ETHZ

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[[Image:Eth_zh_logo_2.png|800px]] __TOC__
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[[Image:ETHZ_banner.png|830px]]
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== Team Members ==
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<center>[[ETHZ | Main Page]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Model | System Modeling]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Simulation | Simulations]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology | System Implementation]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Biology/Lab| Lab Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Meet_the_team | Meet the Team]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Internal | Team Notes]] &nbsp;&nbsp;&nbsp;&nbsp; [[ETHZ/Pictures | Pictures!]]</center><br>
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| class="taxo-image" | [[Image:|thumb|398px|'''Picture:''' The ETH Zuerich iGEM Team 2007 ''(left to right: )'']]
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<script type="text/javascript" src="http://christos.bergeles.net/eth_dropdowntabs.js">
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For the International Competition on Genetically Engineered Machines, the teams should be composed of both biologist and engineers. That way, the engineers will try to develop new systems in a bottom-up fashion and run numerical simulations, while the biologists will be able to assess the feasibility of such systems, and construct them from biological parts.
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<P><b>Instructors:</b></P>
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[https://2007.igem.org/User:sven Sven Panke]
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Joerg Stelling
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<ul>
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<li><a href="https://2007.igem.org/wiki/index.php?title=ETHZ" title="Home" rel="dropmenu_home"><span>Home</span></a></li>
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<li><a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Model" title="Modeling" rel="dropmenu_modeling"><span>System Modeling</span></a></li>
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<li><a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Simulation" title="Simulations" rel="dropmenu_simulation"><span>Simulations</span></a></li>
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<li><a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Biology" title="System Implementation" rel="dropmenu_biology"><span>System Implementation</span></a></li>
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<li><a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Meet_the_team" title="Meet the team" rel="dropmenu_meettheteam"><span>Meet the team</span></a></li>
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<li><a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Pictures" title="Pictures!" rel="dropmenu_pictures"><span>Pictures!</span></a></li>
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<div class="ddcolortabsline">&nbsp;</div>
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<P><b>Students:</b></P>
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[https://2007.igem.org/User:brutsche Martin Brutsche]
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<div id="dropmenu_home" class="dropmenudiv_a">
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ#Introduction">Introduction</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ#Team_Members">Team Members</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ#Acknowledgments">Acknowledgments</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ#Site_Map">Site map</a>
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</div>
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[https://2007.igem.org/User:kdikaiou Katerina Dikaiou]
 
