IGEM:IMPERIAL/2006/project/Oscillator/project browser/Full System/Design

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Super Parts Not applicable
Actual Part Image:J37036 part.png
Sub Parts Prey Molecule Generator Predator Molecule Generator


Design choices

Design features imposed by our specifications:

  • The oscillatory behaviour should be based on a well known and understood dynamic (to be able to gain control over the frequency and amplitude of the oscillattions).
  • The ouput signal of the oscillator should be the variation in time of the concentration of a given chemical compound at the culture level (easier detection and easier connectivity with other systems).
  • To address noisy and instability behaviours from previous oscillators, we have decided to rely on large population of molecules in order to deal with smooth and continuous biochemical reactions.

From the previous constrains a final design is presented:

  • We have decided to design a molecular Lotka-Volterra(LV) oscillator based on the available Quorum sensing/quenching BioBricks.
  • Following LV dynamic we have specified:
    • a Prey Molecule Generator which produces AHL (the prey-molecule).
    • a Predator Molecule Generator which is able to produce the predator-molecule depending on the prey-population (using LuxR to sense AHL) and also allows the predator molecule to degrade the prey-molecule (using aiiA enzyme).
  • In order to get a stable oscillator, both molecule generators should keep their production capacity constant, meaning that the amount of cells expressing them should be stable. We will use a chemostat to achieve it.
  • To introduce flexibility in our design, we have chosen to base our design on a 2 cell population system. A population of cell will express the Prey Molecule Generator, when the other one will express the Predator Molecule Generator. Depending on the ratio between these two populations we have shown through modelling that we can tune the frequency and amplitude of the resulting oscillator.
  • The use of the quorum sensing mechanism has two major advantages:
    • it defines the oscillations at the culture level which helps to gain stability and avoid noise.
    • it enables an easy connectivity with any other system willing to synchronise by using the pLux promoter.
Figure 1. A block diagram of Lotka-Volterra interactions between the prey (A) and predator (B).
Figure 1. A block diagram of Lotka-Volterra interactions between the prey (A) and predator (B).
Figure 1. Design of the experimental set-up of the full oscillator.
Figure 1. Design of the experimental set-up of the full oscillator.
INPUTS biological component comments
OD Cell culture Optical density due to the 2 populations of cells (Prey Molecule Generator + Predator Molecule Generator). this OD should remain constant through-out the use of the oscillator. Need of the Chemostat.
Ratio Cell culture Ratio between the Prey Molecule Generator and Predator Molecule Generator. This ratio should remain constant too.
Wash-out Chemostat The Chemostat wash-out will have an impact on the dilution of expressed molecules.
OUTPUTS biological component(s) comments
Prey molecule AHL AHL synthesized by the LuxI gene <bbpart>BBa_C0061</bbpart>. In our design, this is the only molecule detectable at the population level. Thus the output of the oscillator is fluctuation in [AHL].
FUNCTIONS biological component(s) comments
Oscillations Full System The oscillations in the concentration of [AHL] are function of the inputs parameters (OD, ratio and wash-out). A detailled study of the full system is described in the modelling section.

Overall Bio-chemical System

Figure 1.  Full system overview showing interacting populations of cells. The Prey cell produces a constant amount of LuxR. The lux R expression is controlled by the tet promoter. LuxR binds AHL and this complex activates transcription at the Lux promoter (pLux). This leads to productin of the Enzyme LuxI. LuxI will produce AHL. The AHL will be bound by more LuxR and create a positive feedback loop. AHL can freely diffuse out of the prey cell and into the predator cell. Here it is bound by luxR. The AHL-LuxR complex will bind the pLux Promoter leading to more LuxR being made. The gene Aiia is also transcribed leading to the production of AHL-Lactonase which degrades AHL so the Predator cell senses and kills AHL. This system is based on Lotka Volterra. However there are important diferences. So the system has been re-modeled and is discussed below
Figure 1. Full system overview showing interacting populations of cells. The Prey cell produces a constant amount of LuxR. The lux R expression is controlled by the tet promoter. LuxR binds AHL and this complex activates transcription at the Lux promoter (pLux). This leads to productin of the Enzyme LuxI. LuxI will produce AHL. The AHL will be bound by more LuxR and create a positive feedback loop. AHL can freely diffuse out of the prey cell and into the predator cell. Here it is bound by luxR. The AHL-LuxR complex will bind the pLux Promoter leading to more LuxR being made. The gene Aiia is also transcribed leading to the production of AHL-Lactonase which degrades AHL so the Predator cell senses and kills AHL. This system is based on Lotka Volterra. However there are important diferences. So the system has been re-modeled and is discussed below

