Team:IIT Delhi/Oscillator

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Motivation

Remember ’Mystique’ from the X-men franchise? Albeit prone to a little villainy, she is that amazing character with the blue skin; and possessed the uncanny ability to effortlessly transform into anyone at will. Quite an intriguing capability; her cells had the potency to reconfigure into any individual of choice as per necessity. Now, that may be a far fetched dream, but we can take a lesson or two from Mystique’s biological gifts. For instance, reconfigurable electronic circuits may not be far out of reach [? ];these systems would be the ideal multitaskers, switching between functionalities as the need arises. Synthetic Biology also seems to be vying for a place in this domain; from reconfigurable logic gate systems [2, 4] to systems with switchable dynamical behavior [1, 2, 6]. On an abstract level, the idea of reconfigurablity resonates with Team iGem IIT Delhi. Works like [1, 6] that propose a novel synthetic circuit, which can act both as an oscillator and a toggle switch have been a major influence on our work. The projet is divided into two parts; and at the heart of it all lies a ”Sychnronized Quorum of Genetic Clocks” [3]. First, we develop a reconfigurable circuit that can switch between an oscillator and a toggle switch. We realize this circuit using the oscillator of [3]. In order to achieve a toggle switch behavior, we add the lambda repressor; which is susceptible to thermal denaturation. Thus, at temperatures of around 30 deg. celsius the system acts as a toggle switch, since the lambda repressor is constitutively produced and represses AiiA. However, as the temperature is raised to 37 deg. celsius, the lambda repressor undergoes thermal denaturation and thus the system starts acting an oscillator. The second part ofour project focuses on modifying the oscillator in [3] such that we are able to tune the frequency response. The frequency tuning is under the control of light. Addition of an optogenetic component to the system affords us control over the frequency by simpling shining light on the system; thus, we call the second system ”Highly Optogenetically Tuned Frequency Modulator” or as we like to call it, HOT-FM. We desgined the purported optogenetic system using the Ccas- Ccar system. Simulations support the hypothesis for frequency modulation. The Biological realization for this part of our project is still under work. Throughout the wiki, we explain the different stages of implementation for the two systems. We begin with a very brief review of the work done of oscillators, followed by a detailed description of the synchronized oscillators of [3]. An increasingly crucial component of synthetic circuit design is the use of computational resources. In this regard, first we replicate the results of [3]; and we build on this computational model to study the effects of our proposed modifiations. Simulations support our hypotheses regarding the two systems that we have proposed.

Oscillator Circuits

The aim of synthetic biology is to design and synthesize biological networks that perform desired funtions in a predictable manner. A significant chunk of workin synthetic biology has focused on the construction of two important type of networks: switches and oscillators. Synthetic biology is at the intersection of biology, engineering and computational mathematics. Consequently, there are two distinct aspects to the design, analysis and implementation of a synthetic circuit: in-silico and in-vivo validation. Under the in-silico paradigm, we forego the specific implementational details, rather we are focused on the abstract mathematical model and the dynamical behavior such a model allows. While for in-vivo analysis, we work on identyfing the specific biological components that can be used to implement the identified topology for the synthetic network. Here, we look at both these aspects for the Danino Oscillator [4].

Figure (number intro1) shows some topologies which are known to show oscillatory behavior [8]:

  1. Goodwin Oscillator
  2. Repressilator
  3. Amplified negative feedback oscillator
  4. Fussenegger Oscillator

We’ll give a very brief overview of the Goodwin and the Amplified negative feed- back oscillators . The Danino Oscillator is essentially a quorum sensing version of the Amplified negative feedback oscillator.

  • Goodwin Oscillator - This was the first synthetic genetic oscillator to be studied. It consists of a single gene with negative autoregulation. Models suggest that oscillations occur in the goodwin oscillator if repression is mod- elled by a nonlinear Hill function with a high cooperativity coefficient [3]. Further, this oscillator has been shown to have a robust time period.
  • Amplified negative feedback oscillator Leaving aside the biological im- plementation, this abstract topology is the one used in the Danino Oscillator.Under this architecture, the activator gene A activates it’s own repressor gene B. In the Daanino oscillator there is a latent phase when both the activator and repressor accumulate, thus allowing large amplitudes to be achived for this configuration.The qualitative behavior of this oscillator is discussed in the modelling section using the mathematical model suggested in [4]. One crucial benefit that the Danino oscillator has over this topology is the prop- erty of quorum sensing; the AHL molecule can diffuse across the cell mem- brane and be exchange with neighbouring cells. This offers a mechanism to synchronize the oscillators.

Project

The main focus of team iGem IIT Delhi has been ’reconfigurability’. Keeping this in mind, we have divided the project into two modules; each centred around the idea of reconfigurability. Module I is a tunable oscillator where we can change the frequency of oscillation. Our proposed design focuses on using an optogentic mod- ule to achieve control over frequency. While Module I would switch the frequency of oscillation between two non-zero values, Module II implements a toggle switch over an oscillator. Thus, the idea is to be able to kill the oscillations in a manner that the concentration of one of the components stays at a low value, while that for another component stably stays high.

Module I - HOTFM

We call this module ’Highly Optimized tunable Frequency Modulator (HOTFM)’. The idea is to modify the Danino Oscillator so that we can tune the frequency of os- cillations. Further, we wish to use optogenetics to control this tuning. Oscillators can be used as clock references, and thus, controlling the frequency of oscillation would offer certain advantages. For instance, increasing the frequency might allow faster execution of a process and vice versa. In this regard, one possible medicalapplication could be time-controlled drug delivery. If a drug is known to function optimally if delivered at distinct regular intervals, an oscillator handling the de- livery of the drug would be an attractive option. Further, if under circumstances quie stark from the ordinary, the rate delivery needs to be increased (decreased), our approach could be a viable option.

We require two additions to an oscillator to realize HOTFM. A mechanism to modulate the frequency of oscillation and an optogenetic module to act as a switch for this mechanism.

Frequency Modulation

The Danino oscillator has a latency period where both the activator (LuxI) and the repressor (AiiA) grow in concentration. One possible way to reduce the time period of oscillations would be to reduce this latency period. For the Danino os- cillator, it is known that the time period and amplitude have a linear relationship [4]; thus, we could, in principle, reduce the amplitude to reduce the latency pe- riod and increase the frequency of oscillation. We propose to do this by adding negative autoregulation on AiiA. This transforms the amplified negative feedback oscillator into a smolen oscillator [8]. There are additional benefits of adding negative autoregulaation on AiiA; this makes the oscillator robust to parameter variability [8]. If there is a sudden variation in AiiA’s concentration the negative auto-regulation would automatically bring the concentration back down. Thus, we expect HOTFM to have lesser peak to peak variability compared to the original Danino oscillator. In-silico simulations confirm our intuition about HOTFM (refer to the modelling section for a detailed analysis).

Optogenetic Module

In order to have a tunable frequency modulator, we need to have a switch that controls the negative auto-regulaation (self-repression) on AiiA. To achieve this end, we use optogentics as a flexible and easy mechanism. The Self-repression pathway is put under the control of lambda repressor; and this lambda repressor controlled by the Ccas-Ccar system, which is an optogenetic module. So, whenever the oscillator is required to change it’s frequency of operation, we can, in principle, shine light on the cells and the self-repression pathway (mediated by the lambda repressor) would be activated.

Danino Oscillator: Synchronized quorum Gene oscillatory Networks

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