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[https://2007.igem.org/User:Raphael Raphael Guebeli]
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<div id="dropmenu_modeling" class="dropmenudiv_a" style="width: 150px;">
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Model#Introduction">Introduction</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Model#Model_Overview">Model Overview</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Model#Detailed_Model">Detailed Model</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Model#Final_Model">Final Model</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Modeling_Basics">Modeling Basics Page</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Model#Mathematical_Model">Mathematical Model</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/FSM">FSM View Page</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/FlipFlop">Flip-Flop View Page</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Parameters">Parameters Page</a>
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[https://2007.igem.org/User:hoehnels Sylke Hoehnel]
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<div id="dropmenu_simulation" class="dropmenudiv_a" style="width: 150px;">
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Simulation#Introduction">Introduction</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Simulation#Simulation_of_Test_Cases">Test Cases</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Simulation#Sensitivity_Analysis">Sensitivity Analysis</a>
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[https://2007.igem.org/User:LiNan Nan Li]
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<div id="dropmenu_biology" class="dropmenudiv_a" style="width: 150px;">
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Biology#Introduction">Introduction</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Biology#The_Complete_System">The Complete System</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Biology#System_Phases">System Phases</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Biology#Current_Cloning_Status">Current Cloning Status</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Biology/parts">System Parts Page</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Biology/Lab">Lab Notes Page</a>
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[https://2007.igem.org/User:Stefan Stefan Luzi]
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Meet_the_team#The_ETH_Zurich_07_Team">The ETH Zurich 07 Team</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Meet_the_team#Team_Description">Team Description</a>
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<a href="https://2007.igem.org/wiki/index.php?title=ETHZ/Internal">Brainstorming Page</a>
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</div>
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<P><b>Graduate Students:</b></P>
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<script type="text/javascript">
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[http://christos.bergeles.net Christos Bergeles]
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[http://www.tik.ee.ethz.ch/~sop/people/thohm/ Tim Hohm]
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[http://fm-eth.ethz.ch/eth/peoplefinder/FMPro?-db=phonebook.fp5&-format=pf%5fdetail%5fde.html&-lay=html&-op=cn&Typ=Staff&Suche%5fText=kemmer&Suche%5fText%5fpre=kemmer&-recid=3772770936&-find=/ Christian Kemmer]
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[https://2007.igem.org/User:JoeKnight Joseph Knight]
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<p align="center"><font size="6"><b>ETH Zurich - educatETH <i>E.coli</i> System</b></font></p>
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[https://2007.igem.org/User:uhrm Markus Uhr]
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=Introduction=
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<blockquote>"All <i>E.coli</i> 's are equal, but some <i>E.coli</i> 's are more equal than others..." ''(freely adapted from "Animal Farm" by George Orwell)''</blockquote> 
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[http://www.ricomoeckel.de Rico Möckel]
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... this is what George Orwell would have written, were he a synthetic biologist. In the <i>E.coli</i> colonies on petri dishes, all bacteria are equal; except for some special ones. Our project is about designing such special <i>E.coli</i> that are "more equal" than the rest: they have the ability to be trained in order to memorize and recognize their environment in the future. Their  story will be presented through this wiki ...
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== Introduction to Synthetic Biology ==
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==Motivation==
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In order to get an initial understanding of the concepts of synthetic biology, we read and presented
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publications on various topics. A representative list of the topics that we covered on this “boot-camp” is listed in the following:
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* Introduction to synthetic biology
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[[Image:ethz_main_pic.jpg|right|thumb|<b>Fig. 1</b>: Artist's approach to the different stages of the development. We started by modeling and simulating the system. We continued by specifying the DNA strands for its implementation. Finally in the end, our system should report with different fluorescent proteins (image edited)|600px]]
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* DNA de novo design
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* DNA circuits
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* Hysteresis
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* Oscillators
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* Zinc fingers
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* Noise in single cell measurements
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* Distance communication
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* Parameter manipulations
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* Protein logic
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* Orthogonal systems
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* mRNA engineering
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* RNA regulators
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== Choosing the Project ==
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Our combined team of biologists and engineers is coping with the problem of implementing memory capabilities in bacterial colonies. First, ''E.coli'' are able to respond differently (with distinct fluorescent proteins) to two different inputs (we used chemicals). Second, they remember which input was presented to them. Finally, when confronted with a new input, they are able to recognize whether it is the one that they were trained with or not.
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=== Step 1: Brainstorming ===
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In other words, in this project '''we are educating the <i>E.coli</i>'''!
