances into and out of the bead. This property of Ca<sup>2+</sup> alginate allows cultivation of bacteria inside beads and does not prevent communication via small molecules between colonies of different beads. Substances such as phosphate or EDTA are sequestrating Ca<sup>2+</sup> and thus destabilizing the alginate gel<sup>[[Team:ETH_Zurich/project/references#refEmergence|[25]]]</sup>. This fact should be considered when choosing the cultivation medium. Beads are permeable for our signal molecules HSL, which allows bead to bead communication.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
===Production===<br />
{|<br />
[[File:ETH2014_BeadsProduction.jpg|left|250px|thumb| '''Figure 3''' Bead production. Alginate droplets are gelling in the CaCl<sub>2</sub> solution in the beaker placed under the needle device]]<br />
Bacteria are resuspended and diluted in NaCl-solution (0.9 % in H<sub>2</sub>O) so as to achieve the desired cell density. Here, we aimed at a comparably high concentration of 10<sup>7</sup> bacteria per bead (3 mm diameter). The resuspension was added to alginate (2.5%) to reach a alginate concentration of 2%. The viscous solution is filled into a syringe containing an appropriate needle device and extruded at a constant, slow velocity. Ideally, the droplets should fall form a height of approximately 30 cm into a 100 mM CaCl<sub>2</sub> solution. In the CaCl<sub>2</sub> solution gelling of the alginate droplets will occur instantaneously. To avoid excessive salt stress for the bacteria the beads should be transferred to a 10 mM CaCl<sub>2</sub> solution. <br />
<br><br />
<br><br />
A more detailed protocol can be found [https://2014.igem.org/Team:ETH_Zurich/lab/protocols here.]<br />
|}<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
===Loading the Chip===<br />
{|<br />
[[File:ETH_Zurich_2014_Beadloding_with_mobile_phone.jpg|right|350px|thumb| '''Figure 4''' Loading of the chip. The cell phone light facilitates to locate the beads loaded in the PDMS chip]]<br />
The beads are better visible in liquid when light shines from the side. So it is easier to load the [https://2014.igem.org/Team:ETH_Zurich/lab/chip the chip] with help of a cell phone light. An inoculation loop with a diameter of around 2 mm can be used to conveniently transfer the beads.<br />
|}<br />
<html></article></html><br />
<br />
<br />
<br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T03:27:35Z<p>Clormeau: </p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<center><br />
{{:Team:ETH Zurich/tpl/scrollbuttontworows|Mold|Design|red}}<br />
{{:Team:ETH Zurich/tpl/scrollbuttontworows|3D|Printing|blue}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton|Preparation|green}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton|movies|red}}<br />
</center><br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
Our project aims for the biological implementation of [https://2014.igem.org/Team:ETH_Zurich/project/background/modeling#Cellular_Automata cellular automata], so we had to find a way to create a regular grid of cells with a defined neighborhood as shown in the figures below. On the left side a classical cellular automata is depicted (see figure 1), on the right side an outline of [https://2014.igem.org/Team:ETH_Zurich/project/overview#Implementation_in_E._coli our biological version] consisting of a grid-like polydimethylsiloxane (PDMS) chip filled with [https://2014.igem.org/Team:ETH_Zurich/lab/bead cell colonies encapsulated in alginate beads] (see figure 2).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[File:ETH_Zurich_2014_theoretical_grid.png|300px|thumb|right|'''Figure 2 Outline of a PDMS-made grid loaded with cells confined in alginate beads for the biological implementation of cellular automata''' (ON state=sfGFP/green, OFF state=white).]]<br />
<br />
<br />
In the following, we have investigated the combination of additive manufacturing (3D-printing) and PDMS chip fabrication for applications in synthetic biology. This rapid prototyping approach allowed us to update our chips continuously according to new [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel insights from modeling] or the wet lab and in particular to avoid more intricate photolitographic approaches, which generally require clean room access, relatively expensive raw materials, and in depth knowledge of etching techniques.<br />
<br><br />
<br><br />
As a result, we are convinced that the tinkering with 3D-printing for mold creation is more economical for our applications and measurements. Also it is perfectly in line with the do-it-yourself spirit of iGEM.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article id='Mold'></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
Our custom-made plates and molds were design using a common personal computer (MacBook Air, 13-inch, early 2014, 1.7 GHz Intel Core i7, 8 GB 1600 MHz) and a 3D computer aided design (CAD) software package that is freely available for Mac OS X 10.9.4 ([http://www.123dapp.com/design Autodesk123D Design]). The CAD models were exported as mesh files (.stl) to the 3D printer's software ([http://www.makerbot.com/support/makerware/troubleshooting/ MakerWare]). The dimensions of the device-structures were usually between 1 mm and 5 mm, falling in the range of millifluidics<sup>[[Team:ETH_Zurich/project/references#refKitson|[31]]]</sup>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''Figure 3-b''' The final mold design for our millifluid PDMS chip used for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
'''All mesh files designed during the project will be made available at the [http://3dprint.nih.gov/ NIH 3D Print Exchange] under the category 'Custom Labware' via our [http://3dprint.nih.gov/users/ethzurichigem2014 ETH_Zurich_iGEM2014] account.<br />
'''<br />
<br />
<br />
<br />
{{:Team:ETH Zurich/tpl/topbutton|red}}<br />
<html></article></html><br />
<br />
<html><article id='3D'></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
The mold designs were printed with a commercial 3D-printer (2nd generation MakerBot Replicator with MakerWare software; [http://www.makerbot.com MakerBotIndustries], Brooklyn, US; 5th generation US$2'899) with acrylonitrile butadiene styrene ([http://en.wikipedia.org/wiki/Acrylonitrile_butadiene_styrene ABS], a copolymer of acrylonitrile, butadiene, and styrene). The maximum object size printable is [mm]: 225 x 145 x 150. The precision and minimum feature size are given as [mm]: 0.011 (XY-axis), 0.0025 (Z-axis); and 0.4 (XY-axis), 0.2 (Z-axis) respectively. The printing time varied with the size of the mold but was usually below 4 hours. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
All fabricated structures were ready to use after removing the support structures and did not require additional surface treatments like sonication, curing, painting or silanization. The molds were then directly used for PDMS chip production. In addition, custom made black 96-well plates (connected wells for diffusion assays, plate reader compatible) were printed but found to be leaky over time. The material costs of the molds were in the range of US$2 to US$4 and for the 96-well plates below US$8 (about US$160 per kg of ABS). The maximum resistance to continuous heat is given as 90 ⁰C <sup>[[Team:ETH_Zurich/project/references#refCRC|[23]]]</sup>, as a result autoclaving at 121 ⁰C was not feasible and led to deformation (see the box in figure 5-a).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''Figure 5-c''' Printed 96-well plate, pairs of wells (edge length of 5 mm) are connected by channels of varied length (1 mm to 6 mm).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
<br />
{{:Team:ETH Zurich/tpl/topbutton|blue}}<br />
<br />
<html></article></html><br />
<br />
<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
For the fabrication of millifluidic-chips raw PDMS (Dow Corning Sylgard 184) was prepared by mixing base and curing agent in 10:1 proportion. The PDMS solution was mixed vigorously and degassed (desiccator connected to vacuum) until no further bubble formation could be observed. Subsequently the mixture was poured over the mold and cured in an vacuum oven over night at RT. While the first mold design separated insufficiently from the PDMS due to an inappropriate aspect ratio (see figures 6-a and 7-a), all other PDMS chips were easily removed without additional aids and placed in clear plastic trays (86 x 128 mm; OmniTrays, Thermo Scientific). All the mold shown below were at least used once before the pictures were taken. The PDMS wells were then filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Complex_bead_medium_.28CB_medium.29 CB medium] and loaded with cells encapsulated in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads].<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''Figure 6-b''' Mold design for a diffusion assay with two connected chambers (edge length of 4 mm) with varied channel length (1 mm to 4 mm).<br />
|'''Figure 6-c''' The final mold design for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''Figure 7-a''' The very first PDMS chip. As the close-up shows, the outer parts are well defined, but the middle part did not separate from the mold due to an inappropriate aspect ratio.<br />
|'''Figure 7-b''' PDMS chip for diffusion assays with two connected chambers (edge length of 4 mm) and varied channel length (1 mm to 4 mm).<br />
|'''Figure 7-c''' The final PDMS chip for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
<br />
<br />
{{:Team:ETH Zurich/tpl/topbutton|green}}<br />
<html></article></html><br />
<br />
<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
Below you find an overview of the time-lapse movies taken during the summer. In the very first trial the wells were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB agar], holes were punched with a pipette tip and filled with highlighter-ink ([http://en.wikipedia.org/wiki/Pyranine pyranine]) to visualize diffusion (see video 1). Later, different set-ups were tested: chambers filled with liquid [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB medium] separated by solidified 2% agarose in the connecting channel and [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in liquid [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Complex_bead_medium_.28CB_medium.29 CB medium]. We continued with the 'alginate beads in liquid medium' set-up, as it yielded the most promising intermediate results, and could then finally show cell-to-cell communication of bacteria confined in beads on our millifluid chip.<br />
<br />
<br />
In all videos shown imaging was implemented with a [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Biostep_Dark-Hood_DH-50.E2.84.A2__and_the_Argus-X1.E2.84.A2_software Biostep Dark-Hood DH-50 (Argus X1 software)] fitted with a Canon EOS 500D DSLR camera and a fluorescence filter (545 nm filter). Pictures were taken every 2 min at an excitation wavelength of 470 nm with the standard Canon EOS Utility software. Time-lapse movies were created with Adobe After Effects CC software. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video1|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/c/c7/ETH_Zurich_2014_two_wells_1st_test_with_highlighter.mp4</html>}}<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video2|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/1/18/ETH_Zurich_2014_two_wells_liquid_culture_small.mp4</html>}}<br />
|-<br />
|'''Video 1 The very first diffusion experiment with fluorescent highlighter ink ([http://en.wikipedia.org/wiki/Pyranine pyranine]).''' The wells of the PDMS chip were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB agar]. About 5 μL ink were added in a punched whole on one side of the two wells. ~4500x faster than real-time.<br />
|'''Video 2 Diffusion experiment with liquid cultures.''' The wells of the PDMS chip were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB medium], separated by solidified 2% agarose in the channel. The bottom well contained 3OC6-HSL (~1 mM), the top well ''E. coli'' cells with sfGFP under the control of pLux. ~4500x faster than real-time.<br />
|-<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video3|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/7/7d/ETH_Zurich_2014_AHL_bead_sensor_bead.mp4</html>}}<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video4|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/8/8c/ETH_Zurich_2014_sender_receiver_beads_small.mp4</html>}}<br />
|-<br />
|'''Video 3 Diffusion experiment with [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained beads with 3OC6-HSL (~1 mM), the top well ''E. coli'' cells with [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Sensor_Constructs sfGFP under the control of pLux] confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained ''E. coli'' [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Producer_Constructs cells expressing LuxI], which catalyzes the production of 3OC6-HSL; the top well contained ''E. coli'' cells with [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Sensor_Constructs sfGFP under the control of pLux]. All cells were confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads). ~3450x faster than real-time.<br />
|-<br />
|colspan="2"|{{:Team:ETH_Zurich/Templates/Video|width=600px|id=video5|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/a/a9/ETH_Zurich_2014_signal_propagation.mp4</html>}}<br />
|-<br />
|colspan="2"|'''Video 5 Row wise, self-propagating [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] of ''E. coli'' cells confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads) on a [https://2014.igem.org/Team:ETH_Zurich/lab/chip custom-made millifluidic PDMS chip].''' All cells contained [https://2014.igem.org/Team:ETH_Zurich/expresults/rr#Riboregulators riboregulated] sfGFP followed by [http://parts.igem.org/Part:BBa_C0161 LuxI (BBa_C0161)] together under the control of the [http://parts.igem.org/Part:BBa_R0062 pLux promoter (BBa_R0062)], and [http://parts.igem.org/Part:BBa_J23100 constitutively (BBa_J23100)] expressed [http://parts.igem.org/Part:BBa_C0062 LuxR (BBa_C0062)]. LuxI catalyzes the production of the autoinducer 3OC6-HSL, which is then diffusing from cell to cell. For initialization, the cells in one bead of the top row were induced with 3OC6-HSL before encapsulation. 1750x faster than real-time, the video starts 7 h after the initiation of the experiment.<br />
|}<br />
<br />
<br />
{{:Team:ETH Zurich/tpl/topbutton|red}}<br />
<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T03:27:15Z<p>Clormeau: </p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<center><br />
{{:Team:ETH Zurich/tpl/scrollbuttontworows|Mold|Design|red}}<br />
{{:Team:ETH Zurich/tpl/scrollbuttontworows|3D|Printing|blue}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton|Preparation|green}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton|movies|red}}<br />
</center><br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
Our project aims for the biological implementation of [https://2014.igem.org/Team:ETH_Zurich/project/background/modeling#Cellular_Automata cellular automata], so we had to find a way to create a regular grid of cells with a defined neighborhood as shown in the figures below. On the left side a classical cellular automata is depicted (see figure 1), on the right side an outline of [https://2014.igem.org/Team:ETH_Zurich/project/overview#Implementation_in_E._coli our biological version] consisting of a grid-like polydimethylsiloxane (PDMS) chip filled with [https://2014.igem.org/Team:ETH_Zurich/lab/bead cell colonies encapsulated in alginate beads] (see figure 2).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[File:ETH_Zurich_2014_theoretical_grid.png|300px|thumb|right|'''Figure 2 Outline of a PDMS-made grid loaded with cells confined in alginate beads for the biological implementation of cellular automata''' (ON state=sfGFP/green, OFF state=white).]]<br />
<br />
<br />
In the following, we have investigated the combination of additive manufacturing (3D-printing) and PDMS chip fabrication for applications in synthetic biology. This rapid prototyping approach allowed us to update our chips continuously according to new [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel insights from modeling] or the wet lab and in particular to avoid more intricate photolitographic approaches, which generally require clean room access, relatively expensive raw materials, and in depth knowledge of etching techniques.<br />
<br><br />
<br><br />
As a result, we are convinced that the tinkering with 3D-printing for mold creation is more economical for our applications and measurements. Also it is perfectly in line with the do-it-yourself spirit of iGEM.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article id='Mold'></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
Our custom-made plates and molds were design using a common personal computer (MacBook Air, 13-inch, early 2014, 1.7 GHz Intel Core i7, 8 GB 1600 MHz) and a 3D computer aided design (CAD) software package that is freely available for Mac OS X 10.9.4 ([http://www.123dapp.com/design Autodesk123D Design]). The CAD models were exported as mesh files (.stl) to the 3D printer's software ([http://www.makerbot.com/support/makerware/troubleshooting/ MakerWare]). The dimensions of the device-structures were usually between 1 mm and 5 mm, falling in the range of millifluidics<sup>[[Team:ETH_Zurich/project/references#refKitson|[31]]]</sup>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''Figure 3-b''' The final mold design for our millifluid PDMS chip used for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
'''All mesh files designed during the project will be made available at the [http://3dprint.nih.gov/ NIH 3D Print Exchange] under the category 'Custom Labware' via our [http://3dprint.nih.gov/users/ethzurichigem2014 ETH_Zurich_iGEM2014] account.<br />
'''<br />
<br />
<br />
<br />
{{:Team:ETH Zurich/tpl/topbutton|red}}<br />
<html></article></html><br />
<br />
<html><article id='3D'></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
The mold designs were printed with a commercial 3D-printer (2nd generation MakerBot Replicator with MakerWare software; [http://www.makerbot.com MakerBotIndustries], Brooklyn, US; 5th generation US$2'899) with acrylonitrile butadiene styrene ([http://en.wikipedia.org/wiki/Acrylonitrile_butadiene_styrene ABS], a copolymer of acrylonitrile, butadiene, and styrene). The maximum object size printable is [mm]: 225 x 145 x 150. The precision and minimum feature size are given as [mm]: 0.011 (XY-axis), 0.0025 (Z-axis); and 0.4 (XY-axis), 0.2 (Z-axis) respectively. The printing time varied with the size of the mold but was usually below 4 hours. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
All fabricated structures were ready to use after removing the support structures and did not require additional surface treatments like sonication, curing, painting or silanization. The molds were then directly used for PDMS chip production. In addition, custom made black 96-well plates (connected wells for diffusion assays, plate reader compatible) were printed but found to be leaky over time. The material costs of the molds were in the range of US$2 to US$4 and for the 96-well plates below US$8 (about US$160 per kg of ABS). The maximum resistance to continuous heat is given as 90 ⁰C <sup>[[Team:ETH_Zurich/project/references#refCRC|[23]]]</sup>, as a result autoclaving at 121 ⁰C was not feasible and led to deformation (see the box in figure 5-a).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''Figure 5-c''' Printed 96-well plate, pairs of wells (edge length of 5 mm) are connected by channels of varied length (1 mm to 6 mm).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
<br />
{{:Team:ETH Zurich/tpl/topbutton|blue}}<br />
<br />
<html></article></html><br />
<br />
<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
For the fabrication of millifluidic-chips raw PDMS (Dow Corning Sylgard 184) was prepared by mixing base and curing agent in 10:1 proportion. The PDMS solution was mixed vigorously and degassed (desiccator connected to vacuum) until no further bubble formation could be observed. Subsequently the mixture was poured over the mold and cured in an vacuum oven over night at RT. While the first mold design separated insufficiently from the PDMS due to an inappropriate aspect ratio (see figures 6-a and 7-a), all other PDMS chips were easily removed without additional aids and placed in clear plastic trays (86 x 128 mm; OmniTrays, Thermo Scientific). All the mold shown below were at least used once before the pictures were taken. The PDMS wells were then filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Complex_bead_medium_.28CB_medium.29 CB medium] and loaded with cells encapsulated in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads].<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''Figure 6-b''' Mold design for a diffusion assay with two connected chambers (edge length of 4 mm) with varied channel length (1 mm to 4 mm).<br />
|'''Figure 6-c''' The final mold design for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''Figure 7-a''' The very first PDMS chip. As the close-up shows, the outer parts are well defined, but the middle part did not separate from the mold due to an inappropriate aspect ratio.<br />
|'''Figure 7-b''' PDMS chip for diffusion assays with two connected chambers (edge length of 4 mm) and varied channel length (1 mm to 4 mm).<br />
|'''Figure 7-c''' The final PDMS chip for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
<br />
<br />
{{:Team:ETH Zurich/tpl/topbutton|green}}<br />
<html></article></html><br />
<br />
<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
Below you find an overview of the time-lapse movies taken during the summer. In the very first trial the wells were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB agar], holes were punched with a pipette tip and filled with highlighter-ink ([http://en.wikipedia.org/wiki/Pyranine pyranine]) to visualize diffusion (see video 1). Later, different set-ups were tested: chambers filled with liquid [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB medium] separated by solidified 2% agarose in the connecting channel and [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in liquid [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Complex_bead_medium_.28CB_medium.29 CB medium]. We continued with the 'alginate beads in liquid medium' set-up, as it yielded the most promising intermediate results, and could then finally show cell-to-cell communication of bacteria confined in beads on our millifluid chip.<br />
<br />
<br />
In all videos shown imaging was implemented with a [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Biostep_Dark-Hood_DH-50.E2.84.A2__and_the_Argus-X1.E2.84.A2_software Biostep Dark-Hood DH-50 (Argus X1 software)] fitted with a Canon EOS 500D DSLR camera and a fluorescence filter (545 nm filter). Pictures were taken every 2 min at an excitation wavelength of 470 nm with the standard Canon EOS Utility software. Time-lapse movies were created with Adobe After Effects CC software. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video1|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/c/c7/ETH_Zurich_2014_two_wells_1st_test_with_highlighter.mp4</html>}}<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video2|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/1/18/ETH_Zurich_2014_two_wells_liquid_culture_small.mp4</html>}}<br />
|-<br />
|'''Video 1 The very first diffusion experiment with fluorescent highlighter ink ([http://en.wikipedia.org/wiki/Pyranine pyranine]).''' The wells of the PDMS chip were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB agar]. About 5 μL ink were added in a punched whole on one side of the two wells. ~4500x faster than real-time.<br />
|'''Video 2 Diffusion experiment with liquid cultures.''' The wells of the PDMS chip were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB medium], separated by solidified 2% agarose in the channel. The bottom well contained 3OC6-HSL (~1 mM), the top well ''E. coli'' cells with sfGFP under the control of pLux. ~4500x faster than real-time.<br />
|-<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video3|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/7/7d/ETH_Zurich_2014_AHL_bead_sensor_bead.mp4</html>}}<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video4|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/8/8c/ETH_Zurich_2014_sender_receiver_beads_small.mp4</html>}}<br />
|-<br />
|'''Video 3 Diffusion experiment with [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained beads with 3OC6-HSL (~1 mM), the top well ''E. coli'' cells with [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Sensor_Constructs sfGFP under the control of pLux] confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained ''E. coli'' [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Producer_Constructs cells expressing LuxI], which catalyzes the production of 3OC6-HSL; the top well contained ''E. coli'' cells with [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Sensor_Constructs sfGFP under the control of pLux]. All cells were confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads). ~3450x faster than real-time.<br />
