Barry Canton at 00:43, 4 October 2006

Barry Canton — Wed, 04 Oct 2006 00:43:50 GMT

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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.
{|width="720px" align="center"
|width="100px"|
|align="center"|<math>\frac{}{}A_{corrected} = A_{raw}-A_{media}</math>
|align="right" width="100px"|...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?''].

{|width="720px" align="center"
|width="100px"|
|align="center"|<math>\frac{}{}G_{corrected} = G_{raw}-G_{cells}</math>
|align="right" width="100px"|...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.

{|width="720px" align="center"
|width="100px"|
|align="center"|<math>\frac{}{}CFU = 3.1e8 * A_{corrected} - 1.6e6</math>
|align="right" width="100px"|...Equation 3
|-
|width="100px"|
|align="center"|<math>\frac{}{}GFP = 7.0e8 * G_{corrected} + 6.0e11</math>
|align="right" width="100px"|...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>.
{|width="720px" align="center"
|width="100px"|
|align="center"|<math>\frac{}{}S_{total} = \frac{d[GFP]}{dt}</math>
|align="right" width="100px"|...Equation 5
|-
|width="100px"|
|align="center"|<math>\frac{}{}S_{cell} = \frac{S_{total}}{CFU}</math>
|align="right" width="100px"|...Equation 6
|}

Endy:Victor3 Calculating fluorescent protein synthesis - Revision history

Barry Canton at 00:43, 4 October 2006