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Initially, we wanted to come up with as many ideas as possible, in order to be able to choose among them the best, and find a cool project to carry out. For this reason, we had brainstorming sessions. We split up in three teams, and each team tried to come up with many fancy and showy ideas, which was facilitated by keeping in mind the following brainstorming guidelines:
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# Defer judgment - the rules of nature don't apply
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# Encourage wild ideas
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# Build on the ideas of others
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# Be visual
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# Go for quantity
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# Stay focused on topic
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The ideas that we came up with, as were presented in the following meetings, were:
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===Multipurpose Cell Lines===
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* '''PID Controller''': Design a PID controller out of biological elements. The P component can be a simple output to a regulatory protein, and the I component can be the overall protein production at a time period. What can the D component be?
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* '''Motion Detector''': Cells are grown on a petri dish. Below the dish, moving images are displayed. A 3-state automaton is proposed. Output A is created when light is present. Output B is created when light is absent. Moving patterns will cause some cells to create both outputs over time. This will result in some “inspector” cells producing output C, by collecting outputs A and B.
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* '''Analog-to-Digital Converter''': Compare the level of protein concentration with thresholds, and digitize the output.
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* '''Neural Network''': Create a sort of biological neural network with bacteria. We should address the issue of learning, and find a way to incorporate the feedback in the cell decision making process. Directed evolution can be a sort of feedback, but we want to avoid this. (This idea was the basis for the “learning project”)
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* '''Paramedic Cells''': Some cells are able to detect signals coming from other cells, and create food for them, or create proteins in order to save them and make them function better.
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* '''Cell Batteries''': Cells are able to create and store large quantities of ATP, during a “storing process”. Afterwards, they can detect a signal and give back all the energy they stored, in a short burst, like a capacitor. Other ideas are that the cells can “blow up” and emit large amounts of GFP, based on the ATP that they have accumulated.
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* '''Flashing Bacteria''': Cells are grown on a light pattern. The cells that are on the bright parts of the image are oscillating in phase, while the others are remaining dark. This results in the observation of a flashing pattern.
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* '''Biocam''': Visible to Fluorescent light converter.
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* '''BioCD''': “Print” cells on a film, then read them out and “reconstruct” the original data. Basically, it is an analog to digital converter, followed by a system that can interpret the digitized data. (This idea was the basis for the “Music of life project”, where cells would produce fluorescent proteins based on an analog input. Then, the amount and type of fluorescence would code some music).
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* '''Clock''': A follow-the-leader system. We have to groups of cells. The first group creates something that repels the second group. The second group creates a protein that attracts the first group. This way, they first group wants to “catch” the second group, whereas the second group wants to “avoid” the first group. This results in them moving around. We can say that the second group is the leader, and the first group exhibits a "follow-the-leader” behavior.
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* '''Sensors''': Various systems that can sense PH, pressure, temperature, meat quality, moisture e.t.c. have been proposed.
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=== Step 2: Preferred Projects ===
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Our system has the ability to behave in different ways according to an internal toggle inside it switching states based on the chemical substances that the system is exposed to. The toggle states could generally be used to trigger events such as enzyme synthesis, transcriptional regulation, virion production, or even cell death. Therefore, one may view the bacterial cell line containing this system as a multipurpose cell line. By adding a certain chemical to a cell line, the latter may be trained to exhibit a desired behavior, and then it is not necessary any more to construct two independent cell lines.
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We decided that three of the above ideas were worth a deeper examination. Namely, we split again in three
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This means that one applies an “input engineering” instead of a “DNA engineering” approach. If one extends this idea to several multi-inducible toggle switches being harbored in the same cell line, the number of possible phenotypes increases to 2<i><sup>n</sup></i>, where <i>n</i> equals the number of toggle switches. For example, if one would have 5 such toggle switches inside a cell line, 32 different behavior patterns would be possible.  
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teams, so that we can come up with an initial system, with remarks on its feasibility and coolness. Our results
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were presented on all the team members, so that we could then limit the potential projects down to two and
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one, and proceed with constructing the DNA sequence. The three projects and the presentations that we did
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were:
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# '''Music of Life''': The basic idea is that instead of having an analog-to-digital converter with four outputs (three fluorescent proteins, and no output), we can have two switches. When switch A is on, RFP is produced. When switch B is on, GFP is produced. When both switches A and B are on, a yellowish output is observed. By recording these outputs, we can later create music, by assigning each fluorescent protein to a chord. For example, RFP would correspond to a G chord. The strength of the fluorescence can signify the strength of the chord. If the cells are placed on a spinning disk, we can have something like a vinyl player. A camera is observing the cells, and music is created on the fly.