|-<br />
|colspan="2"|{{:Team:ETH_Zurich/Templates/Video|width=600px|id=video5|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/a/a9/ETH_Zurich_2014_signal_propagation.mp4</html>}}<br />
|-<br />
|colspan="2"|'''Video 5 Row wise, self-propagating [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] of ''E. coli'' cells confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads) on a [https://2014.igem.org/Team:ETH_Zurich/lab/chip custom-made millifluidic PDMS chip].''' All cells contained [https://2014.igem.org/Team:ETH_Zurich/expresults/rr#Riboregulators riboregulated] sfGFP followed by [http://parts.igem.org/Part:BBa_C0161 LuxI (BBa_C0161)] together under the control of the [http://parts.igem.org/Part:BBa_R0062 pLux promoter (BBa_R0062)], and [http://parts.igem.org/Part:BBa_J23100 constitutively (BBa_J23100)] expressed [http://parts.igem.org/Part:BBa_C0062 LuxR (BBa_C0062)]. LuxI catalyzes the production of the autoinducer 3OC6-HSL, which is then diffusing from cell to cell. For initialization, the cells in one bead of the top row were induced with 3OC6-HSL before encapsulation. 1750x faster than real-time, the video starts 7 h after the initiation of the experiment.<br />
|}<br />
<br />
<br />
{{:Team:ETH Zurich/tpl/topbutton|blue}}<br />
<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T03:26:31Z<p>Clormeau: /* Mold Design and 3D Print Exchange */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<center><br />
{{:Team:ETH Zurich/tpl/scrollbuttontworows|Mold|Design|red}}<br />
{{:Team:ETH Zurich/tpl/scrollbuttontworows|3D|Printing|blue}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton|Preparation|green}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton|movies|red}}<br />
</center><br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
Our project aims for the biological implementation of [https://2014.igem.org/Team:ETH_Zurich/project/background/modeling#Cellular_Automata cellular automata], so we had to find a way to create a regular grid of cells with a defined neighborhood as shown in the figures below. On the left side a classical cellular automata is depicted (see figure 1), on the right side an outline of [https://2014.igem.org/Team:ETH_Zurich/project/overview#Implementation_in_E._coli our biological version] consisting of a grid-like polydimethylsiloxane (PDMS) chip filled with [https://2014.igem.org/Team:ETH_Zurich/lab/bead cell colonies encapsulated in alginate beads] (see figure 2).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[File:ETH_Zurich_2014_theoretical_grid.png|300px|thumb|right|'''Figure 2 Outline of a PDMS-made grid loaded with cells confined in alginate beads for the biological implementation of cellular automata''' (ON state=sfGFP/green, OFF state=white).]]<br />
<br />
<br />
In the following, we have investigated the combination of additive manufacturing (3D-printing) and PDMS chip fabrication for applications in synthetic biology. This rapid prototyping approach allowed us to update our chips continuously according to new [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel insights from modeling] or the wet lab and in particular to avoid more intricate photolitographic approaches, which generally require clean room access, relatively expensive raw materials, and in depth knowledge of etching techniques.<br />
<br><br />
<br><br />
As a result, we are convinced that the tinkering with 3D-printing for mold creation is more economical for our applications and measurements. Also it is perfectly in line with the do-it-yourself spirit of iGEM.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article id='Mold'></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
Our custom-made plates and molds were design using a common personal computer (MacBook Air, 13-inch, early 2014, 1.7 GHz Intel Core i7, 8 GB 1600 MHz) and a 3D computer aided design (CAD) software package that is freely available for Mac OS X 10.9.4 ([http://www.123dapp.com/design Autodesk123D Design]). The CAD models were exported as mesh files (.stl) to the 3D printer's software ([http://www.makerbot.com/support/makerware/troubleshooting/ MakerWare]). The dimensions of the device-structures were usually between 1 mm and 5 mm, falling in the range of millifluidics<sup>[[Team:ETH_Zurich/project/references#refKitson|[31]]]</sup>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''Figure 3-b''' The final mold design for our millifluid PDMS chip used for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
'''All mesh files designed during the project will be made available at the [http://3dprint.nih.gov/ NIH 3D Print Exchange] under the category 'Custom Labware' via our [http://3dprint.nih.gov/users/ethzurichigem2014 ETH_Zurich_iGEM2014] account.<br />
'''<br />
<br />
<br />
<br />
{{:Team:ETH Zurich/tpl/topbutton|red}}<br />
<html></article></html><br />
<br />
<html><article id='3D'></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
The mold designs were printed with a commercial 3D-printer (2nd generation MakerBot Replicator with MakerWare software; [http://www.makerbot.com MakerBotIndustries], Brooklyn, US; 5th generation US$2'899) with acrylonitrile butadiene styrene ([http://en.wikipedia.org/wiki/Acrylonitrile_butadiene_styrene ABS], a copolymer of acrylonitrile, butadiene, and styrene). The maximum object size printable is [mm]: 225 x 145 x 150. The precision and minimum feature size are given as [mm]: 0.011 (XY-axis), 0.0025 (Z-axis); and 0.4 (XY-axis), 0.2 (Z-axis) respectively. The printing time varied with the size of the mold but was usually below 4 hours. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
All fabricated structures were ready to use after removing the support structures and did not require additional surface treatments like sonication, curing, painting or silanization. The molds were then directly used for PDMS chip production. In addition, custom made black 96-well plates (connected wells for diffusion assays, plate reader compatible) were printed but found to be leaky over time. The material costs of the molds were in the range of US$2 to US$4 and for the 96-well plates below US$8 (about US$160 per kg of ABS). The maximum resistance to continuous heat is given as 90 ⁰C <sup>[[Team:ETH_Zurich/project/references#refCRC|[23]]]</sup>, as a result autoclaving at 121 ⁰C was not feasible and led to deformation (see the box in figure 5-a).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''Figure 5-c''' Printed 96-well plate, pairs of wells (edge length of 5 mm) are connected by channels of varied length (1 mm to 6 mm).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
For the fabrication of millifluidic-chips raw PDMS (Dow Corning Sylgard 184) was prepared by mixing base and curing agent in 10:1 proportion. The PDMS solution was mixed vigorously and degassed (desiccator connected to vacuum) until no further bubble formation could be observed. Subsequently the mixture was poured over the mold and cured in an vacuum oven over night at RT. While the first mold design separated insufficiently from the PDMS due to an inappropriate aspect ratio (see figures 6-a and 7-a), all other PDMS chips were easily removed without additional aids and placed in clear plastic trays (86 x 128 mm; OmniTrays, Thermo Scientific). All the mold shown below were at least used once before the pictures were taken. The PDMS wells were then filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Complex_bead_medium_.28CB_medium.29 CB medium] and loaded with cells encapsulated in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads].<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''Figure 6-b''' Mold design for a diffusion assay with two connected chambers (edge length of 4 mm) with varied channel length (1 mm to 4 mm).<br />
|'''Figure 6-c''' The final mold design for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''Figure 7-a''' The very first PDMS chip. As the close-up shows, the outer parts are well defined, but the middle part did not separate from the mold due to an inappropriate aspect ratio.<br />
|'''Figure 7-b''' PDMS chip for diffusion assays with two connected chambers (edge length of 4 mm) and varied channel length (1 mm to 4 mm).<br />
|'''Figure 7-c''' The final PDMS chip for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
<html></article></html><br />
<br />
<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
Below you find an overview of the time-lapse movies taken during the summer. In the very first trial the wells were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB agar], holes were punched with a pipette tip and filled with highlighter-ink ([http://en.wikipedia.org/wiki/Pyranine pyranine]) to visualize diffusion (see video 1). Later, different set-ups were tested: chambers filled with liquid [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB medium] separated by solidified 2% agarose in the connecting channel and [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in liquid [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Complex_bead_medium_.28CB_medium.29 CB medium]. We continued with the 'alginate beads in liquid medium' set-up, as it yielded the most promising intermediate results, and could then finally show cell-to-cell communication of bacteria confined in beads on our millifluid chip.<br />
<br />
<br />
In all videos shown imaging was implemented with a [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Biostep_Dark-Hood_DH-50.E2.84.A2__and_the_Argus-X1.E2.84.A2_software Biostep Dark-Hood DH-50 (Argus X1 software)] fitted with a Canon EOS 500D DSLR camera and a fluorescence filter (545 nm filter). Pictures were taken every 2 min at an excitation wavelength of 470 nm with the standard Canon EOS Utility software. Time-lapse movies were created with Adobe After Effects CC software. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video1|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/c/c7/ETH_Zurich_2014_two_wells_1st_test_with_highlighter.mp4</html>}}<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video2|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/1/18/ETH_Zurich_2014_two_wells_liquid_culture_small.mp4</html>}}<br />
|-<br />
|'''Video 1 The very first diffusion experiment with fluorescent highlighter ink ([http://en.wikipedia.org/wiki/Pyranine pyranine]).''' The wells of the PDMS chip were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB agar]. About 5 μL ink were added in a punched whole on one side of the two wells. ~4500x faster than real-time.<br />
|'''Video 2 Diffusion experiment with liquid cultures.''' The wells of the PDMS chip were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB medium], separated by solidified 2% agarose in the channel. The bottom well contained 3OC6-HSL (~1 mM), the top well ''E. coli'' cells with sfGFP under the control of pLux. ~4500x faster than real-time.<br />
|-<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video3|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/7/7d/ETH_Zurich_2014_AHL_bead_sensor_bead.mp4</html>}}<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video4|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/8/8c/ETH_Zurich_2014_sender_receiver_beads_small.mp4</html>}}<br />
|-<br />
|'''Video 3 Diffusion experiment with [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained beads with 3OC6-HSL (~1 mM), the top well ''E. coli'' cells with [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Sensor_Constructs sfGFP under the control of pLux] confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained ''E. coli'' [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Producer_Constructs cells expressing LuxI], which catalyzes the production of 3OC6-HSL; the top well contained ''E. coli'' cells with [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Sensor_Constructs sfGFP under the control of pLux]. All cells were confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads). ~3450x faster than real-time.<br />
|-<br />
|colspan="2"|{{:Team:ETH_Zurich/Templates/Video|width=600px|id=video5|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/a/a9/ETH_Zurich_2014_signal_propagation.mp4</html>}}<br />
|-<br />
|colspan="2"|'''Video 5 Row wise, self-propagating [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] of ''E. coli'' cells confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads) on a [https://2014.igem.org/Team:ETH_Zurich/lab/chip custom-made millifluidic PDMS chip].''' All cells contained [https://2014.igem.org/Team:ETH_Zurich/expresults/rr#Riboregulators riboregulated] sfGFP followed by [http://parts.igem.org/Part:BBa_C0161 LuxI (BBa_C0161)] together under the control of the [http://parts.igem.org/Part:BBa_R0062 pLux promoter (BBa_R0062)], and [http://parts.igem.org/Part:BBa_J23100 constitutively (BBa_J23100)] expressed [http://parts.igem.org/Part:BBa_C0062 LuxR (BBa_C0062)]. LuxI catalyzes the production of the autoinducer 3OC6-HSL, which is then diffusing from cell to cell. For initialization, the cells in one bead of the top row were induced with 3OC6-HSL before encapsulation. 1750x faster than real-time, the video starts 7 h after the initiation of the experiment.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T03:26:17Z<p>Clormeau: /* Mold Design and 3D Print Exchange */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<center><br />
{{:Team:ETH Zurich/tpl/scrollbuttontworows|Mold|Design|red}}<br />
{{:Team:ETH Zurich/tpl/scrollbuttontworows|3D|Printing|blue}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton|Preparation|green}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton|movies|red}}<br />
</center><br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
Our project aims for the biological implementation of [https://2014.igem.org/Team:ETH_Zurich/project/background/modeling#Cellular_Automata cellular automata], so we had to find a way to create a regular grid of cells with a defined neighborhood as shown in the figures below. On the left side a classical cellular automata is depicted (see figure 1), on the right side an outline of [https://2014.igem.org/Team:ETH_Zurich/project/overview#Implementation_in_E._coli our biological version] consisting of a grid-like polydimethylsiloxane (PDMS) chip filled with [https://2014.igem.org/Team:ETH_Zurich/lab/bead cell colonies encapsulated in alginate beads] (see figure 2).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[File:ETH_Zurich_2014_theoretical_grid.png|300px|thumb|right|'''Figure 2 Outline of a PDMS-made grid loaded with cells confined in alginate beads for the biological implementation of cellular automata''' (ON state=sfGFP/green, OFF state=white).]]<br />
<br />
<br />
In the following, we have investigated the combination of additive manufacturing (3D-printing) and PDMS chip fabrication for applications in synthetic biology. This rapid prototyping approach allowed us to update our chips continuously according to new [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel insights from modeling] or the wet lab and in particular to avoid more intricate photolitographic approaches, which generally require clean room access, relatively expensive raw materials, and in depth knowledge of etching techniques.<br />
<br><br />
<br><br />
As a result, we are convinced that the tinkering with 3D-printing for mold creation is more economical for our applications and measurements. Also it is perfectly in line with the do-it-yourself spirit of iGEM.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article id='Mold'></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
Our custom-made plates and molds were design using a common personal computer (MacBook Air, 13-inch, early 2014, 1.7 GHz Intel Core i7, 8 GB 1600 MHz) and a 3D computer aided design (CAD) software package that is freely available for Mac OS X 10.9.4 ([http://www.123dapp.com/design Autodesk123D Design]). The CAD models were exported as mesh files (.stl) to the 3D printer's software ([http://www.makerbot.com/support/makerware/troubleshooting/ MakerWare]). The dimensions of the device-structures were usually between 1 mm and 5 mm, falling in the range of millifluidics<sup>[[Team:ETH_Zurich/project/references#refKitson|[31]]]</sup>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''Figure 3-b''' The final mold design for our millifluid PDMS chip used for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
'''All mesh files designed during the project will be made available at the [http://3dprint.nih.gov/ NIH 3D Print Exchange] under the category 'Custom Labware' via our [http://3dprint.nih.gov/users/ethzurichigem2014 ETH_Zurich_iGEM2014] account.<br />
'''<br />
<br />
{{:Team:ETH Zurich/tpl/topbutton|red}}<br />
<html></article></html><br />
<br />
<html><article id='3D'></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
The mold designs were printed with a commercial 3D-printer (2nd generation MakerBot Replicator with MakerWare software; [http://www.makerbot.com MakerBotIndustries], Brooklyn, US; 5th generation US$2'899) with acrylonitrile butadiene styrene ([http://en.wikipedia.org/wiki/Acrylonitrile_butadiene_styrene ABS], a copolymer of acrylonitrile, butadiene, and styrene). The maximum object size printable is [mm]: 225 x 145 x 150. The precision and minimum feature size are given as [mm]: 0.011 (XY-axis), 0.0025 (Z-axis); and 0.4 (XY-axis), 0.2 (Z-axis) respectively. The printing time varied with the size of the mold but was usually below 4 hours. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
All fabricated structures were ready to use after removing the support structures and did not require additional surface treatments like sonication, curing, painting or silanization. The molds were then directly used for PDMS chip production. In addition, custom made black 96-well plates (connected wells for diffusion assays, plate reader compatible) were printed but found to be leaky over time. The material costs of the molds were in the range of US$2 to US$4 and for the 96-well plates below US$8 (about US$160 per kg of ABS). The maximum resistance to continuous heat is given as 90 ⁰C <sup>[[Team:ETH_Zurich/project/references#refCRC|[23]]]</sup>, as a result autoclaving at 121 ⁰C was not feasible and led to deformation (see the box in figure 5-a).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''Figure 5-c''' Printed 96-well plate, pairs of wells (edge length of 5 mm) are connected by channels of varied length (1 mm to 6 mm).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
For the fabrication of millifluidic-chips raw PDMS (Dow Corning Sylgard 184) was prepared by mixing base and curing agent in 10:1 proportion. The PDMS solution was mixed vigorously and degassed (desiccator connected to vacuum) until no further bubble formation could be observed. Subsequently the mixture was poured over the mold and cured in an vacuum oven over night at RT. While the first mold design separated insufficiently from the PDMS due to an inappropriate aspect ratio (see figures 6-a and 7-a), all other PDMS chips were easily removed without additional aids and placed in clear plastic trays (86 x 128 mm; OmniTrays, Thermo Scientific). All the mold shown below were at least used once before the pictures were taken. The PDMS wells were then filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Complex_bead_medium_.28CB_medium.29 CB medium] and loaded with cells encapsulated in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads].<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''Figure 6-b''' Mold design for a diffusion assay with two connected chambers (edge length of 4 mm) with varied channel length (1 mm to 4 mm).<br />
|'''Figure 6-c''' The final mold design for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''Figure 7-a''' The very first PDMS chip. As the close-up shows, the outer parts are well defined, but the middle part did not separate from the mold due to an inappropriate aspect ratio.<br />
|'''Figure 7-b''' PDMS chip for diffusion assays with two connected chambers (edge length of 4 mm) and varied channel length (1 mm to 4 mm).<br />
|'''Figure 7-c''' The final PDMS chip for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
<html></article></html><br />
<br />
<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
Below you find an overview of the time-lapse movies taken during the summer. In the very first trial the wells were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB agar], holes were punched with a pipette tip and filled with highlighter-ink ([http://en.wikipedia.org/wiki/Pyranine pyranine]) to visualize diffusion (see video 1). Later, different set-ups were tested: chambers filled with liquid [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB medium] separated by solidified 2% agarose in the connecting channel and [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in liquid [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Complex_bead_medium_.28CB_medium.29 CB medium]. We continued with the 'alginate beads in liquid medium' set-up, as it yielded the most promising intermediate results, and could then finally show cell-to-cell communication of bacteria confined in beads on our millifluid chip.<br />
<br />
<br />
In all videos shown imaging was implemented with a [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Biostep_Dark-Hood_DH-50.E2.84.A2__and_the_Argus-X1.E2.84.A2_software Biostep Dark-Hood DH-50 (Argus X1 software)] fitted with a Canon EOS 500D DSLR camera and a fluorescence filter (545 nm filter). Pictures were taken every 2 min at an excitation wavelength of 470 nm with the standard Canon EOS Utility software. Time-lapse movies were created with Adobe After Effects CC software. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video1|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/c/c7/ETH_Zurich_2014_two_wells_1st_test_with_highlighter.mp4</html>}}<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video2|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/1/18/ETH_Zurich_2014_two_wells_liquid_culture_small.mp4</html>}}<br />
|-<br />
|'''Video 1 The very first diffusion experiment with fluorescent highlighter ink ([http://en.wikipedia.org/wiki/Pyranine pyranine]).''' The wells of the PDMS chip were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB agar]. About 5 μL ink were added in a punched whole on one side of the two wells. ~4500x faster than real-time.<br />
|'''Video 2 Diffusion experiment with liquid cultures.''' The wells of the PDMS chip were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB medium], separated by solidified 2% agarose in the channel. The bottom well contained 3OC6-HSL (~1 mM), the top well ''E. coli'' cells with sfGFP under the control of pLux. ~4500x faster than real-time.<br />
|-<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video3|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/7/7d/ETH_Zurich_2014_AHL_bead_sensor_bead.mp4</html>}}<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video4|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/8/8c/ETH_Zurich_2014_sender_receiver_beads_small.mp4</html>}}<br />
|-<br />
|'''Video 3 Diffusion experiment with [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained beads with 3OC6-HSL (~1 mM), the top well ''E. coli'' cells with [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Sensor_Constructs sfGFP under the control of pLux] confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained ''E. coli'' [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Producer_Constructs cells expressing LuxI], which catalyzes the production of 3OC6-HSL; the top well contained ''E. coli'' cells with [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Sensor_Constructs sfGFP under the control of pLux]. All cells were confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads). ~3450x faster than real-time.<br />
|-<br />
|colspan="2"|{{:Team:ETH_Zurich/Templates/Video|width=600px|id=video5|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/a/a9/ETH_Zurich_2014_signal_propagation.mp4</html>}}<br />
|-<br />
|colspan="2"|'''Video 5 Row wise, self-propagating [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] of ''E. coli'' cells confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads) on a [https://2014.igem.org/Team:ETH_Zurich/lab/chip custom-made millifluidic PDMS chip].''' All cells contained [https://2014.igem.org/Team:ETH_Zurich/expresults/rr#Riboregulators riboregulated] sfGFP followed by [http://parts.igem.org/Part:BBa_C0161 LuxI (BBa_C0161)] together under the control of the [http://parts.igem.org/Part:BBa_R0062 pLux promoter (BBa_R0062)], and [http://parts.igem.org/Part:BBa_J23100 constitutively (BBa_J23100)] expressed [http://parts.igem.org/Part:BBa_C0062 LuxR (BBa_C0062)]. LuxI catalyzes the production of the autoinducer 3OC6-HSL, which is then diffusing from cell to cell. For initialization, the cells in one bead of the top row were induced with 3OC6-HSL before encapsulation. 1750x faster than real-time, the video starts 7 h after the initiation of the experiment.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T03:25:35Z<p>Clormeau: </p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<center><br />
