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GFP level (F) Cell count (V) Smoothed Protein Assay Smoothed cell count dm/dt GFP decay rate (m)
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C < D Overview:
This worksheet allows calculation of gene expression levels from GFP data. It implements the GFP model of Brown and Lostroh (2008). Please consult the paper for details on the model's assumptions and interpretation of the output. The example "data" are model-generated and do not represent any specific gene.
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1. In column A, enter the times at which data were collected (e.g. hours after innoculation). The time points do not need to be evenly spaced.
To allow for the "smoothing" of the data, it is necessary to create copies of the first and last data points and place them before and after the real data set.
In the example, the real data runs from t = 0 to t = 20. The first row of data is a copy of the first data point, shifted to an earlier time. The last row of data is a copy of the last data point, shifted to a later time. The times used in these extra data points should correspond to typical time steps in the actual data.
2. In column B, enter the data for GFP fluorescence levels per cell. The first and last values in the column will be copies of the first and last values in the actual data.
3. In column C, enter the cell population data (e.g. OD600). These are relative values only. Include copies of the first and last values, as before.
4. In column D, enter the assumed in vivo decay rate of GFP, in the same units as the time data (e.g. per hour). This may be constant or vary over time. The decay rate can be computed from the half-life H as: LN(2)/H
5. In column E, enter the assumed maturation rate of GFP, in the same units as the time data.
6. In columns F and G, enter the value of the smoothing parameters you wish to use. This must be between 0 and 1, inclusive. If the value is 0, no smoothing is done and the raw data is used. Larger values correspond to more aggressive smoothing, leveling off peaks and valleys.
7. Evaluate columns H-P to determine the inferred dynamics of the reporter protein synthesis, and thus of the gene expression. The units are: relative level of synthesis per unit time. Note that each activity estimate uses data from the previous and the next time step. Thus, there are no values computed for the first time or the last time (if computed, they are not meaningful).
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* List1 Time ~ Verdana
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GFP level (F) ~ Verdana 3 q $ @ pU }* FP ev Cell count (V) ~ Verdana 3 q $ @ pU }* el co GFP decay rate (m) ~ Verdana3 q $ @ pU }* FP ec GFP Maturation rate ~ Verdana q $ @ pU } GFP smoothing parameter ~ Verdana q $ @ pU } Cell count smoothing parameter ~ Verdana q $ @ pU }
Time steps ~ Verdanater ~ Verdana q $ @ pU }* im st Smoothed Protein Assay ~ Verdana Verdana q $ @ pU }* mo he Smoothed cell count ~ Verdanaana Verdana q $ @ pU }* mo he
dF/dt ~ Verdana~ Verdanaana Verdana q $ @ pU }* F/ d2F/dt2 ~ Verdana Verdanaana Verdana q $ @ pU }* 2F t2 dV/dt ~ Verdanana Verdanaana Verdana q $ @ pU }* V/
@ d2V/dt2 ~ Verdana Verdanaana Verdana q $ @ pU }* 2V t2 @ dm/dt ~ Verdanana Verdanaana Verdana q $ @ pU }* m/ @ Inferred activity ~ Verdananaana Verdana q $ @ pU }* nf re
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