Intracellular model

The intracellular model attempts to simulate the synthesis and action of KillerRed using ordinary differential equations over three stages of a typical photobleaching expeiment: induction at 37ºC, storage at 4ºC to allow for protein maturation, and photobleaching at room temperature. It includes:

• mRNA synthesis and degradation
• KillerRed synthesis, maturation, and degradation
• KillerRed states: singlet, singlet excited, triplet excited, and deactivated
• Superoxide production (by KillerRed)
• Superoxide elimination (by superoxide dismutase)

Parameters and assumptions:

• mRNA synthesis: adapted from Kennell's and Riezman's studies of the lac operon¹. Our construct uses a wild-type lac promoter, so we assume that its transcription rates are similar to the lac operon.
• mRNA degradation: Characteristic half-life from Bernstein et. al.²
• KillerRed synthesis: Normal translation rates from Kennell and Riezman². The maximum translation rate in the model is three orders of magnitude lower to reflect the presents of several rare codons. This adjustment is suggested by comparisons of our fluorescence data for KillerRed and mRFP, which have nearly identical brightness.
• Superoxide production: Given by photophysical calculations adapted from Tsien and Waggoner³; Song et. al.⁴; and our own fluorescence data.

References

¹ Kennel, D. and Riezman, H. Transcription and translation initiation frequencies of the Escherichia coli lac operon. J. Mol. Biol. 1977 114: 1-21.

² Bernstein, J.A., Lin, P.-H., Cohen, S.N. and Lin-Chao, S. Global analysis of Escherichia coli RNA degradosome function using DNA microarrays. Proc. Natl. Acad. Sci. U.S.A. 2004 101: 2758–2763.

³ Tsien, R.Y., Ernst, L. and Waggoner A. Fluorophores for Confocal Microscopy: Photophysics and Photochemistry. Handbook of Biological Confocal Microscopy, 3rd ed. SpringerScience+Business Media, New York, 2006.

⁴Song, L., Hennink, E.J., Young, I.T. and Tanke, H.J. Photobleaching Kinetics of Fluorescein in Quantitative Fluorescence Microscopy. Biophys. J. 1995 68: 2588-2600.