{{:Team:ETH Zurich/tpl/scrollbuttontworows|Mold|Design|red}}<br />
{{:Team:ETH Zurich/tpl/scrollbuttontworows|3D|Printing|blue}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton|Preparation|green}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton|movies|red}}<br />
</center><br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
Our project aims for the biological implementation of [https://2014.igem.org/Team:ETH_Zurich/project/background/modeling#Cellular_Automata cellular automata], so we had to find a way to create a regular grid of cells with a defined neighborhood as shown in the figures below. On the left side a classical cellular automata is depicted (see figure 1), on the right side an outline of [https://2014.igem.org/Team:ETH_Zurich/project/overview#Implementation_in_E._coli our biological version] consisting of a grid-like polydimethylsiloxane (PDMS) chip filled with [https://2014.igem.org/Team:ETH_Zurich/lab/bead cell colonies encapsulated in alginate beads] (see figure 2).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[File:ETH_Zurich_2014_theoretical_grid.png|300px|thumb|right|'''Figure 2 Outline of a PDMS-made grid loaded with cells confined in alginate beads for the biological implementation of cellular automata''' (ON state=sfGFP/green, OFF state=white).]]<br />
<br />
<br />
In the following, we have investigated the combination of additive manufacturing (3D-printing) and PDMS chip fabrication for applications in synthetic biology. This rapid prototyping approach allowed us to update our chips continuously according to new [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel insights from modeling] or the wet lab and in particular to avoid more intricate photolitographic approaches, which generally require clean room access, relatively expensive raw materials, and in depth knowledge of etching techniques.<br />
<br><br />
<br><br />
As a result, we are convinced that the tinkering with 3D-printing for mold creation is more economical for our applications and measurements. Also it is perfectly in line with the do-it-yourself spirit of iGEM.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article id='Mold'></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
Our custom-made plates and molds were design using a common personal computer (MacBook Air, 13-inch, early 2014, 1.7 GHz Intel Core i7, 8 GB 1600 MHz) and a 3D computer aided design (CAD) software package that is freely available for Mac OS X 10.9.4 ([http://www.123dapp.com/design Autodesk123D Design]). The CAD models were exported as mesh files (.stl) to the 3D printer's software ([http://www.makerbot.com/support/makerware/troubleshooting/ MakerWare]). The dimensions of the device-structures were usually between 1 mm and 5 mm, falling in the range of millifluidics<sup>[[Team:ETH_Zurich/project/references#refKitson|[31]]]</sup>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''Figure 3-b''' The final mold design for our millifluid PDMS chip used for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
'''All mesh files designed during the project will be made available at the [http://3dprint.nih.gov/ NIH 3D Print Exchange] under the category 'Custom Labware' via our [http://3dprint.nih.gov/users/ethzurichigem2014 ETH_Zurich_iGEM2014] account.<br />
'''<br />
<html></article></html><br />
<br />
<html><article id='3D'></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
The mold designs were printed with a commercial 3D-printer (2nd generation MakerBot Replicator with MakerWare software; [http://www.makerbot.com MakerBotIndustries], Brooklyn, US; 5th generation US$2'899) with acrylonitrile butadiene styrene ([http://en.wikipedia.org/wiki/Acrylonitrile_butadiene_styrene ABS], a copolymer of acrylonitrile, butadiene, and styrene). The maximum object size printable is [mm]: 225 x 145 x 150. The precision and minimum feature size are given as [mm]: 0.011 (XY-axis), 0.0025 (Z-axis); and 0.4 (XY-axis), 0.2 (Z-axis) respectively. The printing time varied with the size of the mold but was usually below 4 hours. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
All fabricated structures were ready to use after removing the support structures and did not require additional surface treatments like sonication, curing, painting or silanization. The molds were then directly used for PDMS chip production. In addition, custom made black 96-well plates (connected wells for diffusion assays, plate reader compatible) were printed but found to be leaky over time. The material costs of the molds were in the range of US$2 to US$4 and for the 96-well plates below US$8 (about US$160 per kg of ABS). The maximum resistance to continuous heat is given as 90 ⁰C <sup>[[Team:ETH_Zurich/project/references#refCRC|[23]]]</sup>, as a result autoclaving at 121 ⁰C was not feasible and led to deformation (see the box in figure 5-a).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''Figure 5-c''' Printed 96-well plate, pairs of wells (edge length of 5 mm) are connected by channels of varied length (1 mm to 6 mm).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
For the fabrication of millifluidic-chips raw PDMS (Dow Corning Sylgard 184) was prepared by mixing base and curing agent in 10:1 proportion. The PDMS solution was mixed vigorously and degassed (desiccator connected to vacuum) until no further bubble formation could be observed. Subsequently the mixture was poured over the mold and cured in an vacuum oven over night at RT. While the first mold design separated insufficiently from the PDMS due to an inappropriate aspect ratio (see figures 6-a and 7-a), all other PDMS chips were easily removed without additional aids and placed in clear plastic trays (86 x 128 mm; OmniTrays, Thermo Scientific). All the mold shown below were at least used once before the pictures were taken. The PDMS wells were then filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Complex_bead_medium_.28CB_medium.29 CB medium] and loaded with cells encapsulated in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads].<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''Figure 6-b''' Mold design for a diffusion assay with two connected chambers (edge length of 4 mm) with varied channel length (1 mm to 4 mm).<br />
|'''Figure 6-c''' The final mold design for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''Figure 7-a''' The very first PDMS chip. As the close-up shows, the outer parts are well defined, but the middle part did not separate from the mold due to an inappropriate aspect ratio.<br />
|'''Figure 7-b''' PDMS chip for diffusion assays with two connected chambers (edge length of 4 mm) and varied channel length (1 mm to 4 mm).<br />
|'''Figure 7-c''' The final PDMS chip for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
<html></article></html><br />
<br />
<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
Below you find an overview of the time-lapse movies taken during the summer. In the very first trial the wells were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB agar], holes were punched with a pipette tip and filled with highlighter-ink ([http://en.wikipedia.org/wiki/Pyranine pyranine]) to visualize diffusion (see video 1). Later, different set-ups were tested: chambers filled with liquid [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB medium] separated by solidified 2% agarose in the connecting channel and [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in liquid [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Complex_bead_medium_.28CB_medium.29 CB medium]. We continued with the 'alginate beads in liquid medium' set-up, as it yielded the most promising intermediate results, and could then finally show cell-to-cell communication of bacteria confined in beads on our millifluid chip.<br />
<br />
<br />
In all videos shown imaging was implemented with a [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Biostep_Dark-Hood_DH-50.E2.84.A2__and_the_Argus-X1.E2.84.A2_software Biostep Dark-Hood DH-50 (Argus X1 software)] fitted with a Canon EOS 500D DSLR camera and a fluorescence filter (545 nm filter). Pictures were taken every 2 min at an excitation wavelength of 470 nm with the standard Canon EOS Utility software. Time-lapse movies were created with Adobe After Effects CC software. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video1|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/c/c7/ETH_Zurich_2014_two_wells_1st_test_with_highlighter.mp4</html>}}<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video2|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/1/18/ETH_Zurich_2014_two_wells_liquid_culture_small.mp4</html>}}<br />
|-<br />
|'''Video 1 The very first diffusion experiment with fluorescent highlighter ink ([http://en.wikipedia.org/wiki/Pyranine pyranine]).''' The wells of the PDMS chip were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB agar]. About 5 μL ink were added in a punched whole on one side of the two wells. ~4500x faster than real-time.<br />
|'''Video 2 Diffusion experiment with liquid cultures.''' The wells of the PDMS chip were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB medium], separated by solidified 2% agarose in the channel. The bottom well contained 3OC6-HSL (~1 mM), the top well ''E. coli'' cells with sfGFP under the control of pLux. ~4500x faster than real-time.<br />
|-<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video3|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/7/7d/ETH_Zurich_2014_AHL_bead_sensor_bead.mp4</html>}}<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video4|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/8/8c/ETH_Zurich_2014_sender_receiver_beads_small.mp4</html>}}<br />
|-<br />
|'''Video 3 Diffusion experiment with [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained beads with 3OC6-HSL (~1 mM), the top well ''E. coli'' cells with [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Sensor_Constructs sfGFP under the control of pLux] confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained ''E. coli'' [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Producer_Constructs cells expressing LuxI], which catalyzes the production of 3OC6-HSL; the top well contained ''E. coli'' cells with [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Sensor_Constructs sfGFP under the control of pLux]. All cells were confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads). ~3450x faster than real-time.<br />
|-<br />
|colspan="2"|{{:Team:ETH_Zurich/Templates/Video|width=600px|id=video5|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/a/a9/ETH_Zurich_2014_signal_propagation.mp4</html>}}<br />
|-<br />
|colspan="2"|'''Video 5 Row wise, self-propagating [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] of ''E. coli'' cells confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads) on a [https://2014.igem.org/Team:ETH_Zurich/lab/chip custom-made millifluidic PDMS chip].''' All cells contained [https://2014.igem.org/Team:ETH_Zurich/expresults/rr#Riboregulators riboregulated] sfGFP followed by [http://parts.igem.org/Part:BBa_C0161 LuxI (BBa_C0161)] together under the control of the [http://parts.igem.org/Part:BBa_R0062 pLux promoter (BBa_R0062)], and [http://parts.igem.org/Part:BBa_J23100 constitutively (BBa_J23100)] expressed [http://parts.igem.org/Part:BBa_C0062 LuxR (BBa_C0062)]. LuxI catalyzes the production of the autoinducer 3OC6-HSL, which is then diffusing from cell to cell. For initialization, the cells in one bead of the top row were induced with 3OC6-HSL before encapsulation. 1750x faster than real-time, the video starts 7 h after the initiation of the experiment.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T03:25:21Z<p>Clormeau: </p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
{{:Team:ETH Zurich/tpl/scrollbuttontworows|Mold|Design|red}}<br />
{{:Team:ETH Zurich/tpl/scrollbuttontworows|3D|Printing|blue}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton|Preparation|green}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton|movies|red}}<br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
Our project aims for the biological implementation of [https://2014.igem.org/Team:ETH_Zurich/project/background/modeling#Cellular_Automata cellular automata], so we had to find a way to create a regular grid of cells with a defined neighborhood as shown in the figures below. On the left side a classical cellular automata is depicted (see figure 1), on the right side an outline of [https://2014.igem.org/Team:ETH_Zurich/project/overview#Implementation_in_E._coli our biological version] consisting of a grid-like polydimethylsiloxane (PDMS) chip filled with [https://2014.igem.org/Team:ETH_Zurich/lab/bead cell colonies encapsulated in alginate beads] (see figure 2).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[File:ETH_Zurich_2014_theoretical_grid.png|300px|thumb|right|'''Figure 2 Outline of a PDMS-made grid loaded with cells confined in alginate beads for the biological implementation of cellular automata''' (ON state=sfGFP/green, OFF state=white).]]<br />
<br />
<br />
In the following, we have investigated the combination of additive manufacturing (3D-printing) and PDMS chip fabrication for applications in synthetic biology. This rapid prototyping approach allowed us to update our chips continuously according to new [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel insights from modeling] or the wet lab and in particular to avoid more intricate photolitographic approaches, which generally require clean room access, relatively expensive raw materials, and in depth knowledge of etching techniques.<br />
<br><br />
<br><br />
As a result, we are convinced that the tinkering with 3D-printing for mold creation is more economical for our applications and measurements. Also it is perfectly in line with the do-it-yourself spirit of iGEM.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article id='Mold'></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
Our custom-made plates and molds were design using a common personal computer (MacBook Air, 13-inch, early 2014, 1.7 GHz Intel Core i7, 8 GB 1600 MHz) and a 3D computer aided design (CAD) software package that is freely available for Mac OS X 10.9.4 ([http://www.123dapp.com/design Autodesk123D Design]). The CAD models were exported as mesh files (.stl) to the 3D printer's software ([http://www.makerbot.com/support/makerware/troubleshooting/ MakerWare]). The dimensions of the device-structures were usually between 1 mm and 5 mm, falling in the range of millifluidics<sup>[[Team:ETH_Zurich/project/references#refKitson|[31]]]</sup>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''Figure 3-b''' The final mold design for our millifluid PDMS chip used for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
'''All mesh files designed during the project will be made available at the [http://3dprint.nih.gov/ NIH 3D Print Exchange] under the category 'Custom Labware' via our [http://3dprint.nih.gov/users/ethzurichigem2014 ETH_Zurich_iGEM2014] account.<br />
'''<br />
<html></article></html><br />
<br />
<html><article id='3D'></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
The mold designs were printed with a commercial 3D-printer (2nd generation MakerBot Replicator with MakerWare software; [http://www.makerbot.com MakerBotIndustries], Brooklyn, US; 5th generation US$2'899) with acrylonitrile butadiene styrene ([http://en.wikipedia.org/wiki/Acrylonitrile_butadiene_styrene ABS], a copolymer of acrylonitrile, butadiene, and styrene). The maximum object size printable is [mm]: 225 x 145 x 150. The precision and minimum feature size are given as [mm]: 0.011 (XY-axis), 0.0025 (Z-axis); and 0.4 (XY-axis), 0.2 (Z-axis) respectively. The printing time varied with the size of the mold but was usually below 4 hours. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
All fabricated structures were ready to use after removing the support structures and did not require additional surface treatments like sonication, curing, painting or silanization. The molds were then directly used for PDMS chip production. In addition, custom made black 96-well plates (connected wells for diffusion assays, plate reader compatible) were printed but found to be leaky over time. The material costs of the molds were in the range of US$2 to US$4 and for the 96-well plates below US$8 (about US$160 per kg of ABS). The maximum resistance to continuous heat is given as 90 ⁰C <sup>[[Team:ETH_Zurich/project/references#refCRC|[23]]]</sup>, as a result autoclaving at 121 ⁰C was not feasible and led to deformation (see the box in figure 5-a).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''Figure 5-c''' Printed 96-well plate, pairs of wells (edge length of 5 mm) are connected by channels of varied length (1 mm to 6 mm).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
For the fabrication of millifluidic-chips raw PDMS (Dow Corning Sylgard 184) was prepared by mixing base and curing agent in 10:1 proportion. The PDMS solution was mixed vigorously and degassed (desiccator connected to vacuum) until no further bubble formation could be observed. Subsequently the mixture was poured over the mold and cured in an vacuum oven over night at RT. While the first mold design separated insufficiently from the PDMS due to an inappropriate aspect ratio (see figures 6-a and 7-a), all other PDMS chips were easily removed without additional aids and placed in clear plastic trays (86 x 128 mm; OmniTrays, Thermo Scientific). All the mold shown below were at least used once before the pictures were taken. The PDMS wells were then filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Complex_bead_medium_.28CB_medium.29 CB medium] and loaded with cells encapsulated in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads].<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''Figure 6-b''' Mold design for a diffusion assay with two connected chambers (edge length of 4 mm) with varied channel length (1 mm to 4 mm).<br />
|'''Figure 6-c''' The final mold design for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''Figure 7-a''' The very first PDMS chip. As the close-up shows, the outer parts are well defined, but the middle part did not separate from the mold due to an inappropriate aspect ratio.<br />
|'''Figure 7-b''' PDMS chip for diffusion assays with two connected chambers (edge length of 4 mm) and varied channel length (1 mm to 4 mm).<br />
|'''Figure 7-c''' The final PDMS chip for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
<html></article></html><br />
<br />
<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
Below you find an overview of the time-lapse movies taken during the summer. In the very first trial the wells were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB agar], holes were punched with a pipette tip and filled with highlighter-ink ([http://en.wikipedia.org/wiki/Pyranine pyranine]) to visualize diffusion (see video 1). Later, different set-ups were tested: chambers filled with liquid [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB medium] separated by solidified 2% agarose in the connecting channel and [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in liquid [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Complex_bead_medium_.28CB_medium.29 CB medium]. We continued with the 'alginate beads in liquid medium' set-up, as it yielded the most promising intermediate results, and could then finally show cell-to-cell communication of bacteria confined in beads on our millifluid chip.<br />
<br />
<br />
In all videos shown imaging was implemented with a [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Biostep_Dark-Hood_DH-50.E2.84.A2__and_the_Argus-X1.E2.84.A2_software Biostep Dark-Hood DH-50 (Argus X1 software)] fitted with a Canon EOS 500D DSLR camera and a fluorescence filter (545 nm filter). Pictures were taken every 2 min at an excitation wavelength of 470 nm with the standard Canon EOS Utility software. Time-lapse movies were created with Adobe After Effects CC software. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video1|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/c/c7/ETH_Zurich_2014_two_wells_1st_test_with_highlighter.mp4</html>}}<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video2|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/1/18/ETH_Zurich_2014_two_wells_liquid_culture_small.mp4</html>}}<br />
|-<br />
|'''Video 1 The very first diffusion experiment with fluorescent highlighter ink ([http://en.wikipedia.org/wiki/Pyranine pyranine]).''' The wells of the PDMS chip were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB agar]. About 5 μL ink were added in a punched whole on one side of the two wells. ~4500x faster than real-time.<br />
|'''Video 2 Diffusion experiment with liquid cultures.''' The wells of the PDMS chip were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB medium], separated by solidified 2% agarose in the channel. The bottom well contained 3OC6-HSL (~1 mM), the top well ''E. coli'' cells with sfGFP under the control of pLux. ~4500x faster than real-time.<br />
|-<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video3|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/7/7d/ETH_Zurich_2014_AHL_bead_sensor_bead.mp4</html>}}<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video4|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/8/8c/ETH_Zurich_2014_sender_receiver_beads_small.mp4</html>}}<br />
|-<br />
|'''Video 3 Diffusion experiment with [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained beads with 3OC6-HSL (~1 mM), the top well ''E. coli'' cells with [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Sensor_Constructs sfGFP under the control of pLux] confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained ''E. coli'' [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Producer_Constructs cells expressing LuxI], which catalyzes the production of 3OC6-HSL; the top well contained ''E. coli'' cells with [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Sensor_Constructs sfGFP under the control of pLux]. All cells were confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads). ~3450x faster than real-time.<br />
|-<br />
|colspan="2"|{{:Team:ETH_Zurich/Templates/Video|width=600px|id=video5|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/a/a9/ETH_Zurich_2014_signal_propagation.mp4</html>}}<br />
|-<br />
|colspan="2"|'''Video 5 Row wise, self-propagating [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] of ''E. coli'' cells confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads) on a [https://2014.igem.org/Team:ETH_Zurich/lab/chip custom-made millifluidic PDMS chip].''' All cells contained [https://2014.igem.org/Team:ETH_Zurich/expresults/rr#Riboregulators riboregulated] sfGFP followed by [http://parts.igem.org/Part:BBa_C0161 LuxI (BBa_C0161)] together under the control of the [http://parts.igem.org/Part:BBa_R0062 pLux promoter (BBa_R0062)], and [http://parts.igem.org/Part:BBa_J23100 constitutively (BBa_J23100)] expressed [http://parts.igem.org/Part:BBa_C0062 LuxR (BBa_C0062)]. LuxI catalyzes the production of the autoinducer 3OC6-HSL, which is then diffusing from cell to cell. For initialization, the cells in one bead of the top row were induced with 3OC6-HSL before encapsulation. 1750x faster than real-time, the video starts 7 h after the initiation of the experiment.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/lab/chipTeam:ETH Zurich/lab/chip2014-10-18T03:22:30Z<p>Clormeau: </p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Millifluidic Chip & Rapid Prototyping}}<br />
<br />
<html><article style='min-height:800px'></html><br />
==Overview==<br />
Our project aims for the biological implementation of [https://2014.igem.org/Team:ETH_Zurich/project/background/modeling#Cellular_Automata cellular automata], so we had to find a way to create a regular grid of cells with a defined neighborhood as shown in the figures below. On the left side a classical cellular automata is depicted (see figure 1), on the right side an outline of [https://2014.igem.org/Team:ETH_Zurich/project/overview#Implementation_in_E._coli our biological version] consisting of a grid-like polydimethylsiloxane (PDMS) chip filled with [https://2014.igem.org/Team:ETH_Zurich/lab/bead cell colonies encapsulated in alginate beads] (see figure 2).<br />
<br />
[[File:ETH Zurich Rule 6.PNG|300px|thumb|left|'''Figure 1''' '''Classical grid from cellular automata theory''' (ON state=back, OFF state=white).]]<br />