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# '''Learning''': Based on the idea of the neural network, we want to create a biological system, where the cells can learn a specific behavior. In order to simplify the system, we decided that the cells can learn to recognize a specific type of other cells. We divide the process in a learning phase, and a recognition phase. First, cells A are put together with cells B. Then, cells A are “learning” to recognize cells B. If afterwards they are put in a petri dish with cells B, they will emit GPF. Otherwise, they will stay dark.
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Idea two, even though it was fancy, was fairly simple, as, creating two switches is a straightforward and well understood process. As a result, it was discarded. We could try to make the system more complex, but making something more complex just for the sake of it, is not a good engineering approach ;)
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For the purpose of creating a toggle switch that is activated in a specific phase only and not always (a multi-inducible toggle switch), as is required for stable biological automatons, we introduced the concept of [https://2007.igem.org/ETHZ/Biology/parts#double_promoters double promoters] to the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts], which can be helpful for engineering systems which exhibit a desired behavior only at specific times.
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== The Project ==
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===Link to Epigenetics===
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Epigenetics refers to features like chromatin or DNA modifications that do not involve changes in the underlying DNA sequence and are stable over many cell divisions [1],[2]. If one has a closer look at our proposed system, one can also view it as a model-system for epigenetics: Although the DNA sequence itself stays the same, two different subpopulations of cells with different phenotypes can develop from it. Put simply, depending in which state (subpopulation) the toggle switch is, the cells will produce different fluorescent proteins upon addition of two different inducer molecules. Therefore, the epigenetic feature here is the binding of specific repressor proteins whose production is dependent on the toggle switch state.
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We decided to proceed with the '''Learning Project'''. Our goal is to design an E.coli strain, able to distinguish between two chemical substances after an assigned learning process, induced by an external chemical signal (teaching substance). After the bacterial strain was taken to a testing phase, the output will result in either yellow or cyan fluorescence, depending on whether the bacteria recognized the same chemical substance in the testing phase as it was trained in the learning phase or a different one.
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===Intelligent Biosensors and Self-Adaptation===
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We aim to implement our system using a toggle switch consisting of different repressor and activator proteins coupled with double regulatory regions.
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Our biological system was inspired from the toggle of [?], and its binary logic equivalent. In order to understand that, one should think of learning as a "switching of behavior". As a response to an external stimulus, the behavior changes. If we approach the problem with the tools of binary logic, we can see that we want to implement a JK flip-flop with a latch. At the learning process, the JK flip-flop will stabilize at its final state Q. At the recognition phase, the flip-flop is latched, and recognition is performed (see figure below):
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The system is capable of sensing different chemicals and producing different fluorescent proteins. Since the cells can be trained to produce one of several specific fluorescent protein types when a certain chemical is present, one can also view those cells as intelligent biosensors which recognize chemical substances according to a training phase. The intelligent biosensors are not limited to detect chemicals; temperature, pH, light, pressure could be detected with an appropriate system as well. Such an application could be especially of interest when the environment to be probed is harmful for humans, for example due to high toxicity.
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[[Image:logic_circuit.png]]
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==Team Members==
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[[Image:ETHZ_Group_photo_6.png|right|thumb|The ETH Zurich iGEM2007 Team|270px]]
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Based on the toggle from [?], we discussed our prototype system (see figure below), which the resulted in the refined system that we describe in the next section.
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The ETH Zurich team consists of a good mixture between biologists and engineering students.
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We are:
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<ul>
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<li><i>Undergraduate students</i>: <br>
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[https://2007.igem.org/User:brutsche Martin Brutsche], [https://2007.igem.org/User:kdikaiou Katerina Dikaiou], <br>[https://2007.igem.org/User:Raphael Raphael Guebeli], [https://2007.igem.org/User:hoehnels Sylke Hoehnel], <br>
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[https://2007.igem.org/Nan_Li Nan Li],
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[https://2007.igem.org/User:Stefan Stefan Luzi]
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<li><i>Graduate students</i>: <br>
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[http://christos.bergeles.net Christos Bergeles],
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[http://www.tik.ee.ethz.ch/~sop/people/thohm/ Tim Hohm], <br>
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[http://www.fussenegger.ethz.ch/people/kemmerc Christian Kemmer],
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[https://2007.igem.org/User:JoeKnight Joseph Knight], <br>
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[http://www.ricomoeckel.de Rico Möckel],
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[http://csb.inf.ethz.ch/people/uhr.html Markus Uhr]
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<li><i>Project advisors</i>: <br>
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[http://www.ipe.ethz.ch/laboratories/bpl/people/panke Sven Panke], <br>
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[http://csb.inf.ethz.ch/people/stelling.html Joerg Stelling]
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</ul>
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(picture of initial draft of the bio system, as taken from Joe, --here--)
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For more information about us, visit our [[ETHZ/Meet_the_team | Meet the Team]] page.
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=== System Models ===
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==Acknowledgments==
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[[Image:ethz_system_1.png]]
