Team:DTU-Denmark/Achievements/Modelling

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Revision as of 21:24, 16 October 2014

Modelling

The main objective of the experimental part of our project was to develop a method for determining promoter activities by measuring fluorescence from the Spinach RNA-fluorophore complex.

Fluorescence Signal is Linearly Dependent on the Concentration of Spinach-DFHBI complex

When the fluorophore DFHBI is bound by Spinach, it fluoresces much more intensely than in its unbound form. The fluorescence of a sample can be assumed to increase linearly with the concentration of Spinach-DFHBI complex:

where the intercept b is the background fluorescence without any Spinach-DFHBI complex, and the slope a is the increase in fluorescence for each concentration unit of Spinach-DFHBI complex.

By adding known concentrations of DFHBI to an excess amount of Spinach RNA, it is possible to make a standard series describing the correlation between fluorescence and DFHBI concentration, and estimate the parameters a and b, if it is assumed that DFHBI is bound completely by the excess Spinach, i.e.:
[EQUATION_2]

This can be used to calculate the concentration of the Spinach-DFHBI complex given a fluorescence measurement:
[EQUATION_3]

If the fluorescence of a Spinach-expressing culture is measured, the standard series can thus be used to calculate the concentration of Spinach-DFHBI complex in the culture.

Calculating Total Spinach Concentration in the Culture

If the fluorescence is measured in the culture with a large excess of DFHBI it can be assumed that the concentration of Spinach-DFHBI is equal to the concentration of correctly folded Spinach, i.e.:

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