The mathematical model for this design is extremely similar to the example of cooperativity given in the [[CellularMemory:Mathematical Models#Cooperativity and Bistability |mathematical modeling]] section of this wiki paper. An activator dilution equation was set equal to an activator production equation in order to determine the stable steady states of the system. The activator production equation took into account the concentration of the activator (YFP) and the concentration of the sensor (RFP), as both are capable of activating the P<sub>CYC1</sub> promoter. It also took into account basal levels of transcription from each promoter as well as the cooperativity of binding of the two activators proteins, a necessary component of the system functionality.
Ajo-Franklin, C.M., Drubin, D., Eskin, J.A., Gee, E.P.S., Landgraf, D., Phillips, I., and Silver, P. 2007. Rational design of memory in eukaryotic cells. Genes & Development 21: 2271-2276.
Overview
The final paper that will be discussed was published in September of 2007 by the lab of Pam Silver at Harvard Medical School. It describes the design and construction of memory in yeast. Using an autoregulatory positive feedback design in conjunction with an accurate mathematical model and quantitatively characterized parts, their work produced yeast that were capable of turning on a GFP positive feedback loop in response to a galactose stimulus. The production of GFP was shown to be able to survive cell division. As was the case with the previous paper, construction of a functioning synthetic network in a eukaryotic organism marks a significant step forward in the design of novel cellular devices.
Specific Biological Design
Figure 1: Biological design of permanent memory in yeast.
The specific biological design of this gene network is a fairly standard autoregulatory positive feedback loop. As can be seen in Figure 1 on the right, two separate plasmids were constructed that each performed separate tasks. The sensor plasmid (on the top of Figure 1) consisted of an galactose-inducible promoter (Pgal) upstream of a hybrid RFP gene (Red Fluorescent Protein). Fused to the RFP gene was a DNA binding domain (DNA BD) that is specific to the PCYC1 promoter, a VP64 activator region, and a nuclear localization signal (NLS). In the presence of galactose, this hybrid RFP protein would be produced and localized to the nucleus of the cell by of the NLS. Once in the nucleus, the DNA BD would allow binding of the hybrid RFP to the PCYC1 promoter, at which point the VP64 activator would turn the PCYC1 promoter on.
The auto-feedback plasmid (on the bottom of Figure 1) consisted of a hybrid YFP gene (Yellow Fluorescent Protein) downstream of the PCYC1 promoter. The same fusions (DNA BD, VP64 activator, and NLS) were made to the YFP gene that had been made the the RFP gene. Thus, the production of the hybrid YFP protein would create an autoregulatory positive feedback loop.
In the presence of galactose, these yeast cells should fluorescence both red and yellow because of activation of both promoters. After galactose is removed from the cells' environment, they should retain their yellow fluorescence but lose their red fluorescence.
Mathematical Modeling
The mathematical model for this design is extremely similar to the example of cooperativity given in the mathematical modeling section of this wiki paper. An activator dilution equation was set equal to an activator production equation in order to determine the stable steady states of the system. The activator production equation took into account the concentration of the activator (YFP) and the concentration of the sensor (RFP), as both are capable of activating the PCYC1 promoter. It also took into account basal levels of transcription from each promoter as well as the cooperativity of binding of the two activators proteins, a necessary component of the system functionality.