[[File:ETH_Zurich_2014_theoretical_grid.png|300px|thumb|right|'''Figure 2 Outline of a PDMS-made grid loaded with cells confined in alginate beads for the biological implementation of cellular automata''' (ON state=sfGFP/green, OFF state=white).]]<br />
<br />
<br />
In the following, we have investigated the combination of additive manufacturing (3D-printing) and PDMS chip fabrication for applications in synthetic biology. This rapid prototyping approach allowed us to update our chips continuously according to new [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel insights from modeling] or the wet lab and in particular to avoid more intricate photolitographic approaches, which generally require clean room access, relatively expensive raw materials, and in depth knowledge of etching techniques.<br />
<br><br />
<br><br />
As a result, we are convinced that the tinkering with 3D-printing for mold creation is more economical for our applications and measurements. Also it is perfectly in line with the do-it-yourself spirit of iGEM.<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article id='Mold'></html><br />
<br />
==Mold Design and 3D Print Exchange==<br />
<br />
Our custom-made plates and molds were design using a common personal computer (MacBook Air, 13-inch, early 2014, 1.7 GHz Intel Core i7, 8 GB 1600 MHz) and a 3D computer aided design (CAD) software package that is freely available for Mac OS X 10.9.4 ([http://www.123dapp.com/design Autodesk123D Design]). The CAD models were exported as mesh files (.stl) to the 3D printer's software ([http://www.makerbot.com/support/makerware/troubleshooting/ MakerWare]). The dimensions of the device-structures were usually between 1 mm and 5 mm, falling in the range of millifluidics<sup>[[Team:ETH_Zurich/project/references#refKitson|[31]]]</sup>. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_mesh_2_20140826.jpg|200px]]<br />
|[[File:ETH Zurich 2014 final mold model 2.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion plate model.jpeg|200px]]<br />
|-<br />
|'''Figure 3-a''' The first design for a gel-comb, a mold for a millifluidic PDMS chip and a corresponding box for the mold.<br />
|'''Figure 3-b''' The final mold design for our millifluid PDMS chip used for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments. <br />
|'''Figure 3-c''' A design for a 96-well plate with connected wells, which allows automated measurements in a plate reader.<br />
|}<br />
<br />
<br />
'''All mesh files designed during the project will be made available at the [http://3dprint.nih.gov/ NIH 3D Print Exchange] under the category 'Custom Labware' via our [http://3dprint.nih.gov/users/ethzurichigem2014 ETH_Zurich_iGEM2014] account.<br />
'''<br />
<html></article></html><br />
<br />
<html><article id='3D'></html><br />
<br />
==3D-Printing and Rapid Prototyping==<br />
<br />
The mold designs were printed with a commercial 3D-printer (2nd generation MakerBot Replicator with MakerWare software; [http://www.makerbot.com MakerBotIndustries], Brooklyn, US; 5th generation US$2'899) with acrylonitrile butadiene styrene ([http://en.wikipedia.org/wiki/Acrylonitrile_butadiene_styrene ABS], a copolymer of acrylonitrile, butadiene, and styrene). The maximum object size printable is [mm]: 225 x 145 x 150. The precision and minimum feature size are given as [mm]: 0.011 (XY-axis), 0.0025 (Z-axis); and 0.4 (XY-axis), 0.2 (Z-axis) respectively. The printing time varied with the size of the mold but was usually below 4 hours. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 MakerBot.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerWare.jpg|200px]]<br />
|[[File:ETH Zurich 2014 MakerBot ABS.jpg|x200px]]<br />
|-<br />
|'''Figure 4-a''' The MakerBot Replicator (2nd generation) we used to print our molds.<br />
|'''Figure 4-b''' 'Screenshot' of the MakerWare software we used to print our molds.<br />
|'''Figure 4-c''' A roll of ABS filament used by the 3D-printer.<br />
|}<br />
<br />
<br />
All fabricated structures were ready to use after removing the support structures and did not require additional surface treatments like sonication, curing, painting or silanization. The molds were then directly used for PDMS chip production. In addition, custom made black 96-well plates (connected wells for diffusion assays, plate reader compatible) were printed but found to be leaky over time. The material costs of the molds were in the range of US$2 to US$4 and for the 96-well plates below US$8 (about US$160 per kg of ABS). The maximum resistance to continuous heat is given as 90 ⁰C <sup>[[Team:ETH_Zurich/project/references#refCRC|[23]]]</sup>, as a result autoclaving at 121 ⁰C was not feasible and led to deformation (see the box in figure 5-a).<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_comb_and_box.jpg|200px]]<br />
|[[File:ETH Zurich 2014 small grid.JPG|200px]]<br />
|[[File:ETH Zurich diffusion plate.JPG|200px]]<br />
|[[File:ETH Zurich 2014 96 well all connected.jpeg|200px]]<br />
|-<br />
|'''Figure 5-a''' Printed gel-comb and box. The box was autoclaved at 121 ⁰C. <br />
|'''Figure 5-b''' Printed millifluid grid with interconnected wells (edge length of 3 mm).<br />
|'''Figure 5-c''' Printed 96-well plate, pairs of wells (edge length of 5 mm) are connected by channels of varied length (1 mm to 6 mm).<br />
|'''Figure 5-d''' Printed 96-well plate, all wells (edge length of 5 mm) are connected.<br />
|}<br />
<br />
<html></article></html><br />
<br />
<html><article id='Preparation'></html><br />
<br />
==PDMS Chip Preparation==<br />
<br />
For the fabrication of millifluidic-chips raw PDMS (Dow Corning Sylgard 184) was prepared by mixing base and curing agent in 10:1 proportion. The PDMS solution was mixed vigorously and degassed (desiccator connected to vacuum) until no further bubble formation could be observed. Subsequently the mixture was poured over the mold and cured in an vacuum oven over night at RT. While the first mold design separated insufficiently from the PDMS due to an inappropriate aspect ratio (see figures 6-a and 7-a), all other PDMS chips were easily removed without additional aids and placed in clear plastic trays (86 x 128 mm; OmniTrays, Thermo Scientific). All the mold shown below were at least used once before the pictures were taken. The PDMS wells were then filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Complex_bead_medium_.28CB_medium.29 CB medium] and loaded with cells encapsulated in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads].<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH_Zurich_2014_first_mold_with_PDMS.jpg|200px]]<br />
|[[File:ETH Zurich 2014 diffusion mold.JPG|200px]]<br />
|[[File:ETH Zurich 2014 final mold.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 final mold closeup.jpeg|200px]]<br />
|-<br />
|'''Figure 6-a''' The very first mold design. PDMS stuck between the wells while removing it. <br />
|'''Figure 6-b''' Mold design for a diffusion assay with two connected chambers (edge length of 4 mm) with varied channel length (1 mm to 4 mm).<br />
|'''Figure 6-c''' The final mold design for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 6-d''' Close up of the final mold design. The separate layers are clearly visible ('additive' manufacturing).<br />
|}<br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:auto; margin: auto;"<br />
|[[File:ETH Zurich 2014 broken chip.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 2 well diffusion chip upsidedown.jpeg|200px]]<br />
|[[File:ETH Zurich 2014 PDMS diffusion chip final.jpeg|200px]]<br />
|[[File:ETH_Zurich_2014_final_chip_zoom.png|200px]]<br />
|-<br />
|'''Figure 7-a''' The very first PDMS chip. As the close-up shows, the outer parts are well defined, but the middle part did not separate from the mold due to an inappropriate aspect ratio.<br />
|'''Figure 7-b''' PDMS chip for diffusion assays with two connected chambers (edge length of 4 mm) and varied channel length (1 mm to 4 mm).<br />
|'''Figure 7-c''' The final PDMS chip for [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] experiments (edge length of 5 mm, channel length of 3 mm). <br />
|'''Figure 7-d''' Close up of the final PDMS chip. The channels are well defined and even small structures separated evenly from the mold.<br />
|}<br />
<html></article></html><br />
<br />
<html><article id='movies'></html><br />
<br />
==Time-Lapse Movies==<br />
<br />
Below you find an overview of the time-lapse movies taken during the summer. In the very first trial the wells were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB agar], holes were punched with a pipette tip and filled with highlighter-ink ([http://en.wikipedia.org/wiki/Pyranine pyranine]) to visualize diffusion (see video 1). Later, different set-ups were tested: chambers filled with liquid [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB medium] separated by solidified 2% agarose in the connecting channel and [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in liquid [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Complex_bead_medium_.28CB_medium.29 CB medium]. We continued with the 'alginate beads in liquid medium' set-up, as it yielded the most promising intermediate results, and could then finally show cell-to-cell communication of bacteria confined in beads on our millifluid chip.<br />
<br />
<br />
In all videos shown imaging was implemented with a [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#Biostep_Dark-Hood_DH-50.E2.84.A2__and_the_Argus-X1.E2.84.A2_software Biostep Dark-Hood DH-50 (Argus X1 software)] fitted with a Canon EOS 500D DSLR camera and a fluorescence filter (545 nm filter). Pictures were taken every 2 min at an excitation wavelength of 470 nm with the standard Canon EOS Utility software. Time-lapse movies were created with Adobe After Effects CC software. <br />
<br />
<br />
{|class="wikitable" style="background-color: white; text-align:center; width:100%; max-width: 650px; margin: auto;"<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video1|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/c/c7/ETH_Zurich_2014_two_wells_1st_test_with_highlighter.mp4</html>}}<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video2|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/1/18/ETH_Zurich_2014_two_wells_liquid_culture_small.mp4</html>}}<br />
|-<br />
|'''Video 1 The very first diffusion experiment with fluorescent highlighter ink ([http://en.wikipedia.org/wiki/Pyranine pyranine]).''' The wells of the PDMS chip were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB agar]. About 5 μL ink were added in a punched whole on one side of the two wells. ~4500x faster than real-time.<br />
|'''Video 2 Diffusion experiment with liquid cultures.''' The wells of the PDMS chip were filled with [https://2014.igem.org/Team:ETH_Zurich/lab/protocols#LB_medium_from_dehydrated_product LB medium], separated by solidified 2% agarose in the channel. The bottom well contained 3OC6-HSL (~1 mM), the top well ''E. coli'' cells with sfGFP under the control of pLux. ~4500x faster than real-time.<br />
|-<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video3|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/7/7d/ETH_Zurich_2014_AHL_bead_sensor_bead.mp4</html>}}<br />
|{{:Team:ETH_Zurich/Templates/Video|width=300px|id=video4|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/8/8c/ETH_Zurich_2014_sender_receiver_beads_small.mp4</html>}}<br />
|-<br />
|'''Video 3 Diffusion experiment with [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained beads with 3OC6-HSL (~1 mM), the top well ''E. coli'' cells with [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Sensor_Constructs sfGFP under the control of pLux] confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads). ~1850x faster than real-time.<br />
|'''Video 4 Cell communication experiment with [ alginate beads] in defined liquid medium.''' The wells of the PDMS chip were filled with CB medium. The bottom well contained ''E. coli'' [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Producer_Constructs cells expressing LuxI], which catalyzes the production of 3OC6-HSL; the top well contained ''E. coli'' cells with [https://2014.igem.org/Team:ETH_Zurich/lab/sequences#Sensor_Constructs sfGFP under the control of pLux]. All cells were confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads). ~3450x faster than real-time.<br />
|-<br />
|colspan="2"|{{:Team:ETH_Zurich/Templates/Video|width=600px|id=video5|ratio=143/100|srcMP4=<html>https://static.igem.org/mediawiki/2014/a/a9/ETH_Zurich_2014_signal_propagation.mp4</html>}}<br />
|-<br />
|colspan="2"|'''Video 5 Row wise, self-propagating [https://2014.igem.org/Team:ETH_Zurich/project/background/biotools#Quorum_Sensing cell-to-cell communication] of ''E. coli'' cells confined in [https://2014.igem.org/Team:ETH_Zurich/lab/bead alginate beads] (d=3 mm, intially 10<sup>7</sup> cell/beads) on a [https://2014.igem.org/Team:ETH_Zurich/lab/chip custom-made millifluidic PDMS chip].''' All cells contained [https://2014.igem.org/Team:ETH_Zurich/expresults/rr#Riboregulators riboregulated] sfGFP followed by [http://parts.igem.org/Part:BBa_C0161 LuxI (BBa_C0161)] together under the control of the [http://parts.igem.org/Part:BBa_R0062 pLux promoter (BBa_R0062)], and [http://parts.igem.org/Part:BBa_J23100 constitutively (BBa_J23100)] expressed [http://parts.igem.org/Part:BBa_C0062 LuxR (BBa_C0062)]. LuxI catalyzes the production of the autoinducer 3OC6-HSL, which is then diffusing from cell to cell. For initialization, the cells in one bead of the top row were induced with 3OC6-HSL before encapsulation. 1750x faster than real-time, the video starts 7 h after the initiation of the experiment.<br />
|}<br />
<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/data/blueTeam:ETH Zurich/data/blue2014-10-18T03:19:02Z<p>Clormeau: </p>
<hr />
<div>{{:Team:ETH Zurich/tpl/head|Enlarged Data Page: Blue Cell Type}}<br />
<html><br />
<script type="text/javascript"><br />
(function($) {<br />
function img(url) {<br />
var i = new Image;<br />
i.src = url;<br />
return i;<br />
}<br />
<br />
if ('naturalWidth' in (new Image)) {<br />
$.fn.naturalWidth = function() { return this[0].naturalWidth; };<br />
$.fn.naturalHeight = function() { return this[0].naturalHeight; };<br />
return;<br />
}<br />
$.fn.naturalWidth = function() { return img(this[0].src).width; };<br />
$.fn.naturalHeight = function() { return img(this[0].src).height; };<br />
})(jQuery);<br />
<br />
<br />
<br />
function onWindowResize() <br />
{<br />
var curWidth = $(window).width(),<br />
curHeight = $(window).height(),<br />
checking=false;<br />
if (checking) {<br />
return;<br />
}<br />
checking = true;<br />
window.setTimeout(<br />
function() {<br />
var newWidth = $(window).width(),<br />
newHeight = $(window).height();<br />
if (!(newWidth !== curWidth ||<br />
newHeight !== curHeight)) {<br />
resize(false); <br />
}<br />
checking=false;<br />
}, 300);<br />
}<br />
<br />
function resize(initial) {<br />
if (!initial)<br />
{<br />
var container = $('#container');<br />
var imgWidth = container.width();<br />
<br />
$( "#map").each(function() {<br />
$(this).css('height', 'auto', 'width', 'auto');<br />
$(this).mapster('resize',Math.min(imgWidth, $(this).naturalWidth()) ,0,0); <br />
});<br />
}<br />
<br />
}<br />
<br />
$(document).ready(function(){<br />
<br />
$('#map').mapster({<br />
fillColor: 'ffffff',<br />
fillOpacity: 0.5,<br />
isSelectable: false,<br />
clickNavigate: true,<br />
});<br />
<br />
<br />
<br />
$(window).resize(<br />
function()<br />
{ <br />
onWindowResize();<br />
});<br />
resize(true);<br />
<br />
});<br />
<br />
</script><br />
</html><br />
<br />
<html><article id = "container"><br />
<img usemap="#map" id="map" src="/wiki/images/6/66/ETH_Zurich_DataPageBlue.png" style="width:100%;"/><br />
<map id="map" name="map"><area shape="poly" alt="" title="" coords="768,500,764,416,676,364,364,368,356,264,728,272,720,320,904,320,892,396,844,396,848,432,784,436,788,500,760,500" href="http://parts.igem.org/Part:BBa_J23100" target="" /><area shape="poly" alt="" title="" coords="1008,660,724,652,712,552,808,568,808,476,872,452,924,484,944,588,1032,572,1044,656" href="http://parts.igem.org/Part:BBa_B0034" target="" /><area shape="poly" alt="" title="" coords="976,476,1248,460,1256,408,1416,516,1264,600,1268,556,948,552,968,556,968,540" href="http://parts.igem.org/Part:BBa_C0062" target="" /><area shape="poly" alt="" title="" coords="1480,496,1480,416,1436,416,1212,372,1208,304,1528,312,1528,372,1560,380,1556,424,1516,420,1512,496,1488,500" href="http://parts.igem.org/Part:BBa_B0015" target="" /><area shape="poly" alt="" title="" coords="1580,500,1568,356,1628,352,1628,276,1976,280,1976,348,1760,372,1756,420,1616,440,1600,504" href="http://parts.igem.org/Part:BBa_J23100" target="" /><area shape="poly" alt="" title="" coords="2136,560,1840,552,1836,464,2140,472,2148,416,2276,508,2144,596" href="http://parts.igem.org/Part:BBa_C0179" target="" /><area shape="poly" alt="" title="" coords="1564,628,1904,640,1904,572,1796,556,1788,472,1720,444,1672,480,1660,532,1668,564,1556,568" href="http://parts.igem.org/Part:BBa_B0034" target="" /><area shape="poly" alt="" title="" coords="2348,496,2348,420,2296,416,2216,360,2224,288,2552,292,2548,376,2416,388,2420,412,2376,420,2376,496,2376,496" href="http://parts.igem.org/Part:BBa_B0015" target="" /><area shape="poly" alt="" title="" coords="488,1472,492,1396,448,1396,284,1352,292,1280,612,1304,600,1368,560,1364,564,1388,508,1396,524,1472" href="http://parts.igem.org/Part:BBa_B0012" target="" /><area shape="poly" alt="" title="" coords="792,1484,732,1388,636,1360,636,1288,700,1292,700,1220,1020,1240,1004,1312,820,1308,808,1484" href="http://parts.igem.org/Part:BBa_R0079" target="" /><area shape="poly" alt="" title="" coords="1252,1484,1240,1348,1112,1348,1112,1224,1416,1236,1416,1312,1280,1308,1344,1376,1344,1376" href="http://parts.igem.org/Part:BBa_R0079" target="" /><area shape="poly" alt="" title="" coords="848,1516,1220,1528,1220,1436,840,1436" href="http://parts.igem.org/Part:BBa_B0040" target="" /><area shape="poly" alt="" title="" coords="216,2092,220,2012,172,2004,172,1976,200,1896,496,1900,496,1964,292,1980,292,2000,252,2004,248,2092,228,2084" href="http://parts.igem.org/Part:BBa_B0012" target="" /><area shape="poly" alt="" title="" coords="768,2156,476,2140,476,2188,332,2104,468,2016,476,2060,768,2056" href="http://parts.igem.org/Part:BBa_I20284" target="" /><area shape="poly" alt="" title="" coords="1048,2088,956,1992,952,1844,1340,1852,1324,1896,1100,1912,1084,1972" href="http://parts.igem.org/Part:BBa_R0062" target="" /><area shape="poly" alt="" title="" coords="1504,2088,1496,1968,1412,1952,1404,1844,1784,1848,1748,1888,1592,1912,1580,2016,1532,2020" href="http://parts.igem.org/Part:BBa_R0062" target="" /><area shape="poly" alt="" title="" coords="1468,2136,1468,2056,1080,2052,1088,2140" href="http://parts.igem.org/Part:BBa_B0040" target="" /><area shape="poly" alt="" title="" coords="1868,2084,1864,2000,1804,1996,1696,1964,1700,1908,2004,1908,1988,1976,1940,1976,1936,2004,1892,2004,1892,2088" href="http://parts.igem.org/Part:BBa_B0012" target="" /><area shape="poly" alt="" title="" coords="1232,2324,828,2320,828,1832,2032,1828,2020,2324,1668,2320,1668,2264,1240,2256,1248,2344,1248,2348,2052,2344,2056,1812,796,1816,808,2340,1244,2348,1236,2348" href="http://parts.igem.org/Part:BBa_K154XXXX" target="" /><area shape="poly" alt="" title="" coords="344,2896,292,2764,292,2624,628,2632,616,2688,484,2700,484,2788,388,2840,368,2900,348,2896" href="http://parts.igem.org/Part:BBa_J23100" target="" /><area shape="poly" alt="" title="" coords="748,2896,744,2820,700,2816,588,2764,604,2704,916,2708,912,2776,816,2780,820,2816,776,2820,776,2904" href="http://parts.igem.org/Part:BBa_B0015" target="" /><area shape="poly" alt="" title="" coords="2052,2876,2320,2848,2344,2856,2336,2808,2516,2900,2344,2992,2336,2956,2044,2952" href="http://parts.igem.org/Part:BBa_C0161" target="" /><area shape="poly" alt="" title="" coords="1932,1572,2068,1476,1928,1396,1928,1440,1544,1440,1540,1528,1932,1528" href="http://parts.igem.org/Part:BBa_K1039012" target="" /><!-- Created by Online Image Map Editor (http://www.maschek.hu/imagemap/index) --></map></html><br />
<br/><br />
<center>Go back on the [https://2014.igem.org/Team:ETH_Zurich/data general data page] and learn more about the features of our system.</center><br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/labblog/20140921modTeam:ETH Zurich/labblog/20140921mod2014-10-18T03:12:01Z<p>Clormeau: </p>
<hr />
<div><html><article class="mix carousel modeling edinburgh" id='edinburgh' date="20140615"></html><br />
<br />
== Let a new collaboration begin! == <br />
==== Sunday, September 21th ====<br />
<br />
After discovering the awesome video of the Edinburgh team on Twitter, we wanted to know more about their project. As it is clear that the three quorum sensing systems we consider are crosstalking, finding a team focused on the development on communication ways between bacteria was really interesting for us. Their topic is metabolic wiring. <br />
<br/><br />
<br/><br />
We had a Skype meeting with them this week to discuss about possible opportunities of collaboration. Given the stages of our experiments, we decided that a theoretical collaboration would be more plausible. Next step will be to talk about the models and design a set-up that could fit our two projects.<br />
{{:Team:ETH Zurich/tpl/rmbutton|blue}}<br />
{{:Team:ETH Zurich/tpl/topbutton|blue}}<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/labblog/20140921modTeam:ETH Zurich/labblog/20140921mod2014-10-18T03:11:49Z<p>Clormeau: /* Let a new collaboration begin! */</p>
<hr />
<div><html><article class="mix modeling edinburgh" id='edinburgh' date="20140615"></html><br />
<br />
== Let a new collaboration begin! == <br />
==== Sunday, September 21th ====<br />
<br />
After discovering the awesome video of the Edinburgh team on Twitter, we wanted to know more about their project. As it is clear that the three quorum sensing systems we consider are crosstalking, finding a team focused on the development on communication ways between bacteria was really interesting for us. Their topic is metabolic wiring. <br />
<br/><br />
<br/><br />
We had a Skype meeting with them this week to discuss about possible opportunities of collaboration. Given the stages of our experiments, we decided that a theoretical collaboration would be more plausible. Next step will be to talk about the models and design a set-up that could fit our two projects.<br />
{{:Team:ETH Zurich/tpl/rmbutton|blue}}<br />
{{:Team:ETH Zurich/tpl/topbutton|blue}}<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/blogTeam:ETH Zurich/blog2014-10-18T03:09:52Z<p>Clormeau: </p>
<hr />
<div>{{:Team:ETH Zurich/tpl/head|Blog}}<br />
{{:Team:ETH Zurich/tpl/read-more}}<br />
<html> <h4 style='color:#FFFFFF; text-align:center;'> We have uploaded our chronological progress in several articles which are classified by subject. Click on the triangles below to filter the category of articles you want to read. If you want an even more precise filtering in that category, you can choose subjects on the mosaic around.</h4> <br/> <br/> </html> <br />
{{:Team:ETH Zurich/labblog/buttons}}<br />
<html><br/> <br/> </html> <br />
<!-- ADD YOUR ARTICLE HERE --><br />
<html><div id='Container'><br />
</html><br />
{{:Team:ETH_Zurich/labblog/20141014hum}}<br />
{{:Team:ETH Zurich/labblog/20141010hum}}<br />
{{:Team:ETH Zurich/labblog/20140930hum}}<br />
{{:Team:ETH_Zurich/labblog/20140929meet}}<br />
{{:Team:ETH_Zurich/labblog/20140922meet}}<br />
{{:Team:ETH_Zurich/labblog/20140921mod}}<br />
{{:Team:ETH Zurich/labblog/20140920hum}}<br />
{{:Team:ETH_Zurich/labblog/20140915meet}}<br />
{{:Team:ETH Zurich/labblog/20140915hum}}<br />
{{:Team:ETH_Zurich/labblog/20140909hum}}<br />
{{:Team:ETH_Zurich/labblog/20140908meet}}<br />
{{:Team:ETH Zurich/labblog/20140906}}<br />
{{:Team:ETH_Zurich/labblog/20140901meet}}<br />
{{:Team:ETH Zurich/labblog/20140829mod}}<br />
{{:Team:ETH_Zurich/labblog/20140825}}<br />
{{:Team:ETH_Zurich/labblog/20140825meet}}<br />
{{:Team:ETH_Zurich/labblog/20140824mod}}<br />
{{:Team:ETH_Zurich/labblog/20140822mod}}<br />
{{:Team:ETH_Zurich/labblog/20140820meet}}<br />
{{:Team:ETH_Zurich/labblog/20140818mod}}<br />
{{:Team:ETH Zurich/labblog/20140813meet}}<br />
{{:Team:ETH_Zurich/labblog/20140811}}<br />
{{:Team:ETH Zurich/labblog/20140806meet}}<br />
{{:Team:ETH Zurich/labblog/20140804}}<br />
{{:Team:ETH Zurich/labblog/20140803mod}}<br />
{{:Team:ETH_Zurich/labblog/20140802}}<br />
{{:Team:ETH Zurich/labblog/20140730meet}}<br />
{{:Team:ETH Zurich/labblog/20140723meet}}<br />
{{:Team:ETH Zurich/labblog/20140716meet}}<br />
{{:Team:ETH Zurich/labblog/20140709meet}}<br />
{{:Team:ETH Zurich/labblog/20140705}}<br />
{{:Team:ETH Zurich/labblog/20140702meet}}<br />
{{:Team:ETH Zurich/labblog/20140625meet}}<br />
{{:Team:ETH Zurich/labblog/20140618meet}}<br />
{{:Team:ETH Zurich/labblog/20140615mod}}<br />
{{:Team:ETH Zurich/labblog/20140611meet}} <br />
{{:Team:ETH Zurich/labblog/20140604meet}}<br />
