"
Team:KU Leuven/Project/StickerSystem
From 2013.igem.org
Secret garden
Congratulations! You've found our secret garden! Follow the instructions below and win a great prize at the World jamboree!
- A video shows that two of our team members are having great fun at our favourite company. Do you know the name of the second member that appears in the video?
- For one of our models we had to do very extensive computations. To prevent our own computers from overheating and to keep the temperature in our iGEM room at a normal level, we used a supercomputer. Which centre maintains this supercomputer? (Dutch abbreviation)
- We organised a symposium with a debate, some seminars and 2 iGEM project presentations. An iGEM team came all the way from the Netherlands to present their project. What is the name of their city?
Now put all of these in this URL:https://2013.igem.org/Team:KU_Leuven/(firstname)(abbreviation)(city), (loose the brackets and put everything in lowercase) and follow the very last instruction to get your special jamboree prize!
The Oscillator
In this part we describe the design of an oscillator that could be useful in biological networks. We designed one ourselves since we have very specific demands and look forward to the challenge. We even tried to create a system that creates synchronized oscillations without depending heavily on the components used. So our proposal oscillates inherently, and only slightly depends on the parameters of the components used. In this text, we start with an explanation of how this oscillating model fits within the framework of our project. Second, we explain several necessities to obtain a synchronized oscillator, and how we managed to incorporate those within our network. For the thorough study of the network and to see what has been achieved in the lab, we refer to the modeling page and the wetlab page respectively.
Modeling
On this page we will talk about some more modeling stuff.
Wetlab
The C1 FFL coming to life.
Determining our requirements.
Why would we use an oscillator?
An autonomous production system for β-farnesene might be a good solution in order to avoid having to put our systems directly on plants. However, a constitutive production of the pheromone, might rapidly render the aphids insensitive to it (Kunert, Reinhold and Gershenzon, 2010). Consequently we need a solution in which the production of β-farnesene is alternatingly on and off. In order to elaborate on the possibility of such a periodical production we investigated biological oscillating networks. A transcriptional network that exhibits oscillating behavior is the repressilator of Elowitz and Leibler (2000). This has been a cornerstone for synthetic biology since they were among the first to successfully introduce a synthetic model in a living organism. However, their paper mentions the lack of colony-wide synchronization. This is a necessity to achieve a periodical production, otherwise the variation will even out, resulting in a de facto constitutive expression. This means the repressilator does not suffice for a bacterial production unit with a periodical output.
Figure xǀ Text
Figure xǀ An ant 'milks' an aphid for his honeydew.
A synchronized oscillator
The iGEM team from Wageningen tried to attain colony wide oscillations in 2011, by using the model proposed by Danino et al. (2010). This model provides a next step in the engineering of genetic circuits and is thoroughly described by the Wageningen 2011 iGEM team. As they mention on their wiki, this model heavily depends on the parameter values. Because we want to use an oscillator as a pace regulator, the eventual system will have multiple other inserted genes. The introduction of another set of genes besides an oscillator means an extra load on the current genetic circuit and this can influence the parameters of the oscillator-network (Shiue and Prather, 2012). To have a synchronized oscillator module that functions ‘independently’ on the presence of other modules, we need a system that gives oscillations for a broad range of parameters. This way it can preserve its oscillating behavior independently of possible other loads on the cell’s metabolism.
Why we designed our own oscillator.
The most important feature of our model is the sustained colony wide oscillations for a broad range of parameters. Meanwhile, for both the amplitude and frequency we do not pose stringent requirements. This is because the production of β-farnesene should definitely not be constitutive, but the height of the peaks in production and the time in between different peaks can vary as long as they remain within reasonable ranges. Since we know very well what we exactly desire from the oscillator we decided to design one ourselves. Another, equally important, reason for this decision is the fact that this design would offer a great journey on its own and give us the opportunity to learn a tremendous amount of new things, which is one of the things iGEM is about.
Figure xǀ Text
Figure xǀ Tekst.
The components.
We will design our model starting from an engineering perspective, inspired by the introduction to systems biology from Alon (2006). As the previously discussed models from the literature, we will also use a transcription factor network that creates oscillations. However, we also try to stretch the features of our model further by having the system not depend too much on what exactly are the used components. With this we mean that the several promoters and transcription factors we use, should be replaceable, without changing the fact that oscillations are produced. This is a very nice feature and is possible because our system will oscillate for a broad range of parameters. Previously we discussed the fact that a broad range of oscillating parameter is a necessity in order to preserve the oscillating behavior despite of other loads on the cell’s metabolism. However allowing components to be changed without altering the qualitative behavior of the system greatly increases the need for a broad range of oscillating parameters.
How we attain synchronization
Quorum Sensing Molecules
TAs for a means of synchronization we will use molecules that oscillate colony-wide, for which the quorum sensing molecules used by Danino et al. (2010) offer a good example. Instead of individual intracellular oscillations that are synchronized by a mechanism that influences the entire colony, we aim for a colony-wide oscillation, which is intrinsically synchronized.
We will use two distinct colony-wide molecules, instead of only one, since that way we can have a less parameter sensitive oscillator. This happens when the two colony-wide molecules (indirectly) repress each other’s production, which will of course be the case in our model. When this is the case then when for instance there is an excess production of the colony-wide molecules, these excesses will partially repress each other. This implicates that a broader spectrum of colony densities should oscillate. As to make sure the evolution towards a stable steady state is avoided for reasonable parameters, we also require delaying steps to ascertain a separation in time of the successive peaks in production. By studying the possibilities for colony-wide transcription factors, we found quorum-sensing molecules as good candidates, since the ones we encountered serve as transcription factors (combined with intracellular receptors) and can diffuse out of the cells (Fugua, Winans and Greenberg, 1994). Because the quorum-sensing molecules are produced by enzymes, instead of being directly transcribed and translated, this extra step in which the enzymes are produced already induces an extra delay. On top of that the processes leading from the activation of a gene towards an active protein also take a finite amount of time, which also adds an extra delay.
Figure xǀ Text
Figure xǀ Tekst.
The logical circuit.
Figure X displays a logical circuit with our proposal for an oscillating model. It consists of two analogous halves that are meant to exhibit sequential peaks. In this system A and B represent the colony-wide molecules. They are produced by enzymes, which is indicated by the dotted lines, and of which the expression is controlled by the logical AND gate. We incorporated this enzyme step already because we will propose a practical implementation that uses quorum-sensing molecules later on. We will first explain how this system produces oscillations and afterwards, with a more thorough explanation of the subsystems and components, we will show how this inherently creates a synchronized oscillation.
