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Team:Cambridge/Modelling
From 2009.igem.org
Categories :
Project :
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Overview
Sensitivity Tuner
--- Characterisation
--- Modelling
Colour Generators
--- Carotenoids (Orange/Red)
--- Melanin (Brown)
--- Violacein (Purple/Green)
The Future
Safety
Notebook :
Team Logistics :
Modelling
Introduction
This project focuses on novel outputs, for example, for environmental sensors. However, there is a need for an 'adaptor'; a middle section to the machine that takes an input and processes it. Our initial work was based around the development of an amplifier that permits a large output that is clearly visible. The next planned stage was the creation of a system that allows switching on of output at different calibrated input signal levels. Creating a model allows the feasibility of the proposed systems to be tested. A basic model of the original amplifier system was put forward, building on both our data and the Cambridge 2007 data.
Modelling the phage activator system
This is the basic 'amplifier' system that consists of an input sensitive promoter system and a protein activator and sensitive promoter. It can therefore be divided into two boxes, the approach taken in putting forward an initial model.
The pBAD promoter
An arabinose input acts as an inducer, permitting transcription, by binding the AraC transcription factor. This is a dual transcription factor; when unbound to arabinose a dimer restricts access of polymersase to reduce basal levels of transcription, upon binding arabinose the conformation changes and the dimer permits binding of polymerase. [1]
To model this situation, araC is first assumed to take the role of a repressor that reversibly binds and unbinds a site on the DNA. If it binds arabinose, it is sequestered and cannot bind the DNA. Here, an input function is created, after Alon []. This gives the rate of transcription from the promoter dependent on the concentration of arabinose. Since mRNA is then ttranslated at a roughly constant rate, it is related within a multiplicative constant to the rate of protein production, in this case activator and RFP.
This gives the rate of transcription as a function of X* which represents the concentration of active repressor, unbound to arabinose. B is the maximum rate of transcription, here this rate is when induced by arabinose at highest concentration. K_d is the dissociation constant (see modelling derivations). Parameters must be found by a parameter scan for sensible values or by comparing to already gathered data.
The concentration of 'active' repressor is given as a function of arabinose concentration by:
equation 2 here
where X^T is the total amount of araC available, bound or unbound. A is arabinose concentration. n is the number of arabinose molecules binding to each molecule of the repressor, and K is a binding constant. n was taken to be two by assuming that each araC dimer needs two molecules to be bound before it can permit transcription.
Combining these two gives the overall input function, which has leaky transcription included at A = 0, seen in the actual results.
The Activator and its Promoter
This is based on a similar idea. Activator is made by transcription from pBAD, the mRNA is then translated (the potential time delays will be taken into account). The activity of the phage promoter is dependent on activator concentration according to:
equation 3
Assuming that translation rates remain constant, the rate of GFP and RFP production would be expected to be multiples of the above promoter activities/ input functions (which represent rate of transcription)
Making a Latch
A switch that remains on once stimulated would be useful if it was only necessary, say, to see if a hazardous contaminant had ever been present in a sample (it could still be there in low levels etc.). A method proposed is positive feedback; an activator placed downstream of its own promoter (as well as the reporter/ pigment) will, in theory, keep pigment production going. The rate of activator production from its own promoter is given in equation 3 above, which is dependent on activator concentration itself.
