aim 4

The Biological Timeline (Empirical Phase in Fibroblasts)

If you transfect this Incoherent Feed-Forward Loop (IFFL) plasmid into fibroblasts (MEFs), the thermodynamic timeline dictated by mammalian synthesis rates will look approximately like this:

• 0 to 12 hours (Latency): The plasmid enters the nucleus. RNA polymerase II machinery and ribosomes begin to assemble. There is no functional protein yet.
• 12 to 36 hours (The Autonomous "Pulse"): Strong transcription of OSK and the repressor begins. Because the repressor must reach a critical concentration threshold (K_D) to bind efficiently to the DNA, it remains ineffective during this window. OSK accumulates almost freely, reaching its maximum peak of cellular expression around 24 to 36 hours.
• 36 to 48 hours (Bifurcation Point): The repressor crosses its critical threshold ([R] > K_D). The binding sites on the plasmid promoter become saturated. The production term α in the thermodynamic equation rapidly collapses to zero.
• 48 to 96+ hours (The Autonomous "Chase"): With the transcriptional "faucet" closed from the inside by the repressor, the system enters free fall. From hour 48 onward, the system is entirely dominated by the empirical degradation constant k_d. Recalling that the half-lives of KLF4 and OCT4 range from 2 to 12 hours, by hour 72 to 96, the OSK pulse will have been "cleared" by the proteasome, leaving the cell in its new state or allowing the somatic identity to attempt to re-establish itself.