{{:Team:ETH Zurich/labblog/20140528meet}}<br />
{{:Team:ETH_Zurich/labblog/20140510hum}}<br />
<html></div></html><br />
<html> </ul> </html><br />
<br />
<br />
<br />
<br />
{{:Team:ETH Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/blogTeam:ETH Zurich/blog2014-10-18T03:09:22Z<p>Clormeau: </p>
<hr />
<div>{{:Team:ETH Zurich/tpl/head|Blog}}<br />
{{:Team:ETH Zurich/tpl/read-more}}<br />
<html> <h4 style='color:#FFFFFF; text-align:center;'> We have uploaded our chronological progress in several articles which are classified by subject. Click on the triangles below to filter the category of articles you want to read. If you want an even more precise filtering in that category, you can choose subjects on the mosaic around.</h4> <br/> <br/> </html> <br />
{{:Team:ETH Zurich/labblog/buttons}}<br />
<html><br/> <br/> </html> <br />
<!-- ADD YOUR ARTICLE HERE --><br />
<html><div id='Container'><br />
</html><br />
{{:Team:ETH_Zurich/labblog/20141014hum}}<br />
{{:Team:ETH Zurich/labblog/20141010hum}}<br />
{{:Team:ETH Zurich/labblog/20140510hum}}<br />
{{:Team:ETH_Zurich/labblog/20140929meet}}<br />
{{:Team:ETH_Zurich/labblog/20140922meet}}<br />
{{:Team:ETH_Zurich/labblog/20140921mod}}<br />
{{:Team:ETH Zurich/labblog/20140920hum}}<br />
{{:Team:ETH_Zurich/labblog/20140915meet}}<br />
{{:Team:ETH Zurich/labblog/20140915hum}}<br />
{{:Team:ETH_Zurich/labblog/20140909hum}}<br />
{{:Team:ETH_Zurich/labblog/20140908meet}}<br />
{{:Team:ETH Zurich/labblog/20140906}}<br />
{{:Team:ETH_Zurich/labblog/20140901meet}}<br />
{{:Team:ETH Zurich/labblog/20140829mod}}<br />
{{:Team:ETH_Zurich/labblog/20140825}}<br />
{{:Team:ETH_Zurich/labblog/20140825meet}}<br />
{{:Team:ETH_Zurich/labblog/20140824mod}}<br />
{{:Team:ETH_Zurich/labblog/20140822mod}}<br />
{{:Team:ETH_Zurich/labblog/20140820meet}}<br />
{{:Team:ETH_Zurich/labblog/20140818mod}}<br />
{{:Team:ETH Zurich/labblog/20140813meet}}<br />
{{:Team:ETH_Zurich/labblog/20140811}}<br />
{{:Team:ETH Zurich/labblog/20140806meet}}<br />
{{:Team:ETH Zurich/labblog/20140804}}<br />
{{:Team:ETH Zurich/labblog/20140803mod}}<br />
{{:Team:ETH_Zurich/labblog/20140802}}<br />
{{:Team:ETH Zurich/labblog/20140730meet}}<br />
{{:Team:ETH Zurich/labblog/20140723meet}}<br />
{{:Team:ETH Zurich/labblog/20140716meet}}<br />
{{:Team:ETH Zurich/labblog/20140709meet}}<br />
{{:Team:ETH Zurich/labblog/20140705}}<br />
{{:Team:ETH Zurich/labblog/20140702meet}}<br />
{{:Team:ETH Zurich/labblog/20140625meet}}<br />
{{:Team:ETH Zurich/labblog/20140618meet}}<br />
{{:Team:ETH Zurich/labblog/20140615mod}}<br />
{{:Team:ETH Zurich/labblog/20140611meet}} <br />
{{:Team:ETH Zurich/labblog/20140604meet}}<br />
{{:Team:ETH Zurich/labblog/20140528meet}}<br />
{{:Team:ETH_Zurich/labblog/20140510hum}}<br />
<html></div></html><br />
<html> </ul> </html><br />
<br />
<br />
<br />
<br />
{{:Team:ETH Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/labblog/20140930humTeam:ETH Zurich/labblog/20140930hum2014-10-18T03:08:57Z<p>Clormeau: Created page with "<html><article class="mix human carousel talks" date="20140930"></html> == Talk for the public== ==== Tuesday, September 30th ==== ===Sharing iGEM with a heterogeneous public=..."</p>
<hr />
<div><html><article class="mix human carousel talks" date="20140930"></html><br />
<br />
== Talk for the public== <br />
==== Tuesday, September 30th ====<br />
===Sharing iGEM with a heterogeneous public===<br />
<br />
Today Max gave a presentation of our project to an open public. Everyone showed great interest and we could start thinking about how to present such a complex project with a single poster or presentation.<br />
<br />
{{:Team:ETH Zurich/tpl/topbutton|red}}<br />
{{:Team:ETH Zurich/tpl/rmbutton|red}}<br />
<br />
<html> </article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/labblog/20140929meetTeam:ETH Zurich/labblog/20140929meet2014-10-18T03:04:28Z<p>Clormeau: /* Monday, September 29th */</p>
<hr />
<div><html><article class="mix meetings integrase carousel biobricks" id='week19' date="20140922"></html><br />
<br />
== Week 19 : Biobricks, where are you? == <br />
<br />
==== Monday, September 29th ====<br />
<br />
After investigation, it is more difficult than expected to put our bricks in the biobrick format.<br />
<br />
To debug the integrase's experiment, we tried to have another insight by using FACS data.<br />
{{:Team:ETH_Zurich/tpl/rmbutton|silver}}<br />
{{:Team:ETH_Zurich/tpl/topbutton|silver}}<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/labblog/20140929meetTeam:ETH Zurich/labblog/20140929meet2014-10-18T03:04:20Z<p>Clormeau: /* Week 19 : Biobricks, where are you? */</p>
<hr />
<div><html><article class="mix meetings integrase carousel biobricks" id='week19' date="20140922"></html><br />
<br />
== Week 19 : Biobricks, where are you? == <br />
<br />
==== Monday, September 29th ====<br />
<br />
After investigation, it is more difficult than expected to put our bricks in the biobrick format.<br />
<br />
To debug the integrase's experiment, we tried to have another insight by using FACS data.<br />
{{:Team:ETH_Zurich/tpl/rmbutton|silver}}<br />
{{:Team:ETH_Zurich/tpl/topbutton|top}}<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/blogTeam:ETH Zurich/blog2014-10-18T03:03:32Z<p>Clormeau: </p>
<hr />
<div>{{:Team:ETH Zurich/tpl/head|Blog}}<br />
{{:Team:ETH Zurich/tpl/read-more}}<br />
<html> <h4 style='color:#FFFFFF; text-align:center;'> We have uploaded our chronological progress in several articles which are classified by subject. Click on the triangles below to filter the category of articles you want to read. If you want an even more precise filtering in that category, you can choose subjects on the mosaic around.</h4> <br/> <br/> </html> <br />
{{:Team:ETH Zurich/labblog/buttons}}<br />
<html><br/> <br/> </html> <br />
<!-- ADD YOUR ARTICLE HERE --><br />
<html><div id='Container'><br />
</html><br />
{{:Team:ETH_Zurich/labblog/20141014hum}}<br />
{{:Team:ETH_Zurich/labblog/20140929meet}}<br />
{{:Team:ETH_Zurich/labblog/20140922meet}}<br />
{{:Team:ETH_Zurich/labblog/20140921mod}}<br />
{{:Team:ETH Zurich/labblog/20140920hum}}<br />
{{:Team:ETH_Zurich/labblog/20140915meet}}<br />
{{:Team:ETH Zurich/labblog/20140915hum}}<br />
{{:Team:ETH_Zurich/labblog/20140909hum}}<br />
{{:Team:ETH_Zurich/labblog/20140908meet}}<br />
{{:Team:ETH Zurich/labblog/20140906}}<br />
{{:Team:ETH_Zurich/labblog/20140901meet}}<br />
{{:Team:ETH Zurich/labblog/20140829mod}}<br />
{{:Team:ETH_Zurich/labblog/20140825}}<br />
{{:Team:ETH_Zurich/labblog/20140825meet}}<br />
{{:Team:ETH_Zurich/labblog/20140824mod}}<br />
{{:Team:ETH_Zurich/labblog/20140822mod}}<br />
{{:Team:ETH_Zurich/labblog/20140820meet}}<br />
{{:Team:ETH_Zurich/labblog/20140818mod}}<br />
{{:Team:ETH Zurich/labblog/20140813meet}}<br />
{{:Team:ETH_Zurich/labblog/20140811}}<br />
{{:Team:ETH Zurich/labblog/20140806meet}}<br />
{{:Team:ETH Zurich/labblog/20140804}}<br />
{{:Team:ETH Zurich/labblog/20140803mod}}<br />
{{:Team:ETH_Zurich/labblog/20140802}}<br />
{{:Team:ETH Zurich/labblog/20140730meet}}<br />
{{:Team:ETH Zurich/labblog/20140723meet}}<br />
{{:Team:ETH Zurich/labblog/20140716meet}}<br />
{{:Team:ETH Zurich/labblog/20140709meet}}<br />
{{:Team:ETH Zurich/labblog/20140705}}<br />
{{:Team:ETH Zurich/labblog/20140702meet}}<br />
{{:Team:ETH Zurich/labblog/20140625meet}}<br />
{{:Team:ETH Zurich/labblog/20140618meet}}<br />
{{:Team:ETH Zurich/labblog/20140615mod}}<br />
{{:Team:ETH Zurich/labblog/20140611meet}} <br />
{{:Team:ETH Zurich/labblog/20140604meet}}<br />
{{:Team:ETH Zurich/labblog/20140528meet}}<br />
{{:Team:ETH_Zurich/labblog/20140510hum}}<br />
<html></div></html><br />
<html> </ul> </html><br />
<br />
<br />
<br />
<br />
{{:Team:ETH Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/labblog/20140915meetTeam:ETH Zurich/labblog/20140915meet2014-10-18T03:02:24Z<p>Clormeau: /* Week 17 : Integrase, again and again */</p>
<hr />
<div><html><article class="mix meetings carousel integrase" id='week17' date="20140528"></html><br />
<br />
== Week 17 : Integrase, again and again == <br />
<br />
==== Monday, September 15th ====<br />
<br />
After characterizing exhaustively crosstalk, it was more than time to begin with the integrases experiments. However, all trials in different experimental set-ups were negative. The construct used was built by our wetlab team. After trying to determine what the difference was between the published plasmid by Bonnet<sup>[[Team:ETH_Zurich/project/references|[9]]]</sup> and ours. No switching was observed after 20 hours with 1% arabinose.<br />
<br />
A diffusion assay with LuxI producers and LuxAHL receivers was done.<br />
<br />
In the model, the description of the quorum sensing module went on.<br />
<br />
{{:Team:ETH_Zurich/tpl/rmbutton|silver}}<br />
{{:Team:ETH_Zurich/tpl/topbutton|silver}}<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/labblog/20140915meetTeam:ETH Zurich/labblog/20140915meet2014-10-18T03:02:04Z<p>Clormeau: /* Week 17 : Integrase, again and again */</p>
<hr />
<div><html><article class="mix meetings carousel integrase" id='week17' date="20140528"></html><br />
<br />
== Week 17 : Integrase, again and again == <br />
<br />
==== Monday, September 15th ====<br />
<br />
After characterizing exhaustively crosstalk, it was more than time to begin with the integrases experiments. However, all trials in different experimental set-ups were negative. The construct used was built by our wetlab team. After trying to determine what the difference was between the published plasmid by Bonnet<sup>[[Team:ETH_Zurich/project/references|[9]]]</sup> and ours. No switching was observed after 20 hours with 1% arabinose.<br />
<br />
A diffusion assay with LuxI producers and LuxAHL receivers was done.<br />
<br />
In the model, the description of the quorum sensing module went on.<br />
{{:Team:ETH_Zurich/tpl/rmbutton|silver}}<br />
{{:Team:ETH_Zurich/tpl/topbutton|silver}}<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/labblog/20140920humTeam:ETH Zurich/labblog/20140920hum2014-10-18T02:58:46Z<p>Clormeau: </p>
<hr />
<div><html><article class="mix carousel survey human" date="20140920hum"></html><br />
<br />
== 500 survey responses reached !== <br />
==== Saturday, September 20th ====<br />
<br />
We reached 500 responses for our [https://2014.igem.org/Team:ETH_Zurich/human/survey survey ] ! If you still want to participate, here are the links in[http://docs.google.com/forms/d/1pvFPVzfH1aiNdy3MdAaaFFzeWysCZyDDybcqgt7LyzY/viewform?usp=send_form ''' English'''], [http://docs.google.com/forms/d/1nLa1qMEAo9QjmIsOJgaN5pEVURDlVDqYKXuOvFnpjW4/viewform?usp=send_form ''' German'''] and [http://docs.google.com/forms/d/1fQ0ZjKvnr-Ssek9fihlDRhmQsVaG8rwxCrNGHDC2JDU/viewform?usp=send_form ''' French'''].<br />
<br />
{{:Team:ETH_Zurich/tpl/topbutton|red}}<br />
{{:Team:ETH_Zurich/tpl/rmbutton|red|week13}}<br />
<html> </article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/labblog/20140922meetTeam:ETH Zurich/labblog/20140922meet2014-10-18T02:57:11Z<p>Clormeau: /* Week 18 : Decisive meeting */</p>
<hr />
<div><html><article class="mix meetings whole carousel biobricks" id='week18' date="20140922"></html><br />
<br />
== Week 18 : Decisive meeting == <br />
<br />
==== Monday, September 22th ====<br />
<br />
This meeting was a turning point of our iGEM adventure. We reviewed all the experiments that had been done so far. Most of them concerns quorum sensing and its non-orthogonality. We summed up the results in a three-layer graphs.<br />
<br />
The dry lab part presented for the first time a whole-cell model. With the parameters estimated, the model actually implemented an XOR gate. However, leakiness of promoters and crosstalk seemed to be affecting the robustness of our design.<br />
<br />
As all our experiments were done in our plasmids, we have to standardize them into the Biobrick format. This year, the deadline corresponds to the delivery in Boston of the biobricks. Thus, it is becoming slowly urgent...<br />
{{:Team:ETH_Zurich/tpl/rmbutton|silver}}<br />
{{:Team:ETH_Zurich/tpl/topbutton|silver|top}}<br />
<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/labblog/20141014humTeam:ETH Zurich/labblog/20141014hum2014-10-18T02:56:09Z<p>Clormeau: /* Tuesday, October 14th */</p>
<hr />
<div><html><article class="mix human school carousel" id='schoolVisit' date="20141014"></html><br />
<br />
== High School Students == <br />
<br />
==== Tuesday, October 14th ====<br />
<br />
On Oct 14 2014 Stefanie and Nadine visited a high school in Bern as a part of our human practice project. The girls had the chance to teach students at the age of 16-17 years. 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. The structure of the lessons was really open for personal inputs from the side of the students. The topics discussed ranged from whether you should or should not express GFP in other organisms (the question of freewill came up), animal trials in research, GMO's in general to the production of artificial meat in bioreactors.<br />
<br />
Furthermore former iGEM projects were quickly outlined and their possibilities and potential discussed. Thus, we increased the level of awareness of synthetic biology and simultaneously teach the students how to approach complex problems. Another aspect of our visit was to promote the studies of science in general. The students were encouraged to ask all questions that came to their minds. <br />
This school visit belongs to the outreach aspect of our human practice project as it aims to share knowledge with the public.<br />
<br />
{{:Team:ETH_Zurich/tpl/rmbutton|red}}<br />
{{:Team:ETH_Zurich/tpl/topbutton|red|}}<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/labblog/20141014humTeam:ETH Zurich/labblog/20141014hum2014-10-18T02:55:38Z<p>Clormeau: /* High School Students */</p>
<hr />
<div><html><article class="mix human school carousel" id='schoolVisit' date="20141014"></html><br />
<br />
== High School Students == <br />
<br />
==== Tuesday, October 14th ====<br />
<br />
On Oct 14 2014 Stefanie and Nadine visited a high school in Bern as a part of our human practice project. The girls had the chance to teach students at the age of 16-17 years. 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. The structure of the lessons was really open for personal inputs from the side of the students. The topics discussed ranged from whether you should or should not express GFP in other organisms (the question of freewill came up), animal trials in research, GMO's in general to the production of artificial meat in bioreactors.<br />
<br />
Furthermore former iGEM projects were quickly outlined and their possibilities and potential discussed. Thus, we increased the level of awareness of synthetic biology and simultaneously teach the students how to approach complex problems. Another aspect of our visit was to promote the studies of science in general. The students were encouraged to ask all questions that came to their minds. <br />
This school visit belongs to the outreach aspect of our human practice project as it aims to share knowledge with the public.<br />
<br />
{{:Team:ETH_Zurich/tpl/rmbutton|red}}<br />
{{:Team:ETH_Zurich/tpl/topbutton|top|}}<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/labblog/20141014humTeam:ETH Zurich/labblog/20141014hum2014-10-18T02:55:19Z<p>Clormeau: </p>
<hr />
<div><html><article class="mix human school carousel" id='schoolVisit' date="20141014"></html><br />
<br />
== High School Students == <br />
<br />
==== Tuesday, October 14th ====<br />
<br />
On Oct 14 2014 Stefanie and Nadine visited a high school in Bern as a part of our human practice project. The girls had the chance to teach students at the age of 16-17 years. 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. The structure of the lessons was really open for personal inputs from the side of the students. The topics discussed ranged from whether you should or should not express GFP in other organisms (the question of freewill came up), animal trials in research, GMO's in general to the production of artificial meat in bioreactors.<br />
<br />
Furthermore former iGEM projects were quickly outlined and their possibilities and potential discussed. Thus, we increased the level of awareness of synthetic biology and simultaneously teach the students how to approach complex problems. Another aspect of our visit was to promote the studies of science in general. The students were encouraged to ask all questions that came to their minds. <br />
This school visit belongs to the outreach aspect of our human practice project as it aims to share knowledge with the public.<br />
<br />
{{:Team:ETH_Zurich/tpl/rmbutton|red}}<br />
{{:Team:ETH_Zurich/tpl/topbutton|top}}<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/labblog/20140920humTeam:ETH Zurich/labblog/20140920hum2014-10-18T02:53:07Z<p>Clormeau: /* 500 survey responses reached ! */</p>
<hr />
<div><html><article class="mix carousel survey" date="20140920hum"></html><br />
<br />
== 500 survey responses reached !== <br />
==== Saturday, September 20th ====<br />
<br />
We reached 500 responses for our [https://2014.igem.org/Team:ETH_Zurich/human/survey survey ] ! If you still want to participate, here are the links in[http://docs.google.com/forms/d/1pvFPVzfH1aiNdy3MdAaaFFzeWysCZyDDybcqgt7LyzY/viewform?usp=send_form ''' English'''], [http://docs.google.com/forms/d/1nLa1qMEAo9QjmIsOJgaN5pEVURDlVDqYKXuOvFnpjW4/viewform?usp=send_form ''' German'''] and [http://docs.google.com/forms/d/1fQ0ZjKvnr-Ssek9fihlDRhmQsVaG8rwxCrNGHDC2JDU/viewform?usp=send_form ''' French'''].<br />
<br />
{{:Team:ETH_Zurich/tpl/topbutton|red}}<br />
{{:Team:ETH_Zurich/tpl/rmbutton|red|week13}}<br />
<html> </article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/blogTeam:ETH Zurich/blog2014-10-18T02:52:49Z<p>Clormeau: </p>
<hr />
<div>{{:Team:ETH Zurich/tpl/head|Blog}}<br />
{{:Team:ETH Zurich/tpl/read-more}}<br />
<html> <h4 style='color:#FFFFFF; text-align:center;'> We have uploaded our chronological progress in several articles which are classified by subject. Click on the triangles below to filter the category of articles you want to read. If you want an even more precise filtering in that category, you can choose subjects on the mosaic around.</h4> <br/> <br/> </html> <br />
{{:Team:ETH Zurich/labblog/buttons}}<br />
<html><br/> <br/> </html> <br />
<!-- ADD YOUR ARTICLE HERE --><br />
<html><div id='Container'><br />
</html><br />
{{:Team:ETH_Zurich/labblog/20141014hum}}<br />
{{:Team:ETH_Zurich/labblog/20140922meet}}<br />
{{:Team:ETH_Zurich/labblog/20140921mod}}<br />
{{:Team:ETH Zurich/labblog/20140920hum}}<br />
{{:Team:ETH_Zurich/labblog/20140915meet}}<br />
{{:Team:ETH Zurich/labblog/20140915hum}}<br />
{{:Team:ETH_Zurich/labblog/20140909hum}}<br />
{{:Team:ETH_Zurich/labblog/20140908meet}}<br />
{{:Team:ETH Zurich/labblog/20140906}}<br />
{{:Team:ETH_Zurich/labblog/20140901meet}}<br />
{{:Team:ETH Zurich/labblog/20140829mod}}<br />
{{:Team:ETH_Zurich/labblog/20140825}}<br />
{{:Team:ETH_Zurich/labblog/20140825meet}}<br />
{{:Team:ETH_Zurich/labblog/20140824mod}}<br />
{{:Team:ETH_Zurich/labblog/20140822mod}}<br />
{{:Team:ETH_Zurich/labblog/20140820meet}}<br />
{{:Team:ETH_Zurich/labblog/20140818mod}}<br />
{{:Team:ETH Zurich/labblog/20140813meet}}<br />
{{:Team:ETH_Zurich/labblog/20140811}}<br />
{{:Team:ETH Zurich/labblog/20140806meet}}<br />
{{:Team:ETH Zurich/labblog/20140804}}<br />
{{:Team:ETH Zurich/labblog/20140803mod}}<br />
{{:Team:ETH_Zurich/labblog/20140802}}<br />
{{:Team:ETH Zurich/labblog/20140730meet}}<br />
{{:Team:ETH Zurich/labblog/20140723meet}}<br />
{{:Team:ETH Zurich/labblog/20140716meet}}<br />
{{:Team:ETH Zurich/labblog/20140709meet}}<br />
{{:Team:ETH Zurich/labblog/20140705}}<br />
{{:Team:ETH Zurich/labblog/20140702meet}}<br />
{{:Team:ETH Zurich/labblog/20140625meet}}<br />
{{:Team:ETH Zurich/labblog/20140618meet}}<br />
{{:Team:ETH Zurich/labblog/20140615mod}}<br />
{{:Team:ETH Zurich/labblog/20140611meet}} <br />
{{:Team:ETH Zurich/labblog/20140604meet}}<br />
{{:Team:ETH Zurich/labblog/20140528meet}}<br />
{{:Team:ETH_Zurich/labblog/20140510hum}}<br />
<html></div></html><br />
<html> </ul> </html><br />
<br />
<br />
<br />
<br />
{{:Team:ETH Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/labblog/20140920humTeam:ETH Zurich/labblog/20140920hum2014-10-18T02:52:15Z<p>Clormeau: Created page with "<html><article class="mix carousel survey" date="20140920hum"></html> == 500 survey responses reached !== ==== Saturday, September 20th ==== We reached 500 responses for our ..."</p>
<hr />
<div><html><article class="mix carousel survey" date="20140920hum"></html><br />
<br />
== 500 survey responses reached !== <br />
==== Saturday, September 20th ====<br />
<br />
We reached 500 responses for our [survey https://2014.igem.org/Team:ETH_Zurich/human/survey] ! If you still want to participate, here are the links in[http://docs.google.com/forms/d/1pvFPVzfH1aiNdy3MdAaaFFzeWysCZyDDybcqgt7LyzY/viewform?usp=send_form ''' English'''], [http://docs.google.com/forms/d/1nLa1qMEAo9QjmIsOJgaN5pEVURDlVDqYKXuOvFnpjW4/viewform?usp=send_form ''' German'''] and [http://docs.google.com/forms/d/1fQ0ZjKvnr-Ssek9fihlDRhmQsVaG8rwxCrNGHDC2JDU/viewform?usp=send_form ''' French'''].<br />
<br />
{{:Team:ETH_Zurich/tpl/topbutton|red}}<br />
{{:Team:ETH_Zurich/tpl/rmbutton|red|week13}}<br />
<html> </article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/labblog/20140820meetTeam:ETH Zurich/labblog/20140820meet2014-10-18T02:46:34Z<p>Clormeau: </p>
<hr />
<div><html><article class="mix meetings carousel survey" id="week13" date="20140820"></html><br />
<br />
== Week 13 : Steady state quorum sensing fitted and human practice survey launched !== <br />
<br />
==== Wednesday, August 20th ====<br />
<br />
* Our first diffusion try with highlighter ink !<br />
<br />
[[File:ETH Zurich Diffusion1.PNG|center|500px|thumb|Camera picture of our punched agar chip glowing highlighter ink]]<br />
<br />
The design of punched agar doesn't seem to be the best but at least we can see something. Diffusion experiments with different designs are running now.<br />
<br />
* We have been fitting quorum sensing experiments at steady state to a Hill function and found some quorum sensing parameters.<br />
<br />
[[File:ETH Zurich Model3.PNG|center|700px|thumb|Fitting quorum sensing Hill function parameters thanks to steady-state data from experiments. Check <html> <a targetid="qsparameters" id="insidelink" href="https://2014.igem.org/Team:ETH_Zurich/blog#qsparameters"> the corresponding article </a> </html> for more details.]]<br />
<br />
* We wrote a [https://docs.google.com/forms/d/1pvFPVzfH1aiNdy3MdAaaFFzeWysCZyDDybcqgt7LyzY/viewform survey in English], [https://docs.google.com/forms/d/1fQ0ZjKvnr-Ssek9fihlDRhmQsVaG8rwxCrNGHDC2JDU/viewform in French] and [https://docs.google.com/forms/d/1nLa1qMEAo9QjmIsOJgaN5pEVURDlVDqYKXuOvFnpjW4/viewform in German] on complexity for our human practice project. We expect 500 responses.<br />
<br />
{{:Team:ETH_Zurich/tpl/topbutton|silver}}<br />
{{:Team:ETH_Zurich/tpl/rmbutton|silver|week13}}<br />
<html> </article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/labblog/20140709meetTeam:ETH Zurich/labblog/20140709meet2014-10-18T02:45:48Z<p>Clormeau: </p>
<hr />
<div><html><article class="mix meetings carousel human" id='week7' date="20140709"></html><br />
<br />
== Week 7: Human practice planning, Plasmid assembly running, Logo design == <br />
<br />
==== Wednesday, July 9th ====<br />
<br />
* We have a logo !<br />
[[File:ETH_Zurich_mosaicoli.png|300px]]<br />
*In the lab, we did some :<br />
**Plasmid preparation<br />
**Sequencing<br />
**Digests<br />
**Purification of backbone fragments needed for GA<br />
<br />
*On the modeling side, we tried to estimate integrase parameters from the paper from Bonnet et al. <sup>[[Team:ETH_Zurich/project/references#refEmergence|[9]]]</sup>, more particularly with the figure S4 :<br />
[[File:ETH Zurich Bonnet figure.PNG|center|500px|thumb|Data we are using from the Bonnet et al. [[Team:ETH_Zurich/project/references#refEmergence| <sup>[9]</sup>]] paper]]<br />
<br />
We are trying several strategies : minimization of the error function and Markov Chain Monte Carlo.<br />
<br />
* We have a precise plan for our human practice project. We would like to study the emergence of complexity in many different fields and investigate how people deal with it. We will do this with interviews of experts in different fields, and with a survey for a wider outreach. We will also reach younger people by organizing talks in schools. We will finally give our own advice on the question by writing an essay on the subject and linking it to our experience with Mosaicoli. <br />
<br />
{{:Team:ETH Zurich/tpl/topbutton|silver}}<br />
{{:Team:ETH Zurich/tpl/rmbutton|silver|week7}}<br />
<br />
<html> </article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/modeling/parametersTeam:ETH Zurich/modeling/parameters2014-10-18T02:34:20Z<p>Clormeau: /* Parameters */</p>
<hr />
<div>{{:Team:ETH Zurich/tpl/head|Parameters and Tools}}<br />