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The idea for the project as well as its implementation was done solely by the ETH iGEM 2007 team. We would like to thank the people in [http://www.ipe.ethz.ch/laboratories/bpl/index Sven Panke's Lab], especially Andreas Meyer who was always there for us when we had a problem. Additionally, we would like to thank [http://www.facs.ethz.ch Alfredo Franco-Obregón's lab] and Oralea Büchi for the help with the flow cytometry.
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<br>
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<br>
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[[Image:ethz_system_2.png]]
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=== Simulations ===
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We would also like to acknowledge the financial support by [http://europa.eu EU], the [http://www.ethz.ch ETH Zurich], and [http://www.geneart.com GeneArt]:
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[[ETHZ/Simulations| detailed information on the simulations]]<BR>
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<center>
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{| border="0"
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=== Sensitivity Analysis ===
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== Lab Work ==
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Here we can write when each one will be available. For example:
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*<b>Christos:</b><br> Can be available everyday after 17:30, till 00:00. Weekends as well. Also 11:00-13:00 everyday. Away 17.08-19.08, 22.08-24.08.
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*<b>Tim:</b><br> 13.08.-15.08. after 17:30<br> away from 17.08. until 02.09.
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*<b>Martin:</b><br> Every day, also weekends, for half a day or even more if it's requiered. The last week of August and the first one in September I have only time for aproximately 5-6 h a day due to learning for the exams.
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*<b>Katerina:</b><br> Not available 5.09-7.09 (final exams). Otherwise available after 17:30. Possible also in the morning, if I know in advance that I will be needed.
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=== Cloning Plan ===
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The system consists of 11 parts. These are:
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{| class="wikitable" border = "1" style="text-align:left"
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|+ '''System parts'''
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|-
|-
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| [http://europa.eu http://www.tik.ee.ethz.ch/~thohm/EU.gif]
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| width="40" |
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| [http://www.ethz.ch http://www.tik.ee.ethz.ch/~thohm/ethlogo.jpg]
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| width="40" |
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| [http://www.geneart.com http://www.tik.ee.ethz.ch/~thohm/geneart.gif]
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|}
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</center>
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==Site Map==
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In this wiki, we will present to you a detailed description of the proposed system: starting with the [[ETHZ/Model | modeling of the system]], we describe both, [[ETHZ/Simulation | simulations and theoretical considerations]] of the system, as well as the actual [[ETHZ/Biology | implementation using bio-bricks]] accompanied by our [[ETHZ/Biology/Lab | lab notes]]. Additionally, you will find further [[ETHZ/Meet_the_team | information on the team]], more details about [[ETHZ/Internal | ideas we developed]] before we came up with the system we finally implemented, and some [[ETHZ/Pictures | pictures]] documenting our work.
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The site map of our wiki is the following:
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! 1
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| TetR production(constitutive part of system)
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{| class="wikitable" width="100%" border="1" cellspacing="0" cellpadding="2" style="text-align:left; margin: 1em 1em 1em 0; background: #f9f9f9; border: 1px #aaa solid; border-collapse: collapse;"
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!width="34%"| Modeling Pages                       
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!width="22%"| Biology Pages
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!width="22%"| ETHZ Team Pages
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!width="22%"| Links
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| [[ETHZ/Model | Modeling of the learning system]]
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! 2
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| [[ETHZ/Biology | Biological implementation]]
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| [[ETHZ/Meet_the_team | Team page]]
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| LacI production (constitutive part of system)
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| [https://2006.igem.org/wiki/index.php/ETH_Zurich_2005 The ETH Zurich 2005 project]
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| [[ETHZ/FlipFlop | Representation using flip-flops]]
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! 3
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| [[ETHZ/Biology/parts | Biobricks/parts]]
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| [[ETHZ/Pictures | Pictures]]
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| LuxR production(constitutive part of system)
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| [https://2006.igem.org/wiki/index.php/ETH_Zurich_2006 The ETH Zurich 2006 project]
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| [[ETHZ/FSM | Representation using finite state machines]]
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! 4
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| [[ETHZ/Biology/Lab | Lab notes]]
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| [[ETHZ/Internal | Brainstorming sessions]]
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| 1st half of p22/YFP production (outer part of system, reporting)
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|-
|-
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| [[ETHZ/Simulation | Model simulations and theoretical considerations]]
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! 5
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|
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|
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| 2nd half of p22/YFP production (outer part of system, reporting)
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|  
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|-
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| [[ETHZ/Parameters | Parameters used in our simulations]]
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!6
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|
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|
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| CI production (inner part of system)
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|  
|-
|-
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! 7
 
