Oscillator based

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'''Meetings:'''
 
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'''Friday, 5. August, 09:00 @ Christophe's bureau (CAB F61.2) [[summary_UCJ_08-05|Summary of the meeting]]'''
 
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'''Monday, 8. August, 15:00 @ Christophe's bureau (CAB F61.2)'''
 
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Back to the [[ETH Zurich]] main page.
Back to the [[ETH Zurich]] main page.
=Scope=
=Scope=
-
This group is focussing on Ideas related to Counters and Oscillators.
+
The general focus of our group is on ideas related to counters and oscillators.
 +
 
 +
Currently, our goal is to create a counter for concentration peaks (e.g. of a protein experiencing oscillatory behaviour) that is robust to variations in phase length, peak width and height, while composed of a minimal number of proteins and regulatory pathways.
 +
 
 +
Ideally, this module should be combined with, on one hand, a cell cycle dependent protein to make a generation counter, and, on the other hand, with the implementation of a synthetic oscillator developed by previous initiatives. If both integration fail, a hand-generated inducer oscillation will hopefully work as a backup and provide entertainment to our audience.
=Motto=
=Motto=
  ASCII is an art form!
  ASCII is an art form!
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=People=
+
=Organisation=
 +
==People==
People currently working on these Ideas:
People currently working on these Ideas:
* [[User:realUACM|Urs A. Müller]]
* [[User:realUACM|Urs A. Müller]]
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Please feel free to add your name.
Please feel free to add your name.
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=Modules=
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==Meetings==
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We try to divide all the Ideas related to our scope in some 5 or 6 Modules. Our Goal on the one hand is to define the modules in a way that allows one to design, implement and test the individual modules independently. On the other hand, we also want to ensure that it is possible to connect them to build bigger components with more functionality.
+
'''Friday, 5th August, 09:00 @ Christophe's bureau (CAB F61.2) [[summary_UCJ_08-05|Summary of the meeting]]'''
 +
'''Monday, 8th August, 15:00 @ Christophe's bureau (CAB F61.2)'''
 +
'''Tuesday, 9th August, 09:00 @ Christophe's bureau (CAB F61.2)[[Summary_UCJ_08-09|Summary of the meeting]]'''
 +
'''Wednesday, 10th August, 09:15 @ Christophe's bureau (CAB F61.2)'''
 +
'''Thurdsay, 11th August, 12:00 @ Mensa
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==Module Tree==
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=Design=
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----------------      -----------
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Below, we divide the concept into modules, to allow independence to the greatest extent in the design, implement and test phase of each of them. Note that all three forms of oscillators might not lead to the fluctuation of the same protein, and thus an adaptor or interface will possibly be needed.
-
|              |      |    |    |
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-
|cell division |-----> |    |    |
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|              |      |    |    |
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-
----------------      |    |    |
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                        |----|    |    ---------------------
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----------------      |    |    |    |                  |      ------------
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|              |      |    |    |    |                  |      |          |
+
-
|  oscillator  |-----> |    |    |---->|  divisor/counter  |----->| reporter |
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|              |      |    |    |    |                  |      |          |
+
-
----------------      |    |    |    |                  |      ------------
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-
                        |----|    |    ---------------------
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----------------      |    |    |
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|  triggering  |      |    |    |
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-
|  by hand    |-----> |    |    |
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|              |      |    |    |
+
-
----------------      -----------
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                        Interface
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==cell division==
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+---------------+
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==oscillator==
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|  OSCILLATOR  |
-
Bad news! I raed the paper about oscillatory behavior in E coli ([[atkinson03]]) and if I did get it right then they didn't completely succed to develop a stable oscillator. Their design enabled them to achieve 4 different behaviors of the circuit by changing the promoters, repressors, copy numbers and cooperativity. Due to theire simulations the circuit should have been able to act as a toggle switch, a damped oscillator and a stable oscillator (with different frequencies and peak concentrations). They succeded in establishing reproducibly a damped oscillation. But by shifting the system parameters into the region of stable oscillation the damping didn't disappear. Pushing it further could possibly help but I don't know if it's worth it and if we have enough time for this.
+
|  ============ |
 +
+---------------+
 +
|              |
 +
| cell division |      +-------------------+      +-------------------+
 +
|              |      |                  |      |                  |
 +
+---------------+      |  DIVISOR/COUNTER  |      |                  |
 +
|  oscillator  |----> |  ===============  |----> |  reporter gene  |
 +
| from previous |      |                  |      |                  |
 +
|    work      |      |                  |      |                  |
 +
+---------------+      +-------------------+      +-------------------+
 +
|  triggering  | 
 +
|  by hand    |
 +
|              |   
 +
+---------------+     
 +
                     