Registry

The full system is based on 2 parts which can be found in the Registry.

  • The Prey Molecule Generator: <bbpart>BBa_J37015</bbpart>

Image:J37015 logo.png

  • The Predator Molecule Generator:: <bbpart>BBa_J37036</bbpart>

Image:J37036 parts pic.PNG


Open issues

  • We need to be able to control the prey's positive feedback loop to ensure that AHL concentration is 0 while cell cultures are growing.

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As of 20 August, we have updated the design of our system due to some complication we overlooked when designing our initial system. First, the prey cell must be controlled so that it does not reach a steady state before we implement it into our system. Ideally, we would like to have a method of control to induce or inhibit the expression of AHL molecules in our system. Thus, we have formulated several designs which we hope will be viable in controlling the exponential expression of AHL.

Following reserach into the riboswitch, we have decided to explore this option. We are using a theophylline induced riboswitch which will be placed between the pLuxR promoter and the LuxI protein coding region (the part which codes for the AHL). Once we inject theophylline into the system, the prey cell will then start the exponential growth.

Another idea we developed to control the exponential expression is the CRE system mentioned by Dr. Mann. Basically, the CRE system is a section of DNA which does not code for anything, but is placed in the middle of a coding region. Only until the necessary proteins to cut the sequence out of the DNA is manufactured, expression is repressed. The only drawback to this system is it requires two plasmids within the same cell, which might not be an easy task to achieve. Read more about the Cre-Lox System Design.

Into the tenth week of the iGEM project, the team has decided to scrap the riboswitch part (J37015RS) in favour of using an acylase to control the AHL levels in the prey cell. If we innoculate the culture with an acylase, it should degrade most of the AHL maintaining the concentration in an exponential growth phase. When putting the system together, we can easily remove the acylase by flushing out and adding the predator cell in it's place. Hence, the oscillator should begin with the prey cell not having reached the peak of the exponential production phase.

The acylase itself cleaves the entire lactone ring from the n-acyl homoserine lactone molecule as seen above producing homoserine lactone and a fatty acid. The products are no longer biologically active; however, the paper on the structure of AiiA has commented that HSL is a competitive inhibitor for AHL. This may lead to some problems depending upon the concentration of HSL left in the chemostat after we flush the acylase out.

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  • We need some method of characterizing the half-life and activity of AiiA. We will use a Flag tag to achieve this

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Furthermore, we have decided to alter the design of the predator cell by adding a FLAG immunotag. The purpose of this was so that we are able to characterise the AiiA halflife and activity before producing the final system construct. When testing is done in conjunction with the modelling, this will provide us a better understanding of the way the system works. Moreover, we will be able to simulate with real values to see what we expect in our biological oscillator. The immunotag is attached to the beginning of the AiiA protein coding sequence, since we are using the AiiA sequence already containing an LVA degradation tag. We do not want the degradation tag to interfere with the immunotag, so we have decided to PCR in the immunotag to the 5' end of the sequence. The 3D design of the AiiA enzyme in one of the papers has suggested that the ends of the sequence are not close to the active site of the enzyme, so there is a small but unlikely chance that the tags will interfere with the activity of the enzyme.

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