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| p22 production (inner part of system)
 
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|-
 
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! 8
 
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| 1st half of CI/CFP production (outer part of system, reporting)
 
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|-
 
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! 9
 
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| 2nd half of CI/CFP production (outer part of system, reporting)
 
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|-
 
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! 10
 
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| RFP production (reporting)
 
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|-
 
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! 11
 
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| GFP production (reporting)
 
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|}
|}
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3 plasmids will be used for the 11 system parts as follows:
+
== References ==
-
 
+
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{| class="wikitable" border = "1" style="text-align:left"
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|+'''Plasmids and contents'''
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|-
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! plasmid !! resistance !! copy type!! contents !! comments
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|-
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| [[ETHZ/pbr322| pbr322]] || ampicillin || insert here || 1,2,3 || constitutive part
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|-
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| [[ETHZ/pck01| pck01]] || chloramphenicol|| insert here || 4,5,8,9 || outer part
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|-
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| [[ETHZ/pacyc177| pacyc177]] || kanamycin|| insert here || 6,7,10,11 || inner part, reporting
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|-
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|}
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The standard BioBrick assembly will be used to put the parts in the plasmids. Detailed information on how the BioBrick part fabrication works can be found  [http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication here]. For a shorter explanation of how to assemble 2 parts together check [http://partsregistry.org/Assembly:Standard_assembly here]. Note that the composite part is constructed from the end to the beginning, i.e. each new part is inserted ''before'' the existing one. In the following, the plasmid containing the new part to be inserted will be referred to as the ''donor'' and the plasmid accepting the new part will be referred to as the ''acceptor''. Composite pars made of parts '''a''' and '''b''' are denoted '''a.b'''.
+
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==== '''Plasmid 1 ''(pbr322ap)'' ''' ====
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# Put parts 1,2,3 in  pbr322ap plasmids.
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# Merge plasmid containing part '''2''' ''(donor)'' with plasmid containing part '''3''' ''(acceptor)''. You should get a plasmid containing a '''2.3''' composite part.
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# Merge plasmid containing part '''1''' ''(donor)'' with plasmid containing composite part '''2.3'''  ''(acceptor)''. You should get a plasmid containing a '''1.2.3''' composite part.
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==== '''Plasmid 2 ''(pck01cm)'' ''' ====
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# Put parts 4,5,8,9 in  pck01cm plasmids.
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# Merge plasmid containing part '''4''' ''(donor)'' with plasmid containing part '''5''' ''(acceptor)''. You should get a plasmid containing a '''4.5''' composite part.
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# Merge plasmid containing part '''8''' ''(donor)'' with plasmid containing part '''9''' ''(acceptor)''. You should get a plasmid containing a '''8.9''' composite part. ''Note'': this step can be done simultaneously with the above.
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# Merge plasmid containing composite part '''4.5''' ''(donor)'' with plasmid containing composite part '''8.9''' ''(acceptor)''. You should get a plasmid containing a '''4.5.8.9''' composite part.
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==== '''Plasmid 3 ''(pacyc177km)'' ''' ====
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# Put parts 6,7,10,11 in  pacyc177km plasmids.
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# Merge plasmid containing part '''6''' ''(donor)'' with plasmid containing part '''7''' ''(acceptor)''. You should get a plasmid containing a '''6.7''' composite part.
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# Merge plasmid containing part '''10''' ''(donor)'' with plasmid containing part '''11''' ''(acceptor)''. You should get a plasmid containing a '''10.11''' composite part. ''Note'': this step can be done simultaneously with the above.
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-
# Merge plasmid containing composite part '''6.7''' ''(donor)'' with plasmid containing composite part '''10.11''' ''(acceptor)''. You should get a plasmid containing a '''6.7.10.11''' composite part.
+
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=== Lab Instructions and Methods ===
+
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=== Lab Schedule ===
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<font face="DejaVu Sans" size="3"><b>Week 1</b></font> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <b>06.08.07 - 12.08.07</b><br>
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[[ETHZ/labnotes| lab schedule week 1]]<BR>
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<font face="DejaVu Sans" size="3"><b>Week 2</b></font> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <b>13.08.07 - 19.08.07</b><br>
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[[ETHZ/labnotes| lab schedule week 2]]<BR>
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<font face="DejaVu Sans" size="3"><b>Week 3</b></font> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <b>20.08.07 - 26.08.07</b><br>
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[[ETHZ/labnotes| lab schedule week 3]]<BR>
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<font face="DejaVu Sans" size="3"><b>Week 4</b></font> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <b>27.08.07 - 02.09.07</b><br>
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[[ETHZ/labnotes| lab schedule week 4]]<BR>
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<font face="DejaVu Sans" size="3"><b>Week 5</b></font> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; <b>03.08.07 - 09.09.07</b><br>
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[[ETHZ/labnotes| lab schedule week 5]]<BR>
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=== Lab Notes / Log Book ===
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Here we can write down every day a detailed version of the work we have done (it should be the same stuff we write in the "real lab book" which stays in the lab). In this way everyone can see in detail what was done in the lab.
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[[ETHZ/Lab book| detailed lab notes]]<BR>
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[http://www.nature.com/nature/journal/v447/n7143/abs/nature05913.html;jsessionid=62903C604764B175945C03DB8639ECBD &#91;1&#93; Bird A] <i>"Perceptions of epigenetics"</i>, Nature 447:396-398, 2007 <br />
 +
[http://linkinghub.elsevier.com/retrieve/pii/S096098220701007X &#91;2&#93; Ptashne M] <i>"On the use of the word ‘epigenetic’"</i>, Current Biology 17(7):R233-R236, 2007 <br />
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== Task List ==
 
