Endy:Victor3 Calculating fluorescent protein synthesis

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Background subtraction

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

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

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

[math]\displaystyle{ \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]\displaystyle{ \frac{}{}CFU = 3.1e8 * A_{corrected} - 1.6e6 }[/math] ...Equation 3
[math]\displaystyle{ \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]\displaystyle{ S_{cell} }[/math], assume the total GFP synthesis rate is equal to the time differential of [math]\displaystyle{ GFP }[/math]. [math]\displaystyle{ S_{cell} }[/math] can be calculated as the total synthesis rate divided by [math]\displaystyle{ CFU }[/math].

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