{{:Team:ETH Zurich/tpl/fortables}}<br />
<br />
<br />
<html><article></html><br />
==Parameters==<br />
No model is complete without parameters. Our exhaustive list of parameters are summarised in the table below. <br />
<br />
{|class="wikitable sortable"<br />
!Parameter!!Value!!Description!!Reference<br />
|-<br />
|α<sub>LuxR</sub>||0.005 μMmin<sup>-1</sup>||Production rate of LuxR||Literature <sup>[[Team:ETH_Zurich/project/references|[20]]]</sup><br />
|-<br />
|k<sub>RLux</sub>||0.1 nM<sup>-1</sup>min<sup>-1</sup>|| Rate of formation of RLux from LuxAHL and LuxR||Literature <sup>[[Team:ETH_Zurich/project/references|[19]]]</sup><br />
|-<br />
|k<sub>-RLux</sub>||10 min<sup>-1</sup>||Dissociation rate of RLux ||Literature <sup>[[Team:ETH_Zurich/project/references|[19]]]</sup><br />
|-<br />
|K<sub>mLux</sub>||10 nM||Lumped parameter for the Lux system|| [https://2014.igem.org/Team:ETH_Zurich/modeling/qs#Parameters Fitted to experimental data]<br />
|-<br />
|d<sub>LuxAHL</sub>||0.004 min<sup>-1</sup>||External degradation rate of LuxAHL (30C6HSL)||[https://2014.igem.org/Team:ETH_Zurich/modeling/qs#Degradation Fitted to experimental data]<br />
|-<br />
|d<sub>LuxR</sub>||0.0231 min<sup>-1</sup>||Degradation rate of LuxR||Literature <sup>[[Team:ETH_Zurich/project/references|[21]]]</sup><br />
|-<br />
|d<sub>RLux</sub>||0.0231 min<sup>-1</sup>||Degradation rate of RLux||Literature <sup>[[Team:ETH_Zurich/project/references|[20]]]</sup><br />
|-<br />
|d<sub>mRNABxb1</sub>||0.2773 min<sup>-1</sup>||Degradation rate of mRNA<sub>Bxb1</sub>||Literature <sup>[[Team:ETH_Zurich/project/references|[22]]]</sup><br />
|-<br />
|d<sub>Bxb1</sub>||0.01 min<sup>-1</sup>||Degradation rate of Bxb1||Assumed<br />
|-<br />
|L<sub>PLux</sub>||0.01463 nMmin<sup>-1</sup>||Leakiness after using riboswitch for P<sub>lux</sub>||[https://2014.igem.org/Team:ETH_Zurich/modeling/qs Fitted to experimental data]<br />
|-<br />
|K<sub>mRNABxb1</sub>||5 nMmin<sup>-1</sup>||Rate of transcription of Bxb1||Estimated<br />
|-<br />
|k<sub>Bxb1</sub>||0.1 min<sup>-1</sup>||Rate of formation of Bxb1||Assumed<br />
|-<br />
|α<sub>LasR</sub>||0.005 μMmin<sup>-1</sup>||Production rate of LasR||Literature <sup>[[Team:ETH_Zurich/project/references|[20]]]</sup>(Assumed to be the same as Lux system)<br />
|-<br />
|k<sub>RLas</sub>||0.1 nM<sup>-1</sup>min<sup>-1</sup>|| Rate of formation of RLas from LasAHL and LasR||Literature <sup>[[Team:ETH_Zurich/project/references|[19]]]</sup>(Assumed to be the same as Lux system)<br />
|-<br />
|k<sub>-RLas</sub>||10 min<sup>-1</sup>||Dissociation rate of RLas ||Literature <sup>[[Team:ETH_Zurich/project/references|[19]]]</sup>(Assumed to be the same as Lux system)<br />
|-<br />
|K<sub>mLas</sub>||0.45 nM||Lumped parameter for the Las system ||[https://2014.igem.org/Team:ETH_Zurich/modeling/qs Fitted to experimental data]<br />
|-<br />
|d<sub>LasAHL</sub>||0.004 min<sup>-1</sup>||Degradation rate of LasAHL (30C12HSL)||Fitted to experimental data<br />
|-<br />
|d<sub>LasR</sub>||0.0231 min<sup>-1</sup>||Degradation rate of LasR||Literature <sup>[[Team:ETH_Zurich/project/references|[21]]]</sup> (Assumed to be the same as Lux system)<br />
|-<br />
|d<sub>RLas</sub>||0.0231 min<sup>-1</sup>||Degradation rate of RLas||Literature <sup>[[Team:ETH_Zurich/project/references|[20]]]</sup> (Assumed to be the same as<br />
Lux system)<br />
|-<br />
|d<sub>mRNAϕc31</sub>||0.2773 min<sup>-1</sup>||Degradation rate of mRNA<sub>ϕc31</sub>||Literature <sup>[[Team:ETH_Zurich/project/references|[22]]]</sup><br />
|-<br />
|d<sub>ϕc31</sub>||0.01 min<sup>-1</sup>||Degradation rate of ϕC31||Assumed<br />
|-<br />
|L<sub>PLas</sub>||0.02461 nMmin<sup>-1</sup>||Leakiness after using riboswitch for P<sub>las</sub>||[https://2014.igem.org/Team:ETH_Zurich/modeling/qs Fitted to experimental data]<br />
|-<br />
|K<sub>mRNAϕc31</sub>||5 nMmin<sup>-1</sup>||Rate of transcription of ϕc31||Estimated<br />
|-<br />
|k<sub>ϕc31</sub>||0.1 min<sup>-1</sup>||Rate of formation of ϕc31||Assumed<br />
|-<br />
|k<sub>DBxb1</sub>||1 nM<sup>-1</sup>min<sup>-1</sup>||Dimerization rate of Bxb1||[https://2014.igem.org/Team:ETH_Zurich/modeling/int Fitted]<br />
|-<br />
|k<sub>-DBxb1</sub>||10<sup>-6</sup> min<sup>-1</sup>||Dissociation rate of DBxb1|| [https://2014.igem.org/Team:ETH_Zurich/modeling/int Fitted]<br />
|-<br />
|k<sub>SABxb1</sub>||1 nM<sup>-1</sup>min<sup>-1</sup>||Rate of formation of SA<sub>Bxb1</sub> from DBxb1 and SI<sub>Bxb1</sub>|| [https://2014.igem.org/Team:ETH_Zurich/modeling/int Fitted]<br />
|-<br />
|k<sub>-SABxb1</sub>||10<sup>-6</sup> min<sup>-1</sup>||Dissociation rate of SA<sub>Bxb1</sub>|| [https://2014.igem.org/Team:ETH_Zurich/modeling/int Fitted]<br />
|-<br />
|d<sub>DBxb1</sub>||0.02 min<sup>-1</sup>||Degradation rate of DBxb1||Assumed<br />
|-<br />
|k<sub>Dϕc31</sub>||1 nM<sup>-1</sup>min<sup>-1</sup>||Dimerization rate of ϕc31|| [https://2014.igem.org/Team:ETH_Zurich/modeling/int Fitted]<br />
|-<br />
|k<sub>-Dϕc31</sub>||10<sup>-6</sup> min<sup>-1</sup>||Rate of dissociation of Dϕc31|| [https://2014.igem.org/Team:ETH_Zurich/modeling/int Fitted]<br />
|-<br />
|k<sub>SAϕc31</sub>||1 nM<sup>-1</sup>min<sup>-1</sup>||Rate of formation of SA<sub>ϕc31</sub> from Dϕc31 and SI<sub>ϕc31</sub>||[https://2014.igem.org/Team:ETH_Zurich/modeling/int Fitted]<br />
|-<br />
|k<sub>-SAϕc31</sub>||10<sup>-6</sup> min<sup>-1</sup>||Rate of dissociation of SA<sub>ϕc31</sub>|| [https://2014.igem.org/Team:ETH_Zurich/modeling/int Fitted]<br />
|-<br />
|d<sub>Dϕc31</sub>||0.02 min<sup>-1</sup>||Degradation rate of Dϕc31||Assumed<br />
|-<br />
|k<sub>ToffBxb1</sub>||0.1 nM<sup>-2</sup>min<sup>-1</sup>||Rate of flipping of T<sub>on,i</sub> to T<sub>offBxb1</sub>||Assumed<br />
|-<br />
|k<sub>-ToffBxb1</sub>||0.1 nM<sup>-2</sup>min<sup>-1</sup>||Rate of flipping of T<sub>offBxb1</sub> to T<sub>on,f</sub>||Assumed<br />
|-<br />
|k<sub>Toffϕc31</sub>||0.1 nM<sup>-2</sup>min<sup>-1</sup>||Rate of flipping of T<sub>on,i</sub> to T<sub>offϕc31</sub>||Assumed<br />
|-<br />
|k<sub>-Toffϕc31</sub>||0.1 nM<sup>-2</sup>min<sup>-1</sup>||Rate of flipping of T<sub>offϕc31</sub> to T<sub>on,f</sub>||Assumed<br />
|-<br />
|k<sub>mRNAGFP</sub>||5 nMmin<sup>-1</sup>||Production rate of mRNA<sub>GFP</sub>||Estimated<br />
|-<br />
|k<sub>GFP</sub>||1 min<sup>-1</sup>||Rate of formation of folded GFP||Estimated<br />
|-<br />
|d<sub>mRNAGFP</sub>||0.2773 min<sup>-1</sup>||Degradation rate of mRNA<sub>GFP</sub>||Literature <sup>[[Team:ETH_Zurich/project/references|[22]]]</sup><br />
|-<br />
|d<sub>GFP</sub>||0.0049 min<sup>-1</sup>||Degradation rate of GFP||Fitted to experimental data<br />
|-<br />
|k<sub>mRNALasI</sub>||5 nMmin<sup>-1</sup>||Production rate of mRNA<sub>LasI</sub>||Estimated<br />
|-<br />
|k<sub>LasI</sub>||20 min<sup>-1</sup>||Rate of formation of LasI||Estimated<br />
|-<br />
|d<sub>mRNALasI</sub>||0.2773 min<sup>-1</sup>||Degradation rate of mRNA<sub>LasI</sub>||Literature <sup>[[Team:ETH_Zurich/project/references|[22]]]</sup><br />
|-<br />
|d<sub>LasI</sub>||0.0167 min<sup>-1</sup>||Degradation rate of LasI||Literature <sup>[[Team:ETH_Zurich/project/references|[21]]]</sup><br />
|-<br />
|k<sub>LasAHL</sub>||0.04 min<sup>-1</sup>||Production rate of LasAHL (30C12HSL) from the LasI||Literature <sup>[[Team:ETH_Zurich/project/references|[19]]]</sup><br />
|-<br />
|θ||0.01 μM||K<sub>m</sub> value for the production of mRNA<sub>GFP</sub> and mRNA<sub>LasI</sub>||Literature <sup>[[Team:ETH_Zurich/project/references|[20]]]</sup> (approximation)<br />
|-<br />
|D<sub>AHLext</sub>||4.9 10<sup>-6</sup> cm<sup>2</sup>/s||Diffusion coefficient of extracellular AHL in liquid||Literature <sup>[[Team:ETH_Zurich/project/references#Stewart|[27]]]</sup> <br />
|-<br />
|D<sub>m</sub>||100 min<sup>-1</sup>||Diffusion rate of AHL through the membrane||[https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel#Estimation Estimated] from literature <sup>[[Team:ETH_Zurich/project/references|[27]]]</sup><br />
|-<br />
|r||0.006 min<sup>-1</sup>||Growth rate of ''E. coli'' in our alginate beads||<br />
|-<br />
|&alpha;||100 min<sup>-1</sup>||Ratio of '' E. coli'' volume to the volume of one bead|| V<sub>'' E. coli''</sub> from literature <sup>[[Team:ETH_Zurich/project/references#Kaplan|[28]]]</sup>, bead volume from [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion experimental setup]<br />
|-<br />
|N<sub>0</sub>||10<sup>7</sup> cells||Initial number of cells per bead|| [https://2014.igem.org/Team:ETH_Zurich/expresults#Diffusion Experimental setup]<br />
|-<br />
|N<sub>m</sub>||8 10<sup>7</sup> cells||Maximum number of cells per bead|| [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel#Estimation Estimated] from literature <sup>[[Team:ETH_Zurich/project/references#Lars | [29]]]</sup><br />
|-<br />
|C<sub>beads</sub>||1||Correction factor (a priori) for diffusion of LuxAHL in alginate beads|| [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel#Estimation Estimated] from literature <sup>[[Team:ETH_Zurich/project/references#Cronenberg | [30]]]</sup><br />
|}<br />
<html></article></html><br />
<br />
<html><article></html><br />
<br />
==Tools==<br />
<br />
We used the following tools for modelling and simulation:<br />
<br />
* MATLAB version 8.3.0.532 (R2014a). Natick, Massachusetts: The MathWorks Inc., 2014. for deterministic model, curve fitting (function: fittype ; robustness option: LAR) and parameter estimation. <br />
* COMSOL Multiphysics software Version 4.4.0.248, COMSOL Ltd, 2014, for diffusion model and simulation.<br />
* MEIGO Toolbox for parameter estimation.<sup>[[Team:ETH_Zurich/project/references|[26]]]</sup><br />
<html></article></html><br />
{{:Team:ETH Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/modeling/qsTeam:ETH Zurich/modeling/qs2014-10-18T02:33:41Z<p>Clormeau: </p>
<hr />
<div>{{:Team:ETH Zurich/tpl/head|Quorum Sensing}}<br />
{{:Team:ETH Zurich/tpl/fortables}}<br />
<br />
<center><br />
{{:Team:ETH Zurich/tpl/scrollbutton3|Model|green}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton2|Parameters|blue}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton3|Leakiness|red}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton|Cross-talk|blue}}<br />
{{:Team:ETH Zurich/tpl/scrollbuttontworows|Alternate|Design|green}}<br />
</center><br />
<br />
<html><article id="Model"></html><br />
== Model ==<br />
<br />
The Quorum sensing module is mainly involved in receiving signals from the sender cells. The sender cells produce some signaling molecules (inducers) which diffuse out of their membrane, then diffuse in receiver cells membrane, and bind to the regulator molecules in the receiver cells, thus activating the transcription of certain genes. In order to characterize the quorum sensing module with a transfer function, we consider different initial inputs of external AHL, and see how much output is produced, as it was done in the [https://2014.igem.org/Team:ETH_Zurich/expresults#Quorum_Sensing quorum sensing experiments].<br />
<br />
<br />
As diffusion through the membrane is very fast<sup>[[Team:ETH_Zurich/project/references|[27]]]</sup>, according to Fick's law of diffusion, internal and external concentration of AHL can always be considered as equal. This can also be observed in the [https://2014.igem.org/Team:ETH_Zurich/modeling/diffmodel#Results diffusion model results]. When an initial external AHL concentration is given, AHL diffuses into the cells very quickly (less than 20 seconds)<sup>[[Team:ETH_Zurich/project/references|[27]]]</sup> until internal AHL concentration equals external concentration. Then as soon as some internal AHL is consumed in the cell, it is taken up again without affecting external concentration, because external volume is very high compared to internal volume. Therefore we can consider in this module that external AHL (which is equal to internal AHL) only degrades, with the rate of extracellular decay.<br />
<br />
<br/><br />
<br/><br />
<br />
=== Chemical Species ===<br />
{| class="wikitable"<br />
|-<br />
! '''Name'''<br />
! Description<br />
|-<br />
|'''LuxAHL'''<br />
|30C6-HSL is an acyl homoserine lactone which diffuses into the cell and mainly binds to LuxR. Here we consider internal concentrations of LuxAHL.<br />
|-<br />
|'''LuxR'''<br />
|Constitutively expressed regulator protein that can bind LuxAHL and stimulate transcription of Bxb1.<br />
|-<br />
|'''RLux'''<br />
|LuxR and LuxAHL complex which can dimerize.<br />
|-<br />
|'''DRLux '''<br />
|Dimerized form of RLux.<br />
|-<br />
|'''mRNA<sub>Bxb1</sub>'''<br />
|mRNA of the Bxb1 integrase being transcribed by the Lux promoter.<br />
|-<br />
|'''Bxb1'''<br />
|Serine integrase that can fold into two conformations - Bxb1a and Bxb1b. We chose to use a common connotation for both conformations - Bxb1.<br />
|-<br />
|'''LasAHL'''<br />
|30C12-HSL is an acyl homoserine lactone which diffuses into the cell and mainly binds to LasR. Here we consider internal concentrations of LasAHL.<br />
|-<br />
|'''LasR'''<br />
|Constitutively expressed regulator protein that can bind LasAHL and stimulate transcription of ΦC31.<br />
|-<br />
|'''RLas'''<br />
|LasR and LasAHL complex which can dimerize.<br />
|-<br />
|'''DRLas'''<br />
|Dimerized form of RLas.<br />
|-<br />
|'''mRNA<sub>ΦC31</sub>'''<br />
|mRNA of the ΦC31 integrase being transcribed by the Lux promoter.<br />
|-<br />
|'''ΦC31'''<br />
|Serine integrase that can fold into two conformations - ΦC31a and ΦC31b. We chose to use a common connotation for both conformations - ΦC31.<br />
|}<br />
<br />
<br/><br />
<br />
=== Reactions ===<br />
<br />
For the Lux system:<br />
$$ \begin{align}<br />
&\rightarrow LuxR \\<br />
LuxAHL+LuxR & \leftrightarrow RLux\\<br />
RLux+RLux &\leftrightarrow DRLux\\<br />
DRLux+P_{luxOFF} & \leftrightarrow P_{luxON}\\<br />
P_{luxON}&\rightarrow P_{luxON}+mRNA_{Bxb1}\\<br />
mRNA_{Bxb1}&\rightarrow Bxb1\\<br />
LuxAHL &\rightarrow \\<br />
LuxR &\rightarrow \\<br />
RLux &\rightarrow\\<br />
DRLux &\rightarrow\\<br />
mRNA_{Bxb1} &\rightarrow\\<br />
Bxb1 &\rightarrow<br />
\end{align}$$<br />
<br />
;For the Las system:<br />
\begin{align}<br />
&\rightarrow LasR \\<br />
LasAHL+LasR & \leftrightarrow RLas \\<br />
RLas+RLas & \leftrightarrow DRLas\\<br />
DRLas+P_{LasOFF} & \leftrightarrow P_{LasON}\\<br />
P_{LasON}&\rightarrow P_{LasON}+mRNA_{\phi C31}\\<br />
mRNA_{\phi C31}&\rightarrow \phi C31\\<br />
Las-AHL &\rightarrow \\<br />
LasR &\rightarrow \\<br />
RLas &\rightarrow\\<br />
DRLas &\rightarrow\\<br />
mRNA_{\phi C31} &\rightarrow \\<br />
\phi C31 &\rightarrow \\<br />
\end{align}<br />
<br />
=== Differential Equations ===<br />
<br />
Applying mass action kinetic laws, we obtain the following set of differential equations.<br />
$$\begin{align*}<br />
\frac{d[LuxAHL]}{dt} &= -d_{LuxAHL}[LuxAHL]\\<br />
\frac{d[LuxR]}{dt} &= \alpha_{LuxR} -k_{RLux}[LuxAHL][LuxR] + k_{-RLux}[RLux] - d_{LuxR}[LuxR] \\<br />
\frac{d[RLux]}{dt} &= k_{RLux}[LuxAHL][LuxR] - k_{-RLux}[RLux] - 2 k_{DRLux} [RLux]^2 + 2 k_{-DRLux} [DRLux] - d_{RLux} [RLux] \\ <br />
\frac{d[DRLux]}{dt} &= k_{DRLux} [RLux]^2 - k_{-DRLux} [DRLux] - d_{DRLux} [DRLux] \\<br />
\frac{d[P_{LuxON}]}{dt} &= k_{P_{LuxON}} [P_{LuxOFF}][DRLux] - k_{-P_{LuxON}} [P_{LuxON}]\\<br />
\frac{d[mRNA_{Bxb1}]}{dt} &= L_{P_{Lux}} + k_{mRNA_{Bxb1}} [P_{LuxON}] - d_{mRNA_{Bxb1}} [mRNA_{Bxb1}]\\<br />
\frac{d[Bxb1]}{dt} &= k_{Bxb1} [mRNA_{Bxb1}] - d_{Bxb1}[Bxb1]\\<br />
\end{align*}$$<br />
<br />
<br />
The same holds true for the Las system.<br />
<br />
'''From the original set of reactions, we reduce the rate of production of mRNA<sub>Bxb1</sub> to a Hill function of RLux instead of Mass action kinetics in terms of P<sub>LuxON</sub> and P<sub>LuxOFF</sub>. For more information please check the [https://2014.igem.org/Team:ETH_Zurich/expresults characterization section].'''<br />
<br />
<br />
<br />
<html></article></html><br />
<br />
<html><article id="Parameters"></html><br />
<br />
== Characterization: K<sub>mLux</sub> and K<sub>mLas</sub> ==<br />
<br />
=== Data ===<br />
<br />
For the Quorum sensing module we used established experimentally determined parameters for the rate of formation of RLux (reference). Since, in the literature the other parameters were estimated or fitted to their data, we decided to determine the parameters specific to our system. Hence, we used our data for the remaining parameters. Our data was mainly a transfer function of normalized GFP concentration as a function of input LuxAHL concentrations. (link to data)<br />
<br />
=== Assumptions ===<br />
<br />
'''Assumption A'''<br />
<br />
We assumed that the dimerization of RLux to DRLux is quick. Quasi steady state approximation (QSSA) as follows<br />
<br />
$$\frac{d[DRLux]}{dt} = k_{DRLux} [RLux]^2 - k_{-DRLux} [DRLux] - d_{DRLux} [DRLux] \approx 0\\$$<br />
<br />
'''Assumption B''' <br />
<br />
Further, from literature, we found that DRLux is specific to DNA and the dissociation constant is low (k<sub>m</sub> = 0.1nM) {Reference}. Therefore, we using QSSA again,<br />
<br />
$$\frac{d[P_{LuxON}]}{dt} = k_{P_{LuxON}} [P_{LuxOFF}][DRLux] - k_{-P_{LuxON}} [P_{LuxON}] \approx 0\\$$<br />
<br />
Solving, we get the rate of production of mRNA<sub>Bxb1</sub> as<br />
<br />
$$\frac{d[mRNA_{Bxb1}]}{dt} = L_{P_{Lux}} + \frac{k_{mRNA_{Bxb1}}[RLux]^2}{K_{mLux}^2 + [RLux]^2 }- d_{mRNA_{Bxb1}} [mRNA_{Bxb1}]\\$$<br />
<br />
where <br />
<br />
$$K_{mLux} = \sqrt{\frac {k_{-P_{LuxON}}}{k_{P_{LuxON}}}.\frac {k_{-DRLux} + d_{DRLux}}{k_{DRLux}}}$$<br />
<br />
is a lumped parameter which we fitted to our data.<br />
<br />
Similarly, lumped parameter K<sub>mLas</sub> was derived for the las system and fitted to a transfer function of normalized GFP concentration as a function of input Las-AHL.<br />
<br />
<!--Further,<br />
$$K_{mRNA_{Bxb1}} = k_{mRNA_{Bxb1}} P_{LuxTOT}$$ which we assumed. --><br />
<br />
=== Parameter fitting ===<br />
<br />
We used MEIGO Toolbox to fit the parameters to the experimental data. We used the concentrations at the end of five hours from each simulation and fit it to the experimental concentrations at the same time.<br />
<br />
<br />
[[File:ETHZ_LuxParameterFitting.png|center|500 px|thumb|'''Figure 1''' Lux QS Module fitted to experimental data from riboregulated Lux system.]]<br />
<br />
<br />
Using the 'DHC' local-solver (Direct search method) in MEIGO, we found the lumped parameters <br />
$$K_{mLux} = 0.45 \pm 0.00051 nM$$ <br />
<br />
and <br />
<br />
$$K_{mLas} = 10 \pm 0.0082 nM$$ <br />
<br />
respectively.<br />
<br />
[[File:ETHZ_LasParameterFitting.png|center|500 px|thumb|'''Figure 2''' Las QS Module fitted to experimental data from riboregulated Las system.]]<br />
<br />
=== Range of validity of the assumptions ===<br />
These assumptions hold true for all input LuxAHL and LasAHL concentrations.<br />
<br />
<html></article></html><br />
<br />
<html><article id='Degradation'></html><br />
== Retrieving degradation rates==<br />
<br />
We are considering quorum sensing experiments with riboregulator, where Plux is induced by LuxAHL or Plas is induced by LasAHL and GFP is produced instead of Bxb1, and this time we look at dynamic curves. <br />
<br />
<br />
[[File:ETH_Zurich_dGFP_Dynamic.png|500px|center|thumb| '''Figure 3''' Dynamic response of the promoter Plux to a dose entry at time t=0.]]<br />
<br />
<br />
By adding an additional quasi steady state assumptions on R<sub>Lux</sub>, and neglecting degradation of R<sub>Lux</sub> compared to its unbinding rate, we can find :<br />
<br />
$$\frac{d[GFP]}{dt}=LeakyLux+\frac{k_{mRNAGFP} k_{GFP} \alpha_{LuxR}^2}{d_{LuxR}^2(Km_{Lux}^2+\alpha_{LuxR})} \frac{[AHL]^2}{K_{mAHL}^2 + [AHL]^2}-d_{GFP}[GFP]$$<br />
<br />
$$\text{with} K_{mAHL}=\frac{K_{mLux}^2 k_{-RLux}}{k_{RLux}(K_{mLux}^2+\alpha_{LuxR}^2/d_{LuxR})}$$<br />
<br />
We have dynamic curves for different initial AHL concentrations. <br />
<br />
We can see in the equation above that for initial AHL concentrations much higher than K<sub>mAHL</sub>=0.3 nM, GFP is only produced and degraded and thus :<br />
<br />
$$\frac{d[GFP]}{dt}=LeakyLux+\frac{k_{mRNAGFP} k_{GFP} \alpha_{LuxR}^2}{d_{LuxR}^2(Km_{Lux}^2+\alpha_{LuxR})}-d_{GFP}[GFP]$$<br />
<br />
so by taking <br />
$$t_{1/2}=\frac{ln(2)}{d_{GFP}}$$<br />
from experimental curves, we find <br />
$$d_{GFP} = 4.9 . 10^{-3} min^{-1}$$<br />
<br />
<br />
For initial AHL concentrations much lower than K<sub>mAHL</sub>=0.3 nM, we find<br />
$$\frac{d[GFP]}{dt}=LeakyLux+\frac{k_{mRNAGFP} k_{GFP} \alpha_{LuxR}^2}{d_{LuxR}^2(Km_{Lux}^2+\alpha_{LuxR})} \frac{[AHL]^2}{K_{mAHL}^2}-d_{GFP}[GFP]$$<br />
<br />
and thus a steady state<br />
$$[GFP]=\frac{Constant}{(d_{GFP}-2d_{AHL})}(e^{-2d_{AHL}t}-e^{-d_{GFP}t})$$<br />
<br />
This curve has a maximum at <br />
$$t_{max}=\frac{1}{d_{GFP}-2d_{AHL}}ln\big(\frac{d_{GFP}}{2d_{AHL}}\big)$$<br />
<br />
This way we can find from experimental curves <br />
$$d_{LuxAHL}=4,0.10^{-3} min^{-1}$$<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article id="Leakiness"></html><br />
<br />
== Leakiness ==<br />
<br />
The leakiness of promoters is a major issue in our system. As the signal propagates row-wise, error diffusion could lead to a totally different pattern. The goal is then to control the leakiness. This issue was particularly observed and adressed in the case of the Lux promoter during our [https://2014.igem.org/Team:ETH_Zurich/expresults experiments set]. This leakiness is dependent on LuxR concentration in the cell(see our biobrick characterization in the [http://parts.igem.org/Part:BBa_R0062:Experience Registry] for more information).<br />
<br/><br />
Leakiness was modeled as an offset in the classical Hill function. <br />
<br />
$$rFluo = a + b \frac{[AHL]^n}{K_m^n + [AHL]^n}$$<br />
$$\text{where rFluo is the relative fluorescence (absolute measured fluorescence value over OD),}$$<br />
$$\text{a the basal expression rate (Leakiness),}$$<br />
$$\text{b the maximum fold expression rate,}$$<br />
$$\text{n the Hill coefficient,}$$<br />
$$K_m\text{ the activation concentration of AHL.}$$<br />
<br />
<br />
Given this offset and the maximal expression, the signal over noise ratio can be derived. This ratio, which can then be compared amongst all curves, characterizes the impact of leakiness on the behavior of a system. The leakier a construct in its native form is, the more impact the riboregulator will have, and the more likely it is for the riboregulator to increase the signal over noise ratio. Our final constructs (Promoters with a [https://2014.igem.org/Team:ETH_Zurich/expresults riboregulating system]) have the following parameters:<br />
<br />
<br/><br />
<br/><br />
{| class="wikitable"<br />
|-<br />
! '''Promoter used'''<br />
! '''Signal over noise ratio'''<br />
|-<br />
|P<sub>Lux</sub> without riboregulating system<br />
|23<br />
|-<br />
|P<sub>Lux</sub> with riboregulating system<br />
|79<br />
|-<br />
|P<sub>Las</sub> without riboregulating system<br />
|84<br />
|-<br />
|P<sub>Las</sub> with riboregulating system<br />
|55<br />
|}<br />
<br/><br />
<br/><br />
We took the leakiness coefficients into account in our [https://2014.igem.org/Team:ETH_Zurich/modeling/whole whole cell model].<br />
<br />
<html></article></html><br />
<br />
<br />
<html><article id="Cross-talk"></html><br />
<br />
== Cross-talk ==<br />
<br />
We investigated the existence of cross-talk between three quorum sensing systems (Lux, Las and Rhl). Each quorum sensing system is based on three components: a signaling molecule, a regulatory protein and a promoter. Cross-talk implies non-orthogonality of the communication systems. It corresponds to the fact that LasAHL can activate the Lux promoter, even if it is not its native communicating pathway. There are 27 combinations possible and only 3 native combinations between signaling molecule, regulatory protein and promoter.<br />
<br/><br />
<br/><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 />
<br/><br />
<br/><br />
From the [https://2014.igem.org/Team:ETH_Zurich/expresults exhaustive experimental data], the central role of regulatory proteins was identified, allowing the characterization of two-levels of cross-talk. The following parts in the Registry are presenting our results:<br />
*[http://parts.igem.org/Part:BBa_R0062:Experience BBa_R0062]<br />
*[http://parts.igem.org/Part:BBa_R0079:Experience BBa_R0079]<br />
*[http://parts.igem.org/Part:BBa_I14017:Experience BBa_I14017]<br />
*[http://parts.igem.org/Part:BBa_C0062:Experience BBa_C0062]<br />
*[http://parts.igem.org/Part:BBa_C0179:Experience BBa_C0179]<br />
*[http://parts.igem.org/Part:BBa_C0171:Experience BBa_C0171]<br />
<br />
Each experimental data set was fitted to an Hill function using the [https://2014.igem.org/Team:ETH_Zurich/modeling/parameters Least Absolute Residual method]. <br />
<br />
$$rFluo = a + b \frac{[AHL]^n}{K_m^n + [AHL]^n}$$<br />
$$\text{where rFluo is the relative fluorescence (absolute measured fluorescence value over OD),}$$<br />
$$\text{a the basal expression rate,}$$<br />
$$\text{b the maximum fold expression rate,}$$<br />