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The things to do, from the most pressing (timewise), to the least pressing (timewise) is below. Please put your name next to the task that you believe that you can undertake.
 
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# Team descriptions (overdue) and team photograph
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<html><body>
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# Team rosters due (1.9.)<br> Martin: Does somebody know, what exactly should be done here?
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<a href="http://www3.clustrmaps.com/counter/maps.php?url=https://2007.igem.org/ETHZ" id="clustrMapsLink"><img src="http://www3.clustrmaps.com/counter/index2.php?url=https://2007.igem.org/ETHZ" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onError="this.onError=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com'" />  
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# Labwork (parts have to be at the registry in Boston on 26.10.) <br> Joe: I can be in the lab at least 2 evenings a week and some times through the weekdays. <br> Martin: From Monday I can work every day for the whole day. At the moment I only work for several hours...
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</a>
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# Simulation and sensitivity analysis<br> Martin: From 10. Sept. I've got plenty time to work on it. I think Markus would join here too ;-)  <br> Tim: I can contribute in running stuff and help identify parameters from literature
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# Presentation <br> Joe: I'm American... I can sale anything.<br> Martin: I'm bad in Layout stuff, but maybe I can help as an idea supplier or so.
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<html><body>
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# Poster <br> Joe: See 5. above
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<a>
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# Wiki (Project and part documentation due on 26.10) <br> Martin: See the points above, from next Monday I will give everything, now I'm doing my best...
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<img src="http://www.easycounter.com/counter.php?stefanluzi"
 +
border="0" alt="Welcome to ETHz - iGEM07"></a>
 +
</body></html>

Latest revision as of 20:20, 26 October 2007

ETHZ banner.png

 


ETH Zurich - educatETH E.coli System

Introduction

"All E.coli 's are equal, but some E.coli 's are more equal than others..." (freely adapted from "Animal Farm" by George Orwell)

... this is what George Orwell would have written, were he a synthetic biologist. In the E.coli colonies on petri dishes, all bacteria are equal; except for some special ones. Our project is about designing such special E.coli that are "more equal" than the rest: they have the ability to be trained in order to memorize and recognize their environment in the future. Their story will be presented through this wiki ...

Motivation

Fig. 1: Artist's approach to the different stages of the development. We started by modeling and simulating the system. We continued by specifying the DNA strands for its implementation. Finally in the end, our system should report with different fluorescent proteins (image edited)

Our combined team of biologists and engineers is coping with the problem of implementing memory capabilities in bacterial colonies. First, E.coli are able to respond differently (with distinct fluorescent proteins) to two different inputs (we used chemicals). Second, they remember which input was presented to them. Finally, when confronted with a new input, they are able to recognize whether it is the one that they were trained with or not. In other words, in this project we are educating the E.coli!

Multipurpose Cell Lines

Our system has the ability to behave in different ways according to an internal toggle inside it switching states based on the chemical substances that the system is exposed to. The toggle states could generally be used to trigger events such as enzyme synthesis, transcriptional regulation, virion production, or even cell death. Therefore, one may view the bacterial cell line containing this system as a multipurpose cell line. By adding a certain chemical to a cell line, the latter may be trained to exhibit a desired behavior, and then it is not necessary any more to construct two independent cell lines.

This means that one applies an “input engineering” instead of a “DNA engineering” approach. If one extends this idea to several multi-inducible toggle switches being harbored in the same cell line, the number of possible phenotypes increases to 2n, where n equals the number of toggle switches. For example, if one would have 5 such toggle switches inside a cell line, 32 different behavior patterns would be possible.

