From 2007.igem.org

Contents 1 Team Members 2 Introduction to Synthetic Biology 3 Choosing the Project 3.1 Step 1: Brainstorming 3.2 Step 2: Preferred Projects 4 The Project 4.1 System Models 4.2 Simulations 4.3 Sensitivity Analysis 5 Lab Work 5.1 Cloning Plan 5.1.1 Plasmid 1 (pbr322ap) 5.1.2 Plasmid 2 (pck01cm) 5.1.3 Plasmid 3 (pacyc177km) 5.1.4 Linkers 5.2 Lab Instructions and Methods 5.3 Lab Schedule 5.4 Lab Notes / Log Book 6 Task List

Team Members

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.

Instructors:

Sven Panke

Joerg Stelling

Students:

Martin Brutsche

Katerina Dikaiou

Raphael Guebeli

Sylke Hoehnel

[http://leemnan.spaces.live.com Nan Li]

Stefan Luzi

Graduate Students:

[http://christos.bergeles.net Christos Bergeles]

[http://www.tik.ee.ethz.ch/~sop/people/thohm/ Tim Hohm]

[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]

Joseph Knight

Markus Uhr

[http://www.ricomoeckel.de Rico Möckel]

Introduction to Synthetic Biology

In order to get an initial understanding of the concepts of synthetic biology, we read and presented publications on various topics. A representative list of the topics that we covered on this “boot-camp” is listed in the following:

• Introduction to synthetic biology
• DNA de novo design
• DNA circuits
• Hysteresis
• Oscillators
• Zinc fingers
• Noise in single cell measurements
• Distance communication
• Parameter manipulations
• Protein logic
• Orthogonal systems
• mRNA engineering
• RNA regulators

Choosing the Project

Step 1: Brainstorming

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:

1. Defer judgment - the rules of nature don't apply
2. Encourage wild ideas
3. Build on the ideas of others
4. Be visual
5. Go for quantity
6. Stay focused on topic

The ideas that we came up with, as were presented in the following meetings, were:

• 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?
• 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.
• Analog-to-Digital Converter: Compare the level of protein concentration with thresholds, and digitize the output.
• 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”)
• 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.
• 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.
• 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.
• Biocam: Visible to Fluorescent light converter.
• 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).
• 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.
• Sensors: Various systems that can sense PH, pressure, temperature, meat quality, moisture e.t.c. have been proposed.

Step 2: Preferred Projects

We decided that three of the above ideas were worth a deeper examination. Namely, we split again in three teams, so that we can come up with an initial system, with remarks on its feasibility and coolness. Our results were presented on all the team members, so that we could then limit the potential projects down to two and one, and proceed with constructing the DNA sequence. The three projects and the presentations that we did were:

1. 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.
2. 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.

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 ;)

The Project

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. We aim to implement our system using a toggle switch consisting of different repressor and activator proteins coupled with double regulatory regions.

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):

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.

(picture of initial draft of the bio system, as taken from Joe, --here--)

System Models

Simulations

detailed information on the simulations

Sensitivity Analysis

Lab Work

Here we can write when each one will be available. For example:

• Christos:
  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.
• Tim:
  13.08.-15.08. after 17:30
  away from 17.08. until 02.09.
• Martin:
  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.
• Katerina:
  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.
• Christian:
  Not available 5.10-24.10.2007 --> holidays, I can do most of the labwork during the day in my lab

Cloning Plan

The system consists of 11 parts. These are:

System parts

1	TetR production(constitutive part of system)
2	LacI production (constitutive part of system)
3	LuxR production(constitutive part of system)
4	1st half of p22/YFP production (outer part of system, reporting)
5	2nd half of p22/YFP production (outer part of system, reporting)
6	CI production (inner part of system)
7	p22 production (inner part of system)
8	1st half of CI/CFP production (outer part of system, reporting)
9	2nd half of CI/CFP production (outer part of system, reporting)
10	RFP production (reporting)
11	GFP production (reporting)

3 plasmids will be used for the 11 system parts as follows:

Plasmids and contents

plasmid	resistance	copy type	contents	comments
pbr322	ampicillin	insert here	1,2,3	constitutive part
pck01	chloramphenicol	insert here	4,5,8,9	outer part
pacyc177	kanamycin	insert here	6,7,10,11	inner part, reporting

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.