$$\text{n the Hill coefficient,}$$<br />
$$K_m\text{ the activation concentration of AHL.}$$<br />
<br />
We took cross-talk into account in our [https://2014.igem.org/Team:ETH_Zurich/modeling/whole whole cell model].<br />
<br />
<html></article></html><br />
<br />
<html><article id="Alternate"></html><br />
<br />
== Alternate Design ==<br />
<br />
As cross-talk is a burning issue in quorum sensing, we thought about a theoretical solution. [https://2014.igem.org/Team:Edinburgh Edinburgh iGEM team 2014] also worked on communication between ''E. coli''. They developed new communication channels via metabolic wiring. By assuming that quorum sensing molecules would not cross-talk with metabolites, we used their idea to develop a model on the molecular level. The idea is finally to combine our [https://2014.igem.org/Team:ETH_Zurich/modeling#Alternate_Design whole-cell model] and their idea on metabolites.<br />
<br />
Metabolic wiring is based on the fact that when a resource is at disposal, a cell will produce an enzyme to break it down to smaller pieces. There is often a chain of metabolites. Using the fact that metabolites can diffuse through the membrane, this can allow communication (for more information, please see the [https://2014.igem.org/Team:Edinburgh Edinburgh iGEM team 2014] wiki).<br />
<br />
=== Chemical Species ===<br />
<br />
The species' names are generic because the implementation with particular metabolites implies biological considerations on cell growth and medium used. <br />
<br />
{| class="wikitable"<br />
|-<br />
! Name<br />
! Description<br />
|-<br />
|'''A'''<br />
|Metabolite<br />
|-<br />
|'''B'''<br />
|Metabolite<br />
|-<br />
|'''Enz'''<br />
|Enzyme that catalyzes the transformation from A to B<br />
|-<br />
|'''[A.Enz]'''<br />
|Complex made of metabolite A and enzyme Enz<br />
|-<br />
|'''P_A'''<br />
|Promoter induced by A. It can be either on or off (rescued or not)<br />
|}<br />
<br />
=== Reactions ===<br />
$$\begin{align*}<br />
&\rightarrow A \\<br />
A + P_{Aoff} &\rightarrow P_{Aon} \\<br />
P_{Aon} &\rightarrow P_{Aon} + Enz \\<br />
A + Enz &\leftrightarrow [A.Enz] \\<br />
[A.Enz] &\rightarrow B \\<br />
A &\rightarrow \\<br />
B &\rightarrow \\<br />
[A.Enz] &\rightarrow \\<br />
Enz &\rightarrow<br />
\end{align*}$$<br />
<br />
=== Parameters ===<br />
{| class="wikitable"<br />
|-<br />
! Name<br />
! Description<br />
|-<br />
|'''α<sub>A</sub>'''<br />
|Production rate of metabolite A<br />
|-<br />
|'''d<sub>A</sub>'''<br />
|Degradation rate of metabolite A<br />
|-<br />
|'''α<sub>Enz</sub>'''<br />
|Production rate of enzyme Enz<br />
|-<br />
|'''d<sub>Enz</sub>'''<br />
|Degradation rate of enzyme Enz<br />
|-<br />
|'''α<sub>B</sub>'''<br />
|Production rate of metabolite B<br />
|-<br />
|'''d<sub>B</sub>'''<br />
|Degradation rate of metabolite B<br />
|-<br />
|'''K<sub>d</sub>'''<br />
|Parameter of the Michaelis-Menten function modeling the action of enzyme Enz on the substrate, metabolite A, in order to produce metabolite B<br />
|-<br />
|'''n'''<br />
|Hill coefficient for the Hill function modeling the activation of the transcription of enzyme Enz with metabolite A as inducer<br />
|-<br />
|'''K<sub>A</sub>'''<br />
|Activation concentration for the Hill function modeling the activation of the transcription of enzyme Enz with metabolite A as inducer<br />
|}<br />
<br />
Parameters are not well-known.<br />
<br />
=== Deterministic Model ===<br />
We derived this model doing the following assumptions:<br />
<br />
;'''Assumption 1'''<br />
:The induction of the promoter P<sub>A</sub> by A is supposed to follow an Hill function.<br />
<br />
;'''Assumption 2'''<br />
:The enzyme-based reaction from A to B is supposed to follow a Michaelis-Menten function.<br />
<br />
$$\begin{align}<br />
\frac{d[A]}{dt} &= \alpha_A - d_{A} [A] \\<br />
\frac{d[Enz]}{dt} &= \alpha_{Enz} \frac{[A]^n}{K_{A}^n + [A]^n} - d_{Enz} [Enz] \\<br />
\frac{d[B]}{dt} &= \alpha_B \frac{[Enz] [A]}{K_d + [A]} - d_{B} [B] <br />
\end{align}$$<br />
<br />
=== Application to our project ===<br />
Populations of bacteria will grow into a medium, which provides them metabolite A. The metabolite B will serve as communicating signal, like LasAHL or LuxAHL. It will be an input for the logic construct. As soon as metabolite B is being made available to the cell, the cell will produce the input for the logic construct.<br />
<br/><br />
Moreover, one type of cell will produce B as output. Therefore, the output of the logic gate signal will correspond to the production (or absence of production) of the enzyme Enz, so that the A contained in the medium can be transformed to B. Enz will play the same role as LasI or LuxI in our original model.<br />
<br/><br />
We separate the promoter activating the production of the enzyme with the production of the enzyme itself. Moreover, cells sense B and want to produce B. In our modules, we have to remplace equations in the sensing module and in the production module.<br />
<br/><br />
<br/><br />
;'''Sensing Module'''<br />
:For example, B would induce the production of the integrase, Bxb1.<br />
$$<br />
\begin{align*}<br />
\frac{d[B]}{dt} &= \alpha_B - d_{B} [B] \\<br />
\frac{d[Bxb1]}{dt} &= \alpha_{Enz} \frac{[B]^n}{K_{B}^n + [B]^n} - d_{Bxb1} [Bxb1]<br />
\end{align*}<br />
$$<br />
<br />
;'''Production Module'''<br />
:Here, output<sub>logic</sub> is the output of the XOR logic gate, factorized in one term for simplicty's sake. For more information on this function, see the [https://2014.igem.org/Team:ETH_Zurich/modeling/xor XOR gate modeling page].<br />
$$\begin{align*}<br />
\frac{d[Enz]}{dt} &= output_{logic} - d_{Enz} [Enz] \\<br />
\frac{d[B]}{dt} &= \alpha_B \frac{[Enz] [A]}{K_d + [A]} - d_{B} [B]<br />
\end{align*}<br />
$$<br />
<br />
=== Simulations ===<br />
<br />
We implemented this solution in our [[Team:ETH_Zurich/modeling/whole#Alternate_Design|whole-cell model]]. As no parameters were known, we assumed their values to be in the range of standard rates. The results indicate that the system could work, the next step would be to test this prediction experimentally.<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/human/essay/answerTeam:ETH Zurich/human/essay/answer2014-10-18T02:32:31Z<p>Clormeau: /* Our reflections */</p>
<hr />
<div><html><article></html><br />
== Our reflections ==<br />
This essay is the third of four pillars towards an understanding of complexity. It brings elements from the survey, from interviews and from further reading together. Thinking on how our project and science, in general, relates to these topics. <br />
<br />
<br />
Our human practice project was guided by the following question:<br />
<br />
<br />
'''“Should scientists consider that subparts of a complex entity are mixed in a both ordered and unorganized way, accept uncertainty, and try to take it into account? Or should they consider that parts are strictly ordered, and that complexity arises from simple parts by following rules?”'''<br />
<br />
<br />
This question splits up into two approaches. The first approach is needed to take into account uncertainty of intrinsic complexity of the parts we consider and of the environment. The second approach is necessary to understand the parts better in order to be able to predict results.<br />
<br />
<br />
On our way of answering the questions coming along with complexity we focused on four different components: Listening, discussing, thinking and sharing. <br />
<br />
<br />
The first component of listening was covered by a survey regarding complexity. We listened to the public and learned about the existing ideas of complexity and how people relate to it. Something that we have observed is a trend of increasing complexity when going from non-living objects to living beings. <br />
<br />
<br />
70% of the participants of our survey have shown an interest to simplify and try to understand complexity instead of avoiding it. Another phenomenon observed was the deviation between languages. Depending on the language spoken, complexity was judged in a different way, which may indicate cultural variation. <br />
<br />
We are grateful for all the people that provided us comments in addition to the questions to answer. No matter whether these were suggestions for publications to read, complaints about the complexity of the survey or encouraging words. <br />
<br />
This survey has taught us how complexity is perceived in the public. From our survey we experienced that in our sample population interest in complexity exists. <br />
<br />
From our results we mainly got an indication of complexity in nature. A point to consider is that often people are not forced to deal with complexity directly. A cell, a dog and a computer exist as items in our daily lives but most of us do not think about their complexity in relation to other items on a daily basis. This means the topic is present but not easily formulated. <br />
<br />
<br />
Our second component involved interviews with experts from different backgrounds. The discussions with experts enabled us to broaden our horizons away from the complexity we are facing in our daily life to the complexity faced by people of other backgrounds. This exchange has enriched our project, as the professional fields of the interview partners were very diverse. <br />
<br />
<br />
The knowledge gained from our survey, the interviews and the thoughts about the two previous ones in combination we wanted to share. Sharing as our fourth pillar was done in lectures at a high school where we aimed at explaining the fundamentals of synthetic biology and how it can be a way of approaching complexity. A science slam is defined as a scientific presentation competition where scientists present their topics in a predefined timeslot and in a funny, accessible way for the open public. <br />
<br />
<br />
<br />
Our human practice has shown us the diversity of approaches of addressing complexity in our daily lives, in our professional fields, in science and when encountering complex situations. The interviews have especially made this point clear e.g. from the talk with the priest we found out that in his opinion religion and believe help us to find a way away from complexity and towards God. Thus we can live a life in trust instead of confusion and desperate. <br />
From Dr. Chikkadi and also from Mr. Veress the philosophy teacher we learned that in their point of view complexity arises from simple phenomena. <br />
<br />
From Dr. Garcia we got the following input on the perception of complexity. „Complexity is a property of a system and it can be measured. It can be shown whether a system is complex or not: for a complex system, the sum of its elements is higher than each one of them independently in superposition.“<br />
<br />
We learned that it is often useful to simplify the complexity to obtain a more accessible approach. In the process of simplification we should not forget the relationship to reality. <br />
<br />
In our outreach part we experienced how important it is to break the complexity of the own down to make it accessible for a broader public. On our way of spreading the word of synthetic biology we had many enriching encounters. We met many different people and encountered the phenomenon already described in our survey. The people we met all showed interest in trying to simplify complex problems and a will to understand what seems complex in first place. <br />
<br />
<br />
We did not find a universal answer to the question guiding our human practice project. What we found are many different approaches to address complexity arising in many different fields. This project helped us to improve our understanding of complexity as a whole and how we could profit from this profound, interdisciplinary knowledge. <br />
<br />
<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/human/essay/answerTeam:ETH Zurich/human/essay/answer2014-10-18T02:32:08Z<p>Clormeau: /* Our reflections */</p>
<hr />
<div><html><article></html><br />
== Our reflections ==<br />
This essay is the third of four pillars towards an understanding of complexity. It brings elements from the survey, from interviews and from further reading together. Thinking on how our project and science, in general, relates to these topics. <br />
<br />
<br />
Our human practice project was guided by the following question:<br />
<br />
<br />
“Should scientists consider that subparts of a complex entity are mixed in a both ordered and unorganized way, accept uncertainty, and try to take it into account? Or should they consider that parts are strictly ordered, and that complexity arises from simple parts by following rules?”<br />
<br />
<br />
This question splits up into two approaches. The first approach is needed to take into account uncertainty of intrinsic complexity of the parts we consider and of the environment. The second approach is necessary to understand the parts better in order to be able to predict results.<br />
<br />
<br />
On our way of answering the questions coming along with complexity we focused on four different components: Listening, discussing, thinking and sharing. <br />
<br />
<br />
The first component of listening was covered by a survey regarding complexity. We listened to the public and learned about the existing ideas of complexity and how people relate to it. Something that we have observed is a trend of increasing complexity when going from non-living objects to living beings. <br />
<br />
<br />
70% of the participants of our survey have shown an interest to simplify and try to understand complexity instead of avoiding it. Another phenomenon observed was the deviation between languages. Depending on the language spoken, complexity was judged in a different way, which may indicate cultural variation. <br />
<br />
We are grateful for all the people that provided us comments in addition to the questions to answer. No matter whether these were suggestions for publications to read, complaints about the complexity of the survey or encouraging words. <br />
<br />
This survey has taught us how complexity is perceived in the public. From our survey we experienced that in our sample population interest in complexity exists. <br />
<br />
From our results we mainly got an indication of complexity in nature. A point to consider is that often people are not forced to deal with complexity directly. A cell, a dog and a computer exist as items in our daily lives but most of us do not think about their complexity in relation to other items on a daily basis. This means the topic is present but not easily formulated. <br />
<br />
<br />
Our second component involved interviews with experts from different backgrounds. The discussions with experts enabled us to broaden our horizons away from the complexity we are facing in our daily life to the complexity faced by people of other backgrounds. This exchange has enriched our project, as the professional fields of the interview partners were very diverse. <br />
<br />
<br />
The knowledge gained from our survey, the interviews and the thoughts about the two previous ones in combination we wanted to share. Sharing as our fourth pillar was done in lectures at a high school where we aimed at explaining the fundamentals of synthetic biology and how it can be a way of approaching complexity. A science slam is defined as a scientific presentation competition where scientists present their topics in a predefined timeslot and in a funny, accessible way for the open public. <br />
<br />
<br />
<br />
Our human practice has shown us the diversity of approaches of addressing complexity in our daily lives, in our professional fields, in science and when encountering complex situations. The interviews have especially made this point clear e.g. from the talk with the priest we found out that in his opinion religion and believe help us to find a way away from complexity and towards God. Thus we can live a life in trust instead of confusion and desperate. <br />
From Dr. Chikkadi and also from Mr. Veress the philosophy teacher we learned that in their point of view complexity arises from simple phenomena. <br />
<br />
From Dr. Garcia we got the following input on the perception of complexity. „Complexity is a property of a system and it can be measured. It can be shown whether a system is complex or not: for a complex system, the sum of its elements is higher than each one of them independently in superposition.“<br />
<br />
We learned that it is often useful to simplify the complexity to obtain a more accessible approach. In the process of simplification we should not forget the relationship to reality. <br />
<br />
In our outreach part we experienced how important it is to break the complexity of the own down to make it accessible for a broader public. On our way of spreading the word of synthetic biology we had many enriching encounters. We met many different people and encountered the phenomenon already described in our survey. The people we met all showed interest in trying to simplify complex problems and a will to understand what seems complex in first place. <br />
<br />
<br />
We did not find a universal answer to the question guiding our human practice project. What we found are many different approaches to address complexity arising in many different fields. This project helped us to improve our understanding of complexity as a whole and how we could profit from this profound, interdisciplinary knowledge. <br />
<br />
<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/human/essay/answerTeam:ETH Zurich/human/essay/answer2014-10-18T02:31:28Z<p>Clormeau: /* Our reflections */</p>
<hr />
<div><html><article></html><br />
== Our reflections ==<br />
This essay is the third of four pillars towards an understanding of complexity. It brings elements from the survey, from interviews and from further reading together. Thinking on how our project and science, in general, relates to these topics. <br />
<br />
<br />
Our human practice project was guided by the following question:<br />
<br />
<br />
“Should scientists consider that subparts of a complex entity are mixed in a both ordered and unorganized way, accept uncertainty, and try to take it into account? <br />
<br />
<br />
Or should they consider that parts are strictly ordered, and that complexity arises from simple parts by following rules?”<br />
<br />
<br />
This question splits up into two approaches. The first approach is needed to take into account uncertainty of intrinsic complexity of the parts we consider and of the environment. The second approach is necessary to understand the parts better in order to be able to predict results.<br />
<br />
<br />
On our way of answering the questions coming along with complexity we focused on four different components: Listening, discussing, thinking and sharing. <br />
<br />
<br />
The first component of listening was covered by a survey regarding complexity. We listened to the public and learned about the existing ideas of complexity and how people relate to it. Something that we have observed is a trend of increasing complexity when going from non-living objects to living beings. <br />
<br />
<br />
70% of the participants of our survey have shown an interest to simplify and try to understand complexity instead of avoiding it. Another phenomenon observed was the deviation between languages. Depending on the language spoken, complexity was judged in a different way, which may indicate cultural variation. <br />
<br />
We are grateful for all the people that provided us comments in addition to the questions to answer. No matter whether these were suggestions for publications to read, complaints about the complexity of the survey or encouraging words. <br />
<br />
This survey has taught us how complexity is perceived in the public. From our survey we experienced that in our sample population interest in complexity exists. <br />
<br />
From our results we mainly got an indication of complexity in nature. A point to consider is that often people are not forced to deal with complexity directly. A cell, a dog and a computer exist as items in our daily lives but most of us do not think about their complexity in relation to other items on a daily basis. This means the topic is present but not easily formulated. <br />
<br />
<br />
Our second component involved interviews with experts from different backgrounds. The discussions with experts enabled us to broaden our horizons away from the complexity we are facing in our daily life to the complexity faced by people of other backgrounds. This exchange has enriched our project, as the professional fields of the interview partners were very diverse. <br />
<br />
<br />
The knowledge gained from our survey, the interviews and the thoughts about the two previous ones in combination we wanted to share. Sharing as our fourth pillar was done in lectures at a high school where we aimed at explaining the fundamentals of synthetic biology and how it can be a way of approaching complexity. A science slam is defined as a scientific presentation competition where scientists present their topics in a predefined timeslot and in a funny, accessible way for the open public. <br />
<br />
<br />
<br />
Our human practice has shown us the diversity of approaches of addressing complexity in our daily lives, in our professional fields, in science and when encountering complex situations. The interviews have especially made this point clear e.g. from the talk with the priest we found out that in his opinion religion and believe help us to find a way away from complexity and towards God. Thus we can live a life in trust instead of confusion and desperate. <br />
From Dr. Chikkadi and also from Mr. Veress the philosophy teacher we learned that in their point of view complexity arises from simple phenomena. <br />
<br />
From Dr. Garcia we got the following input on the perception of complexity. „Complexity is a property of a system and it can be measured. It can be shown whether a system is complex or not: for a complex system, the sum of its elements is higher than each one of them independently in superposition.“<br />
<br />
We learned that it is often useful to simplify the complexity to obtain a more accessible approach. In the process of simplification we should not forget the relationship to reality. <br />
<br />
In our outreach part we experienced how important it is to break the complexity of the own down to make it accessible for a broader public. On our way of spreading the word of synthetic biology we had many enriching encounters. We met many different people and encountered the phenomenon already described in our survey. The people we met all showed interest in trying to simplify complex problems and a will to understand what seems complex in first place. <br />
<br />
<br />
We did not find a universal answer to the question guiding our human practice project. What we found are many different approaches to address complexity arising in many different fields. This project helped us to improve our understanding of complexity as a whole and how we could profit from this profound, interdisciplinary knowledge. <br />
<br />
<br />
<html></article></html></div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/criteriaTeam:ETH Zurich/criteria2014-10-18T02:30:03Z<p>Clormeau: /* Gold medal criteria */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Medal Criteria}}<br />
<center><br />
{{:Team:ETH Zurich/tpl/scrollbutton|Bronze|bronze}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton2|Silver|silver}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton3|Gold|gold}}<br />
</center><br />
<html><article id='Bronze'></html><br />
== Bronze medal criteria ==<br />
<br><br />
<br><br />
<br />
{| class="wikitable resized" style='text-align:center'<br />
|- align="center"<br />
! Team registration<br />
! Complete Judging form<br />
! Team Wiki<br />
|- align="center"<br />
| {{:Team:ETH Zurich/tpl/button |<html>https://igem.org/Team_List.cgi?year=2014</html>|bronze|Done}}<br />
| {{:Team:ETH Zurich/tpl/button |<html>https://igem.org/2014_Judging_Form?id=1541 </html>|bronze|Done}}<br />
| {{:Team:ETH Zurich/tpl/button |<html>https://2014.igem.org/Team:ETH_Zurich </html>|bronze|Done}}<br />
|- align="center"<br />
! Present a poster and a talk at the iGEM Jamboree<br />
! Clearly attribute work<br />
! Document at least one new standard BioBrick Part or Device<br />
|- align="center"<br />
|style="vertical-align:middle"| '''Jamboree, here we come!'''<br />
| {{:Team:ETH Zurich/tpl/button |<html>https://2014.igem.org/Team:ETH_Zurich/acknowledgements </html>|bronze|Done}}<br />
| {{:Team:ETH Zurich/tpl/button |<html>http://parts.igem.org/Part:BBa_K1541009 </html>|bronze|Done}}<br />
|}<br />
<br />
<br />
<br />
<html></article></html><br />
<br />
<html><article id='Silver'></html><br />
<br />
== Silver medal criteria ==<br />
<br />
{| class="wikitable resized" <br />
|-<br />
! Experimental validation of new biobrick<br />