For the purpose of creating a toggle switch that is activated in a specific phase only and not always (a multi-inducible toggle switch), as is required for stable biological automatons, we introduced the concept of double promoters to the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts], which can be helpful for engineering systems which exhibit a desired behavior only at specific times.

Link to Epigenetics

Epigenetics refers to features like chromatin or DNA modifications that do not involve changes in the underlying DNA sequence and are stable over many cell divisions [1],[2]. If one has a closer look at our proposed system, one can also view it as a model-system for epigenetics: Although the DNA sequence itself stays the same, two different subpopulations of cells with different phenotypes can develop from it. Put simply, depending in which state (subpopulation) the toggle switch is, the cells will produce different fluorescent proteins upon addition of two different inducer molecules. Therefore, the epigenetic feature here is the binding of specific repressor proteins whose production is dependent on the toggle switch state.

Intelligent Biosensors and Self-Adaptation

The system is capable of sensing different chemicals and producing different fluorescent proteins. Since the cells can be trained to produce one of several specific fluorescent protein types when a certain chemical is present, one can also view those cells as intelligent biosensors which recognize chemical substances according to a training phase. The intelligent biosensors are not limited to detect chemicals; temperature, pH, light, pressure could be detected with an appropriate system as well. Such an application could be especially of interest when the environment to be probed is harmful for humans, for example due to high toxicity.

Team Members

The ETH Zurich iGEM2007 Team

The ETH Zurich team consists of a good mixture between biologists and engineering students. We are:

  • Undergraduate students:
    Martin Brutsche, Katerina Dikaiou,
    Raphael Guebeli, Sylke Hoehnel,
    Nan Li, Stefan Luzi
  • Graduate students:
    [http://christos.bergeles.net Christos Bergeles], [http://www.tik.ee.ethz.ch/~sop/people/thohm/ Tim Hohm],
    [http://www.fussenegger.ethz.ch/people/kemmerc Christian Kemmer], Joseph Knight,
    [http://www.ricomoeckel.de Rico Möckel], [http://csb.inf.ethz.ch/people/uhr.html Markus Uhr]
  • Project advisors:
    [http://www.ipe.ethz.ch/laboratories/bpl/people/panke Sven Panke],
    [http://csb.inf.ethz.ch/people/stelling.html Joerg Stelling]

For more information about us, visit our Meet the Team page.

Acknowledgments

The idea for the project as well as its implementation was done solely by the ETH iGEM 2007 team. We would like to thank the people in [http://www.ipe.ethz.ch/laboratories/bpl/index Sven Panke's Lab], especially Andreas Meyer who was always there for us when we had a problem. Additionally, we would like to thank [http://www.facs.ethz.ch Alfredo Franco-Obregón's lab] and Oralea Büchi for the help with the flow cytometry.

We would also like to acknowledge the financial support by [http://europa.eu EU], the [http://www.ethz.ch ETH Zurich], and [http://www.geneart.com GeneArt]:

[http://europa.eu http://www.tik.ee.ethz.ch/~thohm/EU.gif] [http://www.ethz.ch http://www.tik.ee.ethz.ch/~thohm/ethlogo.jpg] [http://www.geneart.com http://www.tik.ee.ethz.ch/~thohm/geneart.gif]

Site Map

In this wiki, we will present to you a detailed description of the proposed system: starting with the modeling of the system, we describe both, simulations and theoretical considerations of the system, as well as the actual implementation using bio-bricks accompanied by our lab notes. Additionally, you will find further information on the team, more details about ideas we developed before we came up with the system we finally implemented, and some pictures documenting our work.

The site map of our wiki is the following:

Modeling Pages Biology Pages ETHZ Team Pages Links
Modeling of the learning system Biological implementation Team page The ETH Zurich 2005 project
Representation using flip-flops Biobricks/parts Pictures The ETH Zurich 2006 project
Representation using finite state machines Lab notes Brainstorming sessions
Model simulations and theoretical considerations
Parameters used in our simulations

References

[http://www.nature.com/nature/journal/v447/n7143/abs/nature05913.html;jsessionid=62903C604764B175945C03DB8639ECBD [1] Bird A] "Perceptions of epigenetics", Nature 447:396-398, 2007
[http://linkinghub.elsevier.com/retrieve/pii/S096098220701007X [2] Ptashne M] "On the use of the word ‘epigenetic’", Current Biology 17(7):R233-R236, 2007


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