Team:Aberdeen Scotland/hillinput

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University of Aberdeen iGEM 2009

University of Aberdeen - Pico Plumber

iGEM 2009

Introduction

This section details the derivation of the input functions for repressors and activators that we are using for our differential equations describing the Pico Plumber. For further reading we recommend Uri Alon “An Introduction to Systems Biology”.

Repression of a Promoter

During repression of a promoter a repressor protein; X, binds to a DNA site of the promoter, D. The product of this binding process is [XD]. [XD] can also dissociate into [X] and [D] again:

(I)

Where k_on describes the collisions of X and D that occur per protein per unit time at a given concentration and k_off determines the strength of the chemical bond between X and D. In the form of a differential equation, the rate of change of [XD] is described by

(II)

At steady state the concentration [XD] does not change.

(II.1)

(II.2)

(II.3)

(II.4)

Equation (II.4) is called the chemical equilibrium constant equation, where Kd is the dissociation or equilibrium constant. Kd has units of concentration. Therefore, transcription of a gene only happens whenever the repressor is not bound. That is when D is free. The total concentration of the DNA sites [DT] can be written with the help of the conservation law:

or

(III)

Substituting (III) in (II.4) we find

(IV)

(IV.1)

(IV.2)

(IV.3)

(IV.4)

(IV.5)

(IV.6)

As a result, the probability

describing that the site D is free is dependent on [X]. The promoter activity, p, is defined by

or

(V)

where β is the maximal transcription rate of the promoter. If

then [X] = K_d and the promoter activity is reduced by 50%. This [X] needed to repress the promoter activity by a half is called the repression coefficient. If several protein units of X are binding on D, i.e. dimers, tetramers etc, then we can apply the Hill function for repression of a promoter that is

or

(VI)

Activation of a Promoter

In the case of an activation of a promoter, an activator protein, X, binds to its DNA site of the promoter and increases the rate of transcription of the promoter. Similarly to the repression of the promoter the promoter activity, p, in the case of activation can be derived as

or

(VI)

Input Function for an Inducer Molecule

To turn a repressor system from Off state to On state, we need an input signal (for example a molecule called inducer,S) such that the repressor protein, X, binds off the promoter side DNA. The inducer forms a complex with X varying X’s affinity to D. The total amount of concentration of the repressor, [X_T], can be considered as a product of the repressor protein forming a complex with the inducer, [XS] and the repressor protein in its free form [X], whereby free does not differ between bound to the promoter’s DNA site or not.

or or

(VII)

Writing the mass-action kinetic equation for the S binding with the X leaves us with

(IIX)

In steady state

(IIX.1)

Rearranging (IIX.1), we can write

(IIX.2)

where Kx is the dissociation constant defined as the ration of koff to kon of S binding to X. Substituting (VII) in (IIX.2) leaves us with

(IX)

Rearranging equation (IX) we get the Michaelis – Menten equation

(X)

Or substituting the third form of equation (VII) in (IIX.2)

(XI)

that is the concentration of X not bound to S. Thus, like in the case of LacI, only X unbound to S can bind to D repressing production. However, repressor proteins are binding on D as several protein subunits and activation is fully achieved if the inducer is attached to these subunits. To describe this binding process we consider n molecules of S binding on X and by the conservation law we are left with:

or

(XII)

S binding on X is again described by the mass-action kinetic equation:

(XIII)

In steady state

(XIV)

Substituting (XII) in (XIV)

(XV)

Rearranging (XV) leaves us with the Hill equation describing the probability the DNA site is bound compared to an average over binding and unbinding of S.