Plasmid 1 (pbr322ap)

1. Put parts 1,2,3 in pbr322ap plasmids.
2. Merge plasmid containing part 2 (donor) with plasmid containing part 3 (acceptor). You should get a plasmid containing a 2.3 composite part.
3. 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.

Plasmid 2 (pck01cm)

1. Put parts 4,5,8,9 in pck01cm plasmids.
2. Merge plasmid containing part 4 (donor) with plasmid containing part 5 (acceptor). You should get a plasmid containing a 4.5 composite part.
3. 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.
4. 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.

Plasmid 3 (pacyc177km)

1. Put parts 6,7,10,11 in pacyc177km plasmids.
2. Merge plasmid containing part 6 (donor) with plasmid containing part 7 (acceptor). You should get a plasmid containing a 6.7 composite part.
3. 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.
4. 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.

Linkers

1. pck01-1
2. pck01-2
3. pacyc177-1
4. pacyc177-2
5. pbr322-1
6. pbr322-2
7. pbr322-3
8. pbr322-4

We have got 4 linkers for pbr322, because there are two for the tetracycline version of pbr333 and two for the ampicillin version.

Lab Instructions and Methods

Lab Schedule

Week 1       06.08.07 - 12.08.07
lab schedule week 1
Week 2       13.08.07 - 19.08.07
lab schedule week 2
Week 3       20.08.07 - 26.08.07
lab schedule week 3
Week 4       27.08.07 - 02.09.07
lab schedule week 4
Week 5       03.08.07 - 09.09.07
lab schedule week 5

Lab Notes / Log Book

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.

detailed lab notes

Task List

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.

1. Team descriptions (overdue) and team photograph
   Christian: I guess I am the only semiprofessional photographer of the group. I can do some group pictures etc. but for this we need some ideas... - I would like to do something special. I also made the group-pics of the Synth. Biology 3.0 conference. Some references  ;-)  : [http://www.fotocommunity.de/pc/pc/mypics/461397]
   Raphael: What about a short movie of us?
2. Team rosters due (1.9.)
   Martin: Does somebody know, what exactly should be done here?
   Nan: A list of team members, including some basic personal info.? (e.g. nationality, background, pet peeves...? )
3. Labwork (parts have to be at the registry in Boston on 26.10.)
   Joe: I can be in the lab at least 2 evenings a week and some times through the weekdays.
   Martin: From Monday I can work every day for the whole day. At the moment I only work for several hours...
   Rico: I have my exam on Tuesday. Afterwards I can assist.
   Christian: I can do the introduction of the polylinker into the vectors beside my normal labwork on the Hoenggerberg. I could also do the whole biobrick assembly if you want this (I will go on holiday from the 5.10-24.10.07)
   Raphael: That's the part where I will mainly contribute, from 14.09. on I can work several days/week
   Christos: I can assist at the afternoons, if needed.
4. Simulation and sensitivity analysis
   Martin: From 10. Sept. I've got plenty time to work on it. I think Markus would join here too ;-)
   Tim: I can contribute in running stuff and help identify parameters from literature
   Rico: I can do simulations, sensitivity analysis.
   Nan: I would like to do simulations and sensitivity analysis.
   Christos: Yeap, I guess I can be here too. I will check some toolboxes to automate things, this weekend.
5. Presentation
   Joe: I'm American... I can sale anything.
   Martin: I'm bad in Layout stuff, but maybe I can help as an idea supplier or so.
   Rico: I like giving presentations. For preparation we will need a mixture of different background and excellent pictures!!!
   Christian: I can provide the molecular biology knowledge and part.
   Nan: I can work on the slides.
   Christos: I like this part, I guess everyone will contribute anyway...
6. Poster
   Joe: See 5. above
   Rico: I can help.
   Christian: I can provide the molecular biology knowledge and part.
   Nan: Partly art. I will help.
   Christos: You can sell, but can you trick? lol :)
7. Wiki (Project and part documentation due on 26.10)
   Martin: See the points above, from next Monday I will give everything, now I'm doing my best...
   Rico: I can help. I guess this will have to contain the materials that we will also use for poster and presenttion anyway.
   Nan: Shouldn't it be updated with every going on process?
   Christos: I am trying to put stuff in, as it comes along. I will update the bio pages with the presentation material, this weekend (I hope).