Endy:Victor3 Calculating fluorescent protein synthesis

From OpenWetWare

Revision as of 20:43, 3 October 2006 by Barry Canton (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search
Add to My Links

Background subtraction

Subtract a media background, Amedia, from the raw absorbance data, Araw, and assume that the resulting data, Acorrected, is directly proportional to the number of cells in the well.

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

Subtract a fluorescent protein-free cell background, Gcells, from the the raw fluorescent data, Graw, and assume that the resulting data Gcorrected is proportional to the total number of GFP molecules in the well [immature GFP?].

\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.

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

GFP synthesis rate calculations

To calculate the mean synthesis rate of GFP per cell, Scell, assume the total GFP synthesis rate is equal to the time differential of GFP. Scell can be calculated as the total synthesis rate divided by CFU.

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