# Endy:Victor3 Calculating fluorescent protein synthesis

## Background subtraction

Subtract a media background, $\displaystyle{ A_{media} }$, from the raw absorbance data, $\displaystyle{ A_{raw} }$, and assume that the resulting data, $\displaystyle{ A_{corrected} }$, is directly proportional to the number of cells in the well.

 $\displaystyle{ \frac{}{}A_{corrected} = A_{raw}-A_{media} }$ ...Equation 1

Subtract a fluorescent protein-free cell background, $\displaystyle{ G_{cells} }$, from the the raw fluorescent data, $\displaystyle{ G_{raw} }$, and assume that the resulting data $\displaystyle{ G_{corrected} }$ is proportional to the total number of GFP molecules in the well [immature GFP?].

 $\displaystyle{ \frac{}{}G_{corrected} = G_{raw}-G_{cells} }$ ...Equation 2

## Unit conversion

Use standard calibration curves (see here for absorbance and here for fluorescence) to convert the background-corrected data into absolute units (CFU/well and GFP molecules per well). The calibration equations used are shown in Equations 3 & 4.

 $\displaystyle{ \frac{}{}CFU = 3.1e8 * A_{corrected} - 1.6e6 }$ ...Equation 3 $\displaystyle{ \frac{}{}GFP = 7.0e8 * G_{corrected} + 6.0e11 }$ ...Equation 4

## GFP synthesis rate calculations

To calculate the mean synthesis rate of GFP per cell, $\displaystyle{ S_{cell} }$, assume the total GFP synthesis rate is equal to the time differential of $\displaystyle{ GFP }$. $\displaystyle{ S_{cell} }$ can be calculated as the total synthesis rate divided by $\displaystyle{ CFU }$.

 $\displaystyle{ \frac{}{}S_{total} = \frac{d[GFP]}{dt} }$ ...Equation 5 $\displaystyle{ \frac{}{}S_{cell} = \frac{S_{total}}{CFU} }$ ...Equation 6