Endy:Victor3 Calculating fluorescent protein synthesis

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Background subtraction

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

[math]\frac{}{}A_{corrected} = A_{raw}-A_{media}[/math] ...Equation 1

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

[math]\frac{}{}G_{corrected} = G_{raw}-G_{cells}[/math] ...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.

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

GFP synthesis rate calculations

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

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