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Construction of a Genetic Toggle Switch in E. coli The earliest example of a synthetic cellular memory network is the genetic toggle switch in E. coli. This paper was published in 2000 by the lab of J. J. Collins at Boston University. Their system uses the mutual repression design that was discussed in the biological designs section of this paper. Specific Biological Design Figure 1: Biological design of a genetic toggle switch Figure 1 on the right shows the specific configuration of the two plasmid constructs that were used, each of which confers cellular memory. Each of the plasmids consist of two promoter/repressor pairs, one of which is the Ptrc promoter and the lacI repressor (inhibited by IPTG). P1 and R1 represent the second promoter/repressor pair that was used in conjunction with Ptrc and lacI. Plasmids with the PLs1con promoter and the temperature sensitive lambda repressor as their second promoter/repressor pair were referred to as pTAK constructs. Plasmids with the PLtetO-1 promoter and the tetR repressor (inhibited by aTC) were referred to as pIKE constructs. Multiple types of each plasmid design were constructed by modifying the ribosomal binding site strength of the repressor genes. The constructs were capable of toggling between “on” and “off” stable steady states of GFP production through exposure to a repressor-inhibitors (IPTG, 42 degrees C, or aTc). In this way, the plasmids “remember” the most recent inhibitor that was in the system. Mathematical Modeling The mathematical model for this paper will not be described in detail because most of the tools for modeling memory networks have already been explained in