Team:ETH Zurich/Modeling/Construct2

From 2012.igem.org

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Contents

LovTAP/Cph8

In addition to the primary UVR8-TetRDBD system, we are constructing a second genetic circuit. This circuit consists of well-characterised photoreceptors and transcriptional repressors that have been previously shown to work in-vivo, providing a practical alternative to the UVR8-TetRDBD-based system. Although LovTAP mainly acts as a blue-light photoreceptor, a minority of induced reactions are still caused by UV light (~16%). In our model, strong irradiance with blue light as well as red light serves as an indicator for sun light. The other states of our decoder filter out common light sources.

Boolean logic

Boolean response
Red-Lightinput Blue-Lightinput OutputGreen OutputRed OutputViolet/pabAB Situation
0 0 0 0 0 darkness
0 1 1 0 0 modern interior light source e.g. CCFL
1 0 0 1 0 classical tungsten light bulb
1 1 0 0 1 resembles outdoor light

Circuit

The following scheme illustrates the system:

ETH Construct2 Circuit.png

Modelling assumptions

In order to analyse a number of properties of the system, the model needs to be tractable. For this, a number of assumptions and approximations have been made:

  • Cph8 and LovTAP are conserved. This translates to a model not explicitly accounting for photoreceptor expression/degradation, as this will be the case for most systems in steady state anyway. Biological optimisation will instead focus on choosing the optimal ratio of promoter strength kP to degradation rate kdeg of the photoreceptors that effectively equal the steady state concentration of Cph8 and LovTAP.
  • All photoconversion processes are modelled as 2-state processes.
  • Degradation rates are identical for all proteins. While this obviously does not resemble biological reality, setting a degradation rate for every protein species individually is practically impossible, as such rate parameters are not ubiquitous in literature.
  • The negative feedback has not been modelled explicitly here, as it has been accounted for in the previous simpler UVR8-TetRDBD model. As no oscillations are possible, the negative feedback loop merely reduces the dynamic range of the switching.
  • Basal expression as fraction of complete induction is the same for all promoters (= 5% in our case).

ODEs

With the previous assumptions, the coupled system boils down to:

ETH Construct2 ODEs.png


Find the parameters we used on our parameter page. For the optimisation of the promoter expression strengths, we have targeted steady-state repressor concentrations whose KM values equals the geometric mean of the steady-state concentrations between separating conditions, i.e. given a repressor concentration of conditions that are closest together, their steady-state values should yield a geometric mean equal to the repressor's respective KM values.

Results

ETH Construct2 Plots.png

With 5% basal expression, a satisfactory separation can be achieved. Importantly, the expression of the violet pigment indicates a 2-fold induction when the system is induced both with blue and red light. Note that only these steady-state concentrations have a meaning - dynamics of the system might temporarily lift some pigment concentrations above the indicated values - they will eventually drop to the steady state. The separation between conditions is smaller than that of the UVR8-TetRDBD-system: this is due to the worse separation of Cph8, which only yields a 2.4 fold induction, compared to a near perfect separation for UVR8-TetRDBD.

Biological implications

The system has been tuned manually to maximise the dynamic range between conditions. This has resulted in promoter expression parameters that constrain the model:

ETH Construct2 Constraints.png

This in effect requires the same promoter strengths to be employed in-vivo. Coming from a bioinformatics background, we propose a trial-and-error inspection for finding the correct promoter by using a binary-logarithmic "divide-and-conquer" method. This will reduce the number of different promoters required to be tested.

Considerations

In future, screening the literature for candidate sequences that exhibit less leakiness in practice should be considered.

Some uncertainty comes from the fact that no parameters are known for the Cph8 photoreceptor - such that its optimal concentration range can only be guessed.


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