 +
 
 +
==OSCILLATOR==
 +
===Cell division===
 +
Uwe Sauer suggests to look into ftsZ as a protein that is expressed only once per cycle. We could try to get their promoter and use it to build a peak as input for the counter.
 +
 
 +
Uwe: FtsZ might not be the best choice. Also pivotal for cell cycle, I think a substantial fraction is present all the time, it simply assembles around the cell middle to initiate division. What you need, is actually a protein whose presence is primarily regulated at the genetic level. Perhaps FtsZ works, but I am not so sure. I would recommend to go through the literature and see whether or no there is another Fts gene (or others) that are only transiently present during division. There are a lot more proteins involved in the process and all you need is one that is transiently expressed. Reviews are a good starting point, but also hve a look at chapter 101 of the E. coli book.
 +
 
 +
Christophe: I just found a disturbing article from 1979 that is entitled "Individual Proteins are Synthesized Continuously throughout the E. Coli Cell Cycle" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=218185], but then again, that was in 1979, a time where the sole lab instruments were handcrafted petri dishes and self-grinded magnifying lenses. Later, I found yet another disturbing article ([http://www.cnb.uam.es/~mvicente/Rueda(185)3344.pdf]) that clearly shows that the concentration of FtsZ and a few others remain constant throughout the cell cycle in E. Coli. There are many instances of oscillating proteins in other organisms, in particular caulobacter crescentus or yeasts, but so far i am out of luck in E. Coli. Yet more discouraging results! Arends and Weiss [http://jb.asm.org/cgi/content/full/186/3/880] claim in their paper that "Our results also imply that no E. coli genes are expressed in a division cycle-dependent manner"...
 +
 
 +
Alexander: Perhaps DnaA could be used as a target for creating a generation counter. DnaA binds to OriC and initiates DNA replication and later recruiting several protins (DnaB, DnaC, DnaG, ect.). The initiation of chromosomal replication in E. coli is dependent on availability of DnaA. If the concentration will fluctutate with the cellcycle then DnaA can be used as a signal to count generation.
 +
Since DnaA binds to DNA it would can be used as a operator. The idea would be to use a DnaA binding sequence as an operator in a reporter gene.
 +
 
 +
===== Recommended Readings =====
 +
* Review on Bacterial Cell Division  [http://arjournals.annualreviews.org/doi/pdf/10.1146/annurev.genet.33.1.423;jsessionid=jG4OUnl1knY7]
 +
* Review on Microanalysis of gene expression during cell cycle [http://www.cellandchromosome.com/content/2/1/1]
 +
* Report on Oscillating regulators, but only in C. Crecentus... [http://www.sciencemag.org/cgi/content/short/1095191v1]
 +
* Review on Oscillating regulators with relevant modelling info, but only about S. Pombe [http://www.nature.com/nrm/journal/v2/n12/abs/nrm1201-908a_fs.html]
 +
 