! Documentation of part characterization in the Registry<br />
! Submission of this new part to the Registry<br />
! Articulate at least one "beyond the bench" question encountered by your team<br />
|- align="center"<br />
| {{:Team:ETH Zurich/tpl/button |<html>http://parts.igem.org/Part:BBa_K1541009</html>|silver|Done}}<br />
| {{:Team:ETH Zurich/tpl/button |<html>http://parts.igem.org/Part:BBa_K1541009</html>|silver|Done}}<br />
| {{:Team:ETH Zurich/tpl/button |<html>http://parts.igem.org/Part:BBa_K1541009</html>|silver|Done}}<br />
|{{:Team:ETH Zurich/tpl/button |<html>https://2014.igem.org/Team:ETH_Zurich/human/overview </html>|silver|Done}}<br />
|}<br />
<br />
<br><br />
<br />
<br />
<html></article></html><br />
<br />
<html><article id='Gold'></html><br />
<br />
== Gold medal criteria ==<br />
<br><br />
<br><br />
<br />
{| class="wikitable resized" style='text-align:center'<br />
|- align="center"<br />
! Improve the function OR characterization of an existing BioBrick Part or Device <br />
! Help any registered iGEM team from another school or institution <br />
! Describe an approach that your team used to address at least one of the questions beyond the bench.<br />
|- align="center"<br />
| {{:Team:ETH Zurich/tpl/button |<html>http://parts.igem.org/Part:BBa_R0062:Experience</html>|gold|Done}}<br />
| {{:Team:ETH Zurich/tpl/button |<html>https://2014.igem.org/Team:ETH_Zurich/collaborations</html>|gold|Done}}<br />
| {{:Team:ETH Zurich/tpl/button |<html>https://2014.igem.org/Team:ETH_Zurich/human/overview</html>|gold|Done}}<br />
|}<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/criteriaTeam:ETH Zurich/criteria2014-10-18T02:29:44Z<p>Clormeau: /* Gold medal criteria */</p>
<hr />
<div>{{:Team:ETH_Zurich/tpl/head|Medal Criteria}}<br />
<center><br />
{{:Team:ETH Zurich/tpl/scrollbutton|Bronze|bronze}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton2|Silver|silver}}<br />
{{:Team:ETH Zurich/tpl/scrollbutton3|Gold|gold}}<br />
</center><br />
<html><article id='Bronze'></html><br />
== Bronze medal criteria ==<br />
<br><br />
<br><br />
<br />
{| class="wikitable resized" style='text-align:center'<br />
|- align="center"<br />
! Team registration<br />
! Complete Judging form<br />
! Team Wiki<br />
|- align="center"<br />
| {{:Team:ETH Zurich/tpl/button |<html>https://igem.org/Team_List.cgi?year=2014</html>|bronze|Done}}<br />
| {{:Team:ETH Zurich/tpl/button |<html>https://igem.org/2014_Judging_Form?id=1541 </html>|bronze|Done}}<br />
| {{:Team:ETH Zurich/tpl/button |<html>https://2014.igem.org/Team:ETH_Zurich </html>|bronze|Done}}<br />
|- align="center"<br />
! Present a poster and a talk at the iGEM Jamboree<br />
! Clearly attribute work<br />
! Document at least one new standard BioBrick Part or Device<br />
|- align="center"<br />
|style="vertical-align:middle"| '''Jamboree, here we come!'''<br />
| {{:Team:ETH Zurich/tpl/button |<html>https://2014.igem.org/Team:ETH_Zurich/acknowledgements </html>|bronze|Done}}<br />
| {{:Team:ETH Zurich/tpl/button |<html>http://parts.igem.org/Part:BBa_K1541009 </html>|bronze|Done}}<br />
|}<br />
<br />
<br />
<br />
<html></article></html><br />
<br />
<html><article id='Silver'></html><br />
<br />
== Silver medal criteria ==<br />
<br />
{| class="wikitable resized" <br />
|-<br />
! Experimental validation of new biobrick<br />
! Documentation of part characterization in the Registry<br />
! Submission of this new part to the Registry<br />
! Articulate at least one "beyond the bench" question encountered by your team<br />
|- align="center"<br />
| {{:Team:ETH Zurich/tpl/button |<html>http://parts.igem.org/Part:BBa_K1541009</html>|silver|Done}}<br />
| {{:Team:ETH Zurich/tpl/button |<html>http://parts.igem.org/Part:BBa_K1541009</html>|silver|Done}}<br />
| {{:Team:ETH Zurich/tpl/button |<html>http://parts.igem.org/Part:BBa_K1541009</html>|silver|Done}}<br />
|{{:Team:ETH Zurich/tpl/button |<html>https://2014.igem.org/Team:ETH_Zurich/human/overview </html>|silver|Done}}<br />
|}<br />
<br />
<br><br />
<br />
<br />
<html></article></html><br />
<br />
<html><article id='Gold'></html><br />
<br />
== Gold medal criteria ==<br />
<br><br />
<br><br />
<br />
{| class="wikitable resized" style='text-align:center'<br />
|- align="center"<br />
! Improve the function OR characterization of an existing BioBrick Part or Device <br />
! Help any registered iGEM team from another school or institution <br />
! Describe an approach that your team used to address at least one of the questions beyond the bench.<br />
|- align="center"<br />
| {{:Team:ETH Zurich/tpl/button |<html>http://parts.igem.org/Part:BBa_R0062:Experience</html>|gold|Done}}<br />
| {{:Team:ETH Zurich/tpl/button |<html>https://2014.igem.org/Team:ETH_Zurich/collaborations</html>|gold|Done}}<br />
| {{:Team:ETH Zurich/tpl/button |<html>https://2014.igem.org/Team:ETH_Zurich/human/overview#Our</html>|gold|Done}}<br />
|}<br />
<br />
<html></article></html><br />
<br />
{{:Team:ETH_Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/human/overviewTeam:ETH Zurich/human/overview2014-10-18T02:28:38Z<p>Clormeau: </p>
<hr />
<div>{{:Team:ETH Zurich/tpl/head|Human Practice Overview}}<br />
<br />
<center><br />
{{:Team:ETH_Zurich/tpl/scrollbutton2|Complexity|blue}}<br />
{{:Team:ETH_Zurich/tpl/scrollbuttontworows|Our|Approach|red}}<br />
</center><br />
<br />
<html><article id='Complexity'></html><br />
{{:Team:ETH_Zurich/human/overview/question}}<br />
<html></article></html><br />
<br />
<html><br />
<br />
<script type="text/javascript"><br />
(function($) {<br />
function img(url) {<br />
var i = new Image;<br />
i.src = url;<br />
return i;<br />
}<br />
<br />
if ('naturalWidth' in (new Image)) {<br />
$.fn.naturalWidth = function() { return this[0].naturalWidth; };<br />
$.fn.naturalHeight = function() { return this[0].naturalHeight; };<br />
return;<br />
}<br />
$.fn.naturalWidth = function() { return img(this[0].src).width; };<br />
$.fn.naturalHeight = function() { return img(this[0].src).height; };<br />
})(jQuery);<br />
<br />
<br />
<br />
function onWindowResize() <br />
{<br />
var curWidth = $(window).width(),<br />
curHeight = $(window).height(),<br />
checking=false;<br />
if (checking) {<br />
return;<br />
}<br />
checking = true;<br />
window.setTimeout(<br />
function() {<br />
var newWidth = $(window).width(),<br />
newHeight = $(window).height();<br />
if (!(newWidth !== curWidth ||<br />
newHeight !== curHeight)) {<br />
resize(false); <br />
}<br />
checking=false;<br />
}, 300);<br />
}<br />
<br />
function resize(initial) {<br />
if (!initial)<br />
{<br />
var container = $('#container');<br />
var imgWidth = container.width();<br />
<br />
$( "#map").each(function() {<br />
$(this).css('height', 'auto', 'width', 'auto');<br />
$(this).mapster('resize',Math.min(imgWidth, $(this).naturalWidth()) ,0,0); <br />
});<br />
}<br />
<br />
}<br />
<br />
$(document).ready(function(){<br />
<br />
$('#map').mapster({<br />
fillOpacity: 0.5,<br />
isSelectable: false,<br />
clickNavigate: true,<br />
mapKey: 'name',<br />
areas: [<br />
{<br />
key: "red",<br />
fillColor: 'FF0000'<br />
},<br />
<br />
{<br />
key: "blue",<br />
fillColor: '1ED9E1'<br />
},<br />
<br />
{<br />
key: "green",<br />
fillColor: '00FF00'<br />
},<br />
<br />
{<br />
key: "silver",<br />
fillColor: 'C0C0C0;'<br />
}<br />
]<br />
});<br />
<br />
<br />
<br />
$(window).resize(<br />
function()<br />
{ <br />
onWindowResize();<br />
});<br />
resize(true);<br />
<br />
});<br />
<br />
</script><br />
<br />
<article id='Our'></html><br />
== Our Approach ==<br />
<br />
The main focus of our project lies in complexity. How does it emerge? How can we handle it? Additionally, what can we learn from it and how can we use it? <br />
<br><br />
<br><br />
Our human practice project attempts to answer these questions by focusing on four different components: listening, discussing, thinking and sharing.<br />
<br><br />
<br><br />
<br><br />
<br />
<html><br />
<center><br />
<div id="container"><br />
<IMG SRC="https://static.igem.org/mediawiki/2014/d/d3/ETH_Zurich_Hpbuttons.png" width='500px' usemap="#map" id="map"><br />
<map id="map" name="map"><area shape="poly" alt="" title="" coords="235,38,152,175,320,175" href="https://2014.igem.org/Team:ETH_Zurich/human/interviews" id="blue" name="blue" target="" /><area shape="poly" id="red" name="red" alt="" title="" coords="125,224,43,359,207,358" href="https://2014.igem.org/Team:ETH_Zurich/human/essay" target="" /><area shape="poly" alt="" title="" coords="263,359,430,360,346,221" id="green" name="green" href="https://2014.igem.org/Team:ETH_Zurich/human/survey" target="" /><area shape="poly" alt="" title="" coords="0,347,22,362,223,36,249,36,445,366,471,354,263,3,207,3" href="https://2014.igem.org/Team:ETH_Zurich/human/outreach" target="" name="silver"/></map><br />
</div><br />
</center><br />
</html><br />
<br />
=.=<br />
The first component consists of a survey regarding complexity. By listening to the public we learn about the existing ideas of complexity and how people relate to it.<br />
<br><br />
<br><br />
Our second component involves interviews with experts from different backgrounds. These discussions focus on the complexity existing in their field and how they deal with it which enables us to improve our understanding of complexity as a whole and how we could profit from this profound, interdisciplinary knowledge. The various methods of resolution are summarized and compared in an essay.<br />
<br><br />
<br><br />
In our third component, we bring elements from the survey, from interviews, from further reading together. Thinking on how our project and science, in general, relates to these topics.<br />
<br><br />
<br><br />
Finally, we impart or share knowledge via different platforms like high school lectures and science slams. Here, we aim to explain the fundamentals of synthetic biology and how it can be a way to approach complexity. <br />
<html></article></html><br />
<br />
<br />
{{:Team:ETH Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/human/overviewTeam:ETH Zurich/human/overview2014-10-18T02:28:28Z<p>Clormeau: </p>
<hr />
<div>{{:Team:ETH Zurich/tpl/head|Human Practice Overview}}<br />
<br />
<center><br />
{{:Team:ETH_Zurich/tpl/scrollbutton|Complexity|blue}}<br />
{{:Team:ETH_Zurich/tpl/scrollbuttontworows|Our|Approach|red}}<br />
</center><br />
<br />
<html><article id='Complexity'></html><br />
{{:Team:ETH_Zurich/human/overview/question}}<br />
<html></article></html><br />
<br />
<html><br />
<br />
<script type="text/javascript"><br />
(function($) {<br />
function img(url) {<br />
var i = new Image;<br />
i.src = url;<br />
return i;<br />
}<br />
<br />
if ('naturalWidth' in (new Image)) {<br />
$.fn.naturalWidth = function() { return this[0].naturalWidth; };<br />
$.fn.naturalHeight = function() { return this[0].naturalHeight; };<br />
return;<br />
}<br />
$.fn.naturalWidth = function() { return img(this[0].src).width; };<br />
$.fn.naturalHeight = function() { return img(this[0].src).height; };<br />
})(jQuery);<br />
<br />
<br />
<br />
function onWindowResize() <br />
{<br />
var curWidth = $(window).width(),<br />
curHeight = $(window).height(),<br />
checking=false;<br />
if (checking) {<br />
return;<br />
}<br />
checking = true;<br />
window.setTimeout(<br />
function() {<br />
var newWidth = $(window).width(),<br />
newHeight = $(window).height();<br />
if (!(newWidth !== curWidth ||<br />
newHeight !== curHeight)) {<br />
resize(false); <br />
}<br />
checking=false;<br />
}, 300);<br />
}<br />
<br />
function resize(initial) {<br />
if (!initial)<br />
{<br />
var container = $('#container');<br />
var imgWidth = container.width();<br />
<br />
$( "#map").each(function() {<br />
$(this).css('height', 'auto', 'width', 'auto');<br />
$(this).mapster('resize',Math.min(imgWidth, $(this).naturalWidth()) ,0,0); <br />
});<br />
}<br />
<br />
}<br />
<br />
$(document).ready(function(){<br />
<br />
$('#map').mapster({<br />
fillOpacity: 0.5,<br />
isSelectable: false,<br />
clickNavigate: true,<br />
mapKey: 'name',<br />
areas: [<br />
{<br />
key: "red",<br />
fillColor: 'FF0000'<br />
},<br />
<br />
{<br />
key: "blue",<br />
fillColor: '1ED9E1'<br />
},<br />
<br />
{<br />
key: "green",<br />
fillColor: '00FF00'<br />
},<br />
<br />
{<br />
key: "silver",<br />
fillColor: 'C0C0C0;'<br />
}<br />
]<br />
});<br />
<br />
<br />
<br />
$(window).resize(<br />
function()<br />
{ <br />
onWindowResize();<br />
});<br />
resize(true);<br />
<br />
});<br />
<br />
</script><br />
<br />
<article id='Our'></html><br />
== Our Approach ==<br />
<br />
The main focus of our project lies in complexity. How does it emerge? How can we handle it? Additionally, what can we learn from it and how can we use it? <br />
<br><br />
<br><br />
Our human practice project attempts to answer these questions by focusing on four different components: listening, discussing, thinking and sharing.<br />
<br><br />
<br><br />
<br><br />
<br />
<html><br />
<center><br />
<div id="container"><br />
<IMG SRC="https://static.igem.org/mediawiki/2014/d/d3/ETH_Zurich_Hpbuttons.png" width='500px' usemap="#map" id="map"><br />
<map id="map" name="map"><area shape="poly" alt="" title="" coords="235,38,152,175,320,175" href="https://2014.igem.org/Team:ETH_Zurich/human/interviews" id="blue" name="blue" target="" /><area shape="poly" id="red" name="red" alt="" title="" coords="125,224,43,359,207,358" href="https://2014.igem.org/Team:ETH_Zurich/human/essay" target="" /><area shape="poly" alt="" title="" coords="263,359,430,360,346,221" id="green" name="green" href="https://2014.igem.org/Team:ETH_Zurich/human/survey" target="" /><area shape="poly" alt="" title="" coords="0,347,22,362,223,36,249,36,445,366,471,354,263,3,207,3" href="https://2014.igem.org/Team:ETH_Zurich/human/outreach" target="" name="silver"/></map><br />
</div><br />
</center><br />
</html><br />
<br />
=.=<br />
The first component consists of a survey regarding complexity. By listening to the public we learn about the existing ideas of complexity and how people relate to it.<br />
<br><br />
<br><br />
Our second component involves interviews with experts from different backgrounds. These discussions focus on the complexity existing in their field and how they deal with it which enables us to improve our understanding of complexity as a whole and how we could profit from this profound, interdisciplinary knowledge. The various methods of resolution are summarized and compared in an essay.<br />
<br><br />
<br><br />
In our third component, we bring elements from the survey, from interviews, from further reading together. Thinking on how our project and science, in general, relates to these topics.<br />
<br><br />
<br><br />
Finally, we impart or share knowledge via different platforms like high school lectures and science slams. Here, we aim to explain the fundamentals of synthetic biology and how it can be a way to approach complexity. <br />
<html></article></html><br />
<br />
<br />
{{:Team:ETH Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/human/overviewTeam:ETH Zurich/human/overview2014-10-18T02:27:50Z<p>Clormeau: </p>
<hr />
<div>{{:Team:ETH Zurich/tpl/head|Human Practice Overview}}<br />
<br />
{{:Team:ETH_Zurich/tpl/scrollbutton|Complexity|blue}}<br />
{{:Team:ETH_Zurich/tpl/scrollbuttontworows|Our|Approach|red}}<br />
<br />
<html><article></html><br />
{{:Team:ETH_Zurich/human/overview/question}}<br />
<html></article></html><br />
<br />
<html><br />
<br />
<script type="text/javascript"><br />
(function($) {<br />
function img(url) {<br />
var i = new Image;<br />
i.src = url;<br />
return i;<br />
}<br />
<br />
if ('naturalWidth' in (new Image)) {<br />
$.fn.naturalWidth = function() { return this[0].naturalWidth; };<br />
$.fn.naturalHeight = function() { return this[0].naturalHeight; };<br />
return;<br />
}<br />
$.fn.naturalWidth = function() { return img(this[0].src).width; };<br />
$.fn.naturalHeight = function() { return img(this[0].src).height; };<br />
})(jQuery);<br />
<br />
<br />
<br />
function onWindowResize() <br />
{<br />
var curWidth = $(window).width(),<br />
curHeight = $(window).height(),<br />
checking=false;<br />
if (checking) {<br />
return;<br />
}<br />
checking = true;<br />
window.setTimeout(<br />
function() {<br />
var newWidth = $(window).width(),<br />
newHeight = $(window).height();<br />
if (!(newWidth !== curWidth ||<br />
newHeight !== curHeight)) {<br />
resize(false); <br />
}<br />
checking=false;<br />
}, 300);<br />
}<br />
<br />
function resize(initial) {<br />
if (!initial)<br />
{<br />
var container = $('#container');<br />
var imgWidth = container.width();<br />
<br />
$( "#map").each(function() {<br />
$(this).css('height', 'auto', 'width', 'auto');<br />
$(this).mapster('resize',Math.min(imgWidth, $(this).naturalWidth()) ,0,0); <br />
});<br />
}<br />
<br />
}<br />
<br />
$(document).ready(function(){<br />
<br />
$('#map').mapster({<br />
fillOpacity: 0.5,<br />
isSelectable: false,<br />
clickNavigate: true,<br />
mapKey: 'name',<br />
areas: [<br />
{<br />
key: "red",<br />
fillColor: 'FF0000'<br />
},<br />
<br />
{<br />
key: "blue",<br />
fillColor: '1ED9E1'<br />
},<br />
<br />
{<br />
key: "green",<br />
fillColor: '00FF00'<br />
},<br />
<br />
{<br />
key: "silver",<br />
fillColor: 'C0C0C0;'<br />
}<br />
]<br />
});<br />
<br />
<br />
<br />
$(window).resize(<br />
function()<br />
{ <br />
onWindowResize();<br />
});<br />
resize(true);<br />
<br />
});<br />
<br />
</script><br />
<br />
<article></html><br />
== Our Approach ==<br />
<br />
The main focus of our project lies in complexity. How does it emerge? How can we handle it? Additionally, what can we learn from it and how can we use it? <br />
<br><br />
<br><br />
Our human practice project attempts to answer these questions by focusing on four different components: listening, discussing, thinking and sharing.<br />
<br><br />
<br><br />
<br><br />
<br />
<html><br />
<center><br />
<div id="container"><br />
<IMG SRC="https://static.igem.org/mediawiki/2014/d/d3/ETH_Zurich_Hpbuttons.png" width='500px' usemap="#map" id="map"><br />
<map id="map" name="map"><area shape="poly" alt="" title="" coords="235,38,152,175,320,175" href="https://2014.igem.org/Team:ETH_Zurich/human/interviews" id="blue" name="blue" target="" /><area shape="poly" id="red" name="red" alt="" title="" coords="125,224,43,359,207,358" href="https://2014.igem.org/Team:ETH_Zurich/human/essay" target="" /><area shape="poly" alt="" title="" coords="263,359,430,360,346,221" id="green" name="green" href="https://2014.igem.org/Team:ETH_Zurich/human/survey" target="" /><area shape="poly" alt="" title="" coords="0,347,22,362,223,36,249,36,445,366,471,354,263,3,207,3" href="https://2014.igem.org/Team:ETH_Zurich/human/outreach" target="" name="silver"/></map><br />
</div><br />
</center><br />
</html><br />
<br />
=.=<br />
The first component consists of a survey regarding complexity. By listening to the public we learn about the existing ideas of complexity and how people relate to it.<br />
<br><br />
<br><br />
Our second component involves interviews with experts from different backgrounds. These discussions focus on the complexity existing in their field and how they deal with it which enables us to improve our understanding of complexity as a whole and how we could profit from this profound, interdisciplinary knowledge. The various methods of resolution are summarized and compared in an essay.<br />
<br><br />
<br><br />
In our third component, we bring elements from the survey, from interviews, from further reading together. Thinking on how our project and science, in general, relates to these topics.<br />
<br><br />
<br><br />
Finally, we impart or share knowledge via different platforms like high school lectures and science slams. Here, we aim to explain the fundamentals of synthetic biology and how it can be a way to approach complexity. <br />
<html></article></html><br />
<br />
<br />
{{:Team:ETH Zurich/tpl/foot}}</div>Clormeauhttp://2014.igem.org/Team:ETH_Zurich/human/overviewTeam:ETH Zurich/human/overview2014-10-18T02:27:34Z<p>Clormeau: </p>
<hr />
<div>{{:Team:ETH Zurich/tpl/head|Human Practice Overview}}<br />
<br />
{{:https://2014.igem.org/Team:ETH_Zurich/tpl/scrollbutton|Complexity|blue}}<br />
{{:https://2014.igem.org/Team:ETH_Zurich/tpl/scrollbuttontworows|Our|Approach|red}}<br />
<html><article></html><br />
{{:Team:ETH_Zurich/human/overview/question}}<br />
<html></article></html><br />
<br />
<html><br />
<br />
<script type="text/javascript"><br />
(function($) {<br />
function img(url) {<br />
var i = new Image;<br />
i.src = url;<br />
return i;<br />
}<br />
<br />
if ('naturalWidth' in (new Image)) {<br />
$.fn.naturalWidth = function() { return this[0].naturalWidth; };<br />
$.fn.naturalHeight = function() { return this[0].naturalHeight; };<br />
return;<br />
}<br />
$.fn.naturalWidth = function() { return img(this[0].src).width; };<br />
$.fn.naturalHeight = function() { return img(this[0].src).height; };<br />
})(jQuery);<br />
<br />
<br />
<br />
function onWindowResize() <br />
{<br />
var curWidth = $(window).width(),<br />
curHeight = $(window).height(),<br />
checking=false;<br />
if (checking) {<br />
return;<br />
}<br />
checking = true;<br />
window.setTimeout(<br />
function() {<br />
var newWidth = $(window).width(),<br />
newHeight = $(window).height();<br />
if (!(newWidth !== curWidth ||<br />
newHeight !== curHeight)) {<br />
resize(false); <br />
}<br />
checking=false;<br />
}, 300);<br />
}<br />
<br />
function resize(initial) {<br />
if (!initial)<br />
{<br />
var container = $('#container');<br />
var imgWidth = container.width();<br />
<br />
$( "#map").each(function() {<br />
$(this).css('height', 'auto', 'width', 'auto');<br />
$(this).mapster('resize',Math.min(imgWidth, $(this).naturalWidth()) ,0,0); <br />
});<br />
}<br />
<br />
}<br />
<br />
$(document).ready(function(){<br />
<br />
$('#map').mapster({<br />
fillOpacity: 0.5,<br />
isSelectable: false,<br />
clickNavigate: true,<br />
mapKey: 'name',<br />
areas: [<br />
{<br />
key: "red",<br />
fillColor: 'FF0000'<br />
},<br />
<br />
{<br />
key: "blue",<br />
fillColor: '1ED9E1'<br />
},<br />
<br />
{<br />
key: "green",<br />
fillColor: '00FF00'<br />
},<br />
<br />
{<br />
key: "silver",<br />
fillColor: 'C0C0C0;'<br />
}<br />
]<br />
});<br />
<br />
<br />
<br />
$(window).resize(<br />
function()<br />
{ <br />
onWindowResize();<br />
});<br />
resize(true);<br />
<br />
});<br />
<br />
</script><br />
<br />
<article></html><br />
== Our Approach ==<br />
<br />
The main focus of our project lies in complexity. How does it emerge? How can we handle it? Additionally, what can we learn from it and how can we use it? <br />
<br><br />
<br><br />
Our human practice project attempts to answer these questions by focusing on four different components: listening, discussing, thinking and sharing.<br />
<br><br />
<br><br />
<br><br />
<br />
<html><br />
<center><br />
<div id="container"><br />
<IMG SRC="https://static.igem.org/mediawiki/2014/d/d3/ETH_Zurich_Hpbuttons.png" width='500px' usemap="#map" id="map"><br />
<map id="map" name="map"><area shape="poly" alt="" title="" coords="235,38,152,175,320,175" href="https://2014.igem.org/Team:ETH_Zurich/human/interviews" id="blue" name="blue" target="" /><area shape="poly" id="red" name="red" alt="" title="" coords="125,224,43,359,207,358" href="https://2014.igem.org/Team:ETH_Zurich/human/essay" target="" /><area shape="poly" alt="" title="" coords="263,359,430,360,346,221" id="green" name="green" href="https://2014.igem.org/Team:ETH_Zurich/human/survey" target="" /><area shape="poly" alt="" title="" coords="0,347,22,362,223,36,249,36,445,366,471,354,263,3,207,3" href="https://2014.igem.org/Team:ETH_Zurich/human/outreach" target="" name="silver"/></map><br />
</div><br />
</center><br />
</html><br />
<br />
=.=<br />
The first component consists of a survey regarding complexity. By listening to the public we learn about the existing ideas of complexity and how people relate to it.<br />
<br><br />
<br><br />
Our second component involves interviews with experts from different backgrounds. These discussions focus on the complexity existing in their field and how they deal with it which enables us to improve our understanding of complexity as a whole and how we could profit from this profound, interdisciplinary knowledge. The various methods of resolution are summarized and compared in an essay.<br />
<br><br />
<br><br />
In our third component, we bring elements from the survey, from interviews, from further reading together. Thinking on how our project and science, in general, relates to these topics.<br />
<br><br />
<br><br />
Finally, we impart or share knowledge via different platforms like high school lectures and science slams. Here, we aim to explain the fundamentals of synthetic biology and how it can be a way to approach complexity. <br />
<html></article></html><br />
<br />
<br />
{{:Team:ETH Zurich/tpl/foot}}</div>Clormeau