 +
===Oscillator===
 +
====Attenuated Oscillator from Cell Article====
 +
Bad news! I read the paper about oscillatory behavior in E coli ([[atkinson03]]) and if I did get it right then they didn't completely succed to develop a stable oscillator. Their design enabled them to achieve 4 different behaviors of the circuit by changing the promoters, repressors, copy numbers and cooperativity. Due to theire simulations the circuit should have been able to act as a toggle switch, a damped oscillator and a stable oscillator (with different frequencies and peak concentrations). They succeded in establishing reproducibly a damped oscillation. But by shifting the system parameters into the region of stable oscillation the damping didn't disappear. Pushing it further could possibly help but I don't know if it's worth it and if we have enough time for this.
Furthermore they didn't include all the genes into peptides but a part also in the DNA of the E coli itself. I don't know if this would be a problem but I wanted to mention it anyway.
Furthermore they didn't include all the genes into peptides but a part also in the DNA of the E coli itself. I don't know if this would be a problem but I wanted to mention it anyway.
-
==triggering by hand==
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====Repressilator====
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==interface==
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The repressilator is working but as we know it has its drawbacks. On one hand it isn't oscillation isn't really very regullary and on the other hand it's amplitude is increasing with time. With this oscillator you could perhaps count to 2 or 3. Because if the minimum amplitude is to high thecounter wouldn't recognice the difference anymore. But instead of using the simple hand triggering this could be a alternative to test the counter.
-
==divisor / counter ==
+
====Another Project from the Registry of Standard Parts====
 +
 
 +
===Triggering by Hand===
 +
Let's brainstorm about stuff that could be used to generate an oscillation by hand.
 +
* IPTG (deactivates the LacI repressor)? What about degradation time?
 +
* Heat Shock Protein? Think about it, the second time we cook the bacteria, it reacts differently :-)
 +
* AHL quorum sensing? But then, can we order AHL? at which price?
 +
* ...
 +
 
 +
Uwe: I like this back-up idea very much. likely you will have to use it..... Chemical variations are always tricky because the compounds tend to linger around and you'll typicaly end up with a single on-off cycle. IPTG and AHL (but also pH etc) fall into this category unless you add degrading enzymes (..which then effect the next cycle in some way). Physical parameters offer the benefit that they can easily be modified from the outside without a 'memory' effect. I think Temp. is a great choice!
 +
 
 +
Don't worry about cooking. Heat shock is elecited by a relative raise in T, e. g. going from 30 to 37 (or perhaps 40°C). The cells will be fine and the heatshock protein-based reporter is under your external control. You can even set an automated T cycle in a PCR (or similar) machine, which will help you a lot by finging the right set of parameters for optimal performance. Also it looks cooler than working with your hands on vials..
 +
 
 +
Christophe: I looked into sigma 32 heat-shock promoters. That could work, but we still need to find some people who have experience with that, as it is likely that there will be a lot of small details required to have a proper response. Further promoters to evaluate/test: ibp, cspA,
 +
 
 +
====Recommended Readings====
 +
* Introduction to Heat-Shock Response in Bacteria [http://www.tau.ac.il/lifesci/departments/biotech/members/segalg/GS-R2.pdf]
 +
* Consensus sequence for E. Coli Heat Shock gene promoters [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=3887408]
 +
 
 +
==DIVISOR/COUNTER==
This module will get an input concentration from the interface module and will count, due to the changing input, to 4. Also a reporter (for example different colors) will be produced which could serve as an indicater for the state the circuit is currently occupiing.
This module will get an input concentration from the interface module and will count, due to the changing input, to 4. Also a reporter (for example different colors) will be produced which could serve as an indicater for the state the circuit is currently occupiing.
-
==='4 genes' counter===
+
 
-
====Idea====
+
===Idea and Implementation===
-
During our discussion about the divisor implementation we noticed that the previous one wasn't quite stable. Talking about toggle switches we suddenly had the idea of combining the base structur of the toggle switch we learned to know during the crash course with the allready existing design. The follwing drawings show this new design which is basically a combination of two such toggle switches.  
+
During our discussion about the divisor implementation we noticed that the previous one wasn't quite stable. Talking about toggle switches we suddenly had the idea of combining the base structur of the toggle switch we learned to know during the crash course with the allready existing design. The following drawings show this new design which is basically a combination of two such toggle switches.
 +
 
  All substances which are involved should be produced and decomposed quickly
  All substances which are involved should be produced and decomposed quickly
  S = concentration signal from the interface
  S = concentration signal from the interface
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       ----------------        ----------------
       ----------------        ----------------
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===Toggle switch===
 
-
====Idea====
 
-
During our discussion, it became apparent that another possibiliy to build a counter / divisor would be to use a toggle switch.
 
-
It seems that the term "toggle switch" is not well-defined, i.e. it can stand for a few different functionalities of electronic circuits.
 
-
When I use the term "toggle switch" in this section, it shall stand for a genetic device with one input and one output, where both of them have two possible states (''high'' and ''low''). When the input is ''low'', the output doesn't change (it keeps its current state). When there is a peak in the input signal (i.e. it jumps to the high state but doesn't stays there to long, where the length of "to long" depends on the parameters of the circuit), the output changes to the state opposite to its current state (from ''low'' to ''high'' or from ''high'' to ''low''). It "toggles".
+
===Simulation===
 +
We did some early matlab simulations.
 +
A first plot (although kind of overly optimistic regarding the parameter setting options) are showed here: [[Image:CounterSimulationPlot1.png|none]]
 +
The bottom plot is the input signal S.
 +
The four plot above are the concentration of the R[1,2,3,4].
 +
As one can see, the cascade is clear and stable!
-
====Concept for Implementation====
 
-
A possible implementation of this concept could look as following:
 
-
The first device needed is just a ''bistable switch'', as described in the lecture by Jörg (if someones remembers the paper where this came from, ''please add a reference to that paper here'')
+
===Challenges===
-
                 
+
*The combination of promoters, repressing and activating substances will be quite a challenge. They should change the state rather quick. Therefore most of the substances have to be expressed and decomposed quickly. But on the other hand the repression at certain points should stay long enough to ensure that there is only one state the system can fall in.
-
                                           
+
*I think the repression by the input signal S will affect the expression of R2 and R0 quickly. But will the decreasing concentration of S have the same affect on R1 and R3 in comparable time? Or is a second input needed with the reverse effect of S?
-
      R1                                  R2   
+
-
      \                                  /     
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-
        \/|____                    ____|\/     
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    ___/  (1) |_______    _______| (2)  \___
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        \  ____|  = =       = = |____  /   
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        \|      |  |        | |      |/       
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                  |  |        | |                 
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                R2  R3      R4 R1                 
+
-
                                             
+
-
The ''bistable switch'' produces either R1 or R2. When R1 is present, it represses the production of R2. If R2 is present, it represses the production of R1. Thus, this device has two stable states. I will refer to the state where R1 is produced as ''State 1'' and to the state where R2 is produced as ''State 2''. The repressors R3 and R4 should be ignored in this stage, they get their relevance when the second component of the toggle switch is introduced below.
+
-
Now, we need another device that allows us to switch the bistable switch from one state to the other. I call it ''switcher''.
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==REPORTER==
-
This could be achieved in the following way:
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This will probably be one or more colors to indicate the current state of the counter.
-
    S  R1        R3        S  R2        R4
+
=Discussions=
-
    | |        /          |  |        /
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We invite anyone to make comments and participate in discussions [[Talk:Oscillator_based|here]]
-
    |  |  ___|\/            |  |  ___|\/
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-
    _|__|__| (3) \___        _|__|__| (4) \___
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          |___  /                  |___  /
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-
              |/                      |/
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The idea of the ''switcher'' is to repress the production of the repressor (R1 or R2)  that is currently produced by the ''bistable switch''. It does so only when the input signal S is present. The concept of the dynamics of a toggling is as folloeing:
+
==Assessment==
-
Given the ''bistable switch'' is currently in ''state 1'' with gene (1) activated and gene (2) repressed. When the concentration
+
* Modularity
-
of S rises, S in combination with R1 leads to the production of R3, which itself cuts of the production of R1, allowing
+
** The project is divided in modules that can be implemented and tested independently.
-
  R2 to be produced. The ''bistable switch'' has been toggled.  
+
** The counter module itself is also very symmetrical.
 +
** Very powerful combinatorial behaviour: combining two counter units could track 16 states, and so on!
 +
** Reuse: the project is based on parts that have already been developed, such as the oscillator, or toggle switches in the counter design.
 +
* Usefulness
 +
** Counters and divisors have all kinds of interesting applications.
 +
** Similar technology could be used to implement more complex finite state machines.
 +
* Feasibility
 +
** We have 4 genes and 12 regulatory pathways in the counter part. That is obviously not trivial.
 +
** But the design is highly symmetrical, which mean that some parts could be reused, and relatively few parameters need to be tuned.
 +
** Besides, the ''triggering by hand'' part is quite feasible and could allow us some last minute face-saving type of maneuver. We could limit our contribution to that, and rename the project,  ''A H. Sapiens Powered Oscillator for Bacteria''.
 +
* Coolness
 +
** All in all, i think everybody would agree that the project is in fact extremely nerdy. However, in a technical school, nerdy is cool. Therefore, the project is extremely cool.
-
====Challenges====
+
In all fairness, this project also have some weaknesses:
-
To make this device work, one has to adjust the time constants. There are some challenges to face:
+
* The philosophical implications are unclear.
-
* Assume the ''bistable switch'' has just been toggled from state 1 to state 2. When S is still present, R2 produced in state 2 can lead to a ''retoggling'' to state 1. This behavior is clearly undesired. To avoid this, S should be a substance that is degraded quickly.
+
* A stochastic counter with fuzzy logic unit could possibly be cooler.
-
* Even when S has sharp peaks, there is still a problem during transition. Given the ''toggle switch'' is in state 1. S rises, R3 is produced, and the production of R1 in reality is rather first lowered than directly cut off. This leads to a lower production of R3, thus slowing down the toggle process. To avoid this, R3 should be stable on a longer timescale than the timescale of the processes in the ''toggle switch''. If this is ensured, a short peak of S is sufficient to produce some persistent R3, that is able to stop the production of R1. This allows one to make the peak short enough to guarantee that the above described "double toggling" does not happen.  
+
* The counter cannot toast bread.
-
====Using the toggle switch as a counter====
+
==Intersection with [[Quorum_Sensing_based]] projects==
-
If you have such a device working, it is possible to convert an input signal with frequency to a signal with frequency 2f. This is a divisor. If you combine n of these divisors, you are able to count to 2^n. That's a counter!
+
* Triggering by Hand could be implemented using a quorum sensing inducer.
 +
* Reporting would need the same type of visual stuff. We have not thought much about it, have you?
 +
* Maybe there would be a bit of overlap with the ''cell division sensing'' mentioned on your page. Could you perhaps elaborate?
-
====Evaluation of the idea====
+
=Attic=
-
I think that this concept can be implemented. The problems described above should be possible to deal with. I expect the toggle switch to be quite stable and noise resistant, and yet sufficiently simple regarding the different interactions. The catenation of the toggle switches to a counter may prove to be not so easy.
+
Have a look at [[Oscillator_based_Attic]] for old toggle stuff.
-
A toggle switch is a device, that can be used in a wide area of applications, given the importance of the concept in electrical engineering and computer science.
+
-
A test-tube that changes its color from red to green when some substance is added, is not so spectacular, since we all saw this in our high school chemical class. A tube changes back fron green to red when another chemical is added, is slightly better. But what a cool demo, when the tube switches it's color again and again, every time ONE SINGLE SUBSTANCE is added! (for comments and opinions to the coolness of such a demo, please use the [[talk:Oscillator_based|talk page]]...))
+
----
-
==reporter==
+
Back to the [[ETH Zurich]] main page.
-
This will probably be one or more colors to indicate the current state of the counter.
+
-
=Discussions=
+
<div class=exf align='center' style='overflow:auto; height: 1px;'>
-
We invite anyone to make comments and participate in discussions [[Talk:Oscillator_based|here]]
+
[http://lucky7.to/timex/ timex watch co]
-
 
+
</div>
-
 
+
-
Back to the [[ETH Zurich]] main page.
+

Latest revision as of 01:42, 11 December 2005

Back to the ETH Zurich main page.

Contents

Scope

The general focus of our group is on ideas related to counters and oscillators.

Currently, our goal is to create a counter for concentration peaks (e.g. of a protein experiencing oscillatory behaviour) that is robust to variations in phase length, peak width and height, while composed of a minimal number of proteins and regulatory pathways.

Ideally, this module should be combined with, on one hand, a cell cycle dependent protein to make a generation counter, and, on the other hand, with the implementation of a synthetic oscillator developed by previous initiatives. If both integration fail, a hand-generated inducer oscillation will hopefully work as a backup and provide entertainment to our audience.

Motto

ASCII is an art form!

Organisation

People

People currently working on these Ideas:

Please feel free to add your name.

Meetings

Friday, 5th August, 09:00 @ Christophe's bureau (CAB F61.2) Summary of the meeting
Monday, 8th August, 15:00 @ Christophe's bureau (CAB F61.2)
Tuesday, 9th August, 09:00 @ Christophe's bureau (CAB F61.2)Summary of the meeting
Wednesday, 10th August, 09:15 @ Christophe's bureau (CAB F61.2)
Thurdsay, 11th August, 12:00 @ Mensa

Design

Below, we divide the concept into modules, to allow independence to the greatest extent in the design, implement and test phase of each of them. Note that all three forms of oscillators might not lead to the fluctuation of the same protein, and thus an adaptor or interface will possibly be needed. 

+---------------+
|   OSCILLATOR  |
|  ============ |
+---------------+ 
|               |
| cell division |      +-------------------+      +-------------------+
|               |      |                   |      |                   |
+---------------+      |  DIVISOR/COUNTER  |      |                   |
|  oscillator   |----> |  ===============  |----> |   reporter gene   |
| from previous |      |                   |      |                   |
|     work      |      |                   |      |                   |
+---------------+      +-------------------+      +-------------------+
|  triggering   |  
|   by hand     |
|               |    
+---------------+

OSCILLATOR

Cell division

Uwe Sauer suggests to look into ftsZ as a protein that is expressed only once per cycle. We could try to get their promoter and use it to build a peak as input for the counter.

Uwe: FtsZ might not be the best choice. Also pivotal for cell cycle, I think a substantial fraction is present all the time, it simply assembles around the cell middle to initiate division. What you need, is actually a protein whose presence is primarily regulated at the genetic level. Perhaps FtsZ works, but I am not so sure. I would recommend to go through the literature and see whether or no there is another Fts gene (or others) that are only transiently present during division. There are a lot more proteins involved in the process and all you need is one that is transiently expressed. Reviews are a good starting point, but also hve a look at chapter 101 of the E. coli book.

Christophe: I just found a disturbing article from 1979 that is entitled "Individual Proteins are Synthesized Continuously throughout the E. Coli Cell Cycle" [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=218185], but then again, that was in 1979, a time where the sole lab instruments were handcrafted petri dishes and self-grinded magnifying lenses. Later, I found yet another disturbing article ([http://www.cnb.uam.es/~mvicente/Rueda(185)3344.pdf]) that clearly shows that the concentration of FtsZ and a few others remain constant throughout the cell cycle in E. Coli. There are many instances of oscillating proteins in other organisms, in particular caulobacter crescentus or yeasts, but so far i am out of luck in E. Coli. Yet more discouraging results! Arends and Weiss [http://jb.asm.org/cgi/content/full/186/3/880] claim in their paper that "Our results also imply that no E. coli genes are expressed in a division cycle-dependent manner"...

Alexander: Perhaps DnaA could be used as a target for creating a generation counter. DnaA binds to OriC and initiates DNA replication and later recruiting several protins (DnaB, DnaC, DnaG, ect.). The initiation of chromosomal replication in E. coli is dependent on availability of DnaA. If the concentration will fluctutate with the cellcycle then DnaA can be used as a signal to count generation. Since DnaA binds to DNA it would can be used as a operator. The idea would be to use a DnaA binding sequence as an operator in a reporter gene.

Recommended Readings
  • Review on Bacterial Cell Division [http://arjournals.annualreviews.org/doi/pdf/10.1146/annurev.genet.33.1.423;jsessionid=jG4OUnl1knY7]
  • Review on Microanalysis of gene expression during cell cycle [http://www.cellandchromosome.com/content/2/1/1]
  • Report on Oscillating regulators, but only in C. Crecentus... [http://www.sciencemag.org/cgi/content/short/1095191v1]
  • Review on Oscillating regulators with relevant modelling info, but only about S. Pombe [http://www.nature.com/nrm/journal/v2/n12/abs/nrm1201-908a_fs.html]

Oscillator

Attenuated Oscillator from Cell Article

Bad news! I read the paper about oscillatory behavior in E coli (atkinson03) and if I did get it right then they didn't completely succed to develop a stable oscillator. Their design enabled them to achieve 4 different behaviors of the circuit by changing the promoters, repressors, copy numbers and cooperativity. Due to theire simulations the circuit should have been able to act as a toggle switch, a damped oscillator and a stable oscillator (with different frequencies and peak concentrations). They succeded in establishing reproducibly a damped oscillation. But by shifting the system parameters into the region of stable oscillation the damping didn't disappear. Pushing it further could possibly help but I don't know if it's worth it and if we have enough time for this.

Furthermore they didn't include all the genes into peptides but a part also in the DNA of the E coli itself. I don't know if this would be a problem but I wanted to mention it anyway.

Repressilator

The repressilator is working but as we know it has its drawbacks. On one hand it isn't oscillation isn't really very regullary and on the other hand it's amplitude is increasing with time. With this oscillator you could perhaps count to 2 or 3. Because if the minimum amplitude is to high thecounter wouldn't recognice the difference anymore. But instead of using the simple hand triggering this could be a alternative to test the counter.

Another Project from the Registry of Standard Parts

Triggering by Hand

Let's brainstorm about stuff that could be used to generate an oscillation by hand.

  • IPTG (deactivates the LacI repressor)? What about degradation time?
  • Heat Shock Protein? Think about it, the second time we cook the bacteria, it reacts differently :-)
  • AHL quorum sensing? But then, can we order AHL? at which price?
  • ...

Uwe: I like this back-up idea very much. likely you will have to use it..... Chemical variations are always tricky because the compounds tend to linger around and you'll typicaly end up with a single on-off cycle. IPTG and AHL (but also pH etc) fall into this category unless you add degrading enzymes (..which then effect the next cycle in some way). Physical parameters offer the benefit that they can easily be modified from the outside without a 'memory' effect. I think Temp. is a great choice!

Don't worry about cooking. Heat shock is elecited by a relative raise in T, e. g. going from 30 to 37 (or perhaps 40°C). The cells will be fine and the heatshock protein-based reporter is under your external control. You can even set an automated T cycle in a PCR (or similar) machine, which will help you a lot by finging the right set of parameters for optimal performance. Also it looks cooler than working with your hands on vials..

Christophe: I looked into sigma 32 heat-shock promoters. That could work, but we still need to find some people who have experience with that, as it is likely that there will be a lot of small details required to have a proper response. Further promoters to evaluate/test: ibp, cspA,

Recommended Readings

  • Introduction to Heat-Shock Response in Bacteria [http://www.tau.ac.il/lifesci/departments/biotech/members/segalg/GS-R2.pdf]
  • Consensus sequence for E. Coli Heat Shock gene promoters [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=3887408]

DIVISOR/COUNTER

This module will get an input concentration from the interface module and will count, due to the changing input, to 4. Also a reporter (for example different colors) will be produced which could serve as an indicater for the state the circuit is currently occupiing.

Idea and Implementation

During our discussion about the divisor implementation we noticed that the previous one wasn't quite stable. Talking about toggle switches we suddenly had the idea of combining the base structur of the toggle switch we learned to know during the crash course with the allready existing design. The following drawings show this new design which is basically a combination of two such toggle switches. 

All substances which are involved should be produced and decomposed quickly
S = concentration signal from the interface
R1 is produced by gene (1) and represses gene (0) and (3)
R2 is produced by gene (2) and represses gene (0) and (1)
R3 is produced by gene (3) and represses gene (1) and (2)
R0 is produced by gene (0) and represses gene (2) and (3)

                 
                     ---S---                         
     R1             /       \              R3              R3 R0         R1 R2
      \             |       |             /                 | |           | |
       \/|____      |       |      ____|\/          /|___   | |           | |  ____|\ 
    ___/  (1) |_____v__   __v_____| (3)  \___   ___/  (2) |_=_=____   ____=_=_|(0=4) \___
       \  ____| = =           = = |____  /         \  ____|     =       =     |____  /
        \|      | |           | |      |/          /\|          |       |          |/\
                | |           | |                 /             |       |             \
               R2 R3         R0 R1               R2             \       /              R0
                                                                 ---S---

The following time lines should be the result of this gene circuit:
S:
    -----       -----       -----       -----          Let's assume that the counter is in state 0 
   |     |     |     |     |     |     |     |         (expressing R0). As soon as the concentration
   |     |     |     |     |     |     |     |         of S goes up (0) and (2) are repressed
---       -----       -----       -----       ----     (no further expressing of R0). Now R1 can be
R1:                                                    expressed while (3) is still repressed 
     ----                    ----                      by R0. The production of R1 ensures that (3) 
    /    \                  /    \                     won'tbe active when R0 is totally decomposed.
   /      \                /      \                    -> We are in state (1).
---        ----------------        ---------------     When the S goes down again R1 can't be
R2:                                                    expressed anymore. Now R2 can be 
           ----                    ----                expressed while (0) is still repressed by R1. 
          /    \                  /    \               The production of R2 ensures that (0) won't 
         /      \                /      \              be active when R1 is totally decomposed.
---------        ----------------        ---------     -> We are in state (2)
R3:                                                    And so on...
                 ----                    ----
                /    \                  /    \
               /      \                /      \
---------------        ----------------        ---
R0:
---                    ----                    ---
   \                  /    \                  /
    \                /      \                /
     ----------------        ----------------


Simulation

We did some early matlab simulations.

A first plot (although kind of overly optimistic regarding the parameter setting options) are showed here:
CounterSimulationPlot1.png

The bottom plot is the input signal S. The four plot above are the concentration of the R[1,2,3,4]. As one can see, the cascade is clear and stable!


Challenges

  • The combination of promoters, repressing and activating substances will be quite a challenge. They should change the state rather quick. Therefore most of the substances have to be expressed and decomposed quickly. But on the other hand the repression at certain points should stay long enough to ensure that there is only one state the system can fall in.
  • I think the repression by the input signal S will affect the expression of R2 and R0 quickly. But will the decreasing concentration of S have the same affect on R1 and R3 in comparable time? Or is a second input needed with the reverse effect of S?

REPORTER

This will probably be one or more colors to indicate the current state of the counter.

Discussions

We invite anyone to make comments and participate in discussions here

Assessment

  • Modularity
    • The project is divided in modules that can be implemented and tested independently.
    • The counter module itself is also very symmetrical.
    • Very powerful combinatorial behaviour: combining two counter units could track 16 states, and so on!
    • Reuse: the project is based on parts that have already been developed, such as the oscillator, or toggle switches in the counter design.
  • Usefulness
    • Counters and divisors have all kinds of interesting applications.
    • Similar technology could be used to implement more complex finite state machines.
  • Feasibility
    • We have 4 genes and 12 regulatory pathways in the counter part. That is obviously not trivial.
    • But the design is highly symmetrical, which mean that some parts could be reused, and relatively few parameters need to be tuned.
    • Besides, the triggering by hand part is quite feasible and could allow us some last minute face-saving type of maneuver. We could limit our contribution to that, and rename the project, A H. Sapiens Powered Oscillator for Bacteria.
  • Coolness
    • All in all, i think everybody would agree that the project is in fact extremely nerdy. However, in a technical school, nerdy is cool. Therefore, the project is extremely cool.

In all fairness, this project also have some weaknesses:

  • The philosophical implications are unclear.
  • A stochastic counter with fuzzy logic unit could possibly be cooler.
  • The counter cannot toast bread.

Intersection with Quorum_Sensing_based projects

  • Triggering by Hand could be implemented using a quorum sensing inducer.
  • Reporting would need the same type of visual stuff. We have not thought much about it, have you?
  • Maybe there would be a bit of overlap with the cell division sensing mentioned on your page. Could you perhaps elaborate?

Attic

Have a look at Oscillator_based_Attic for old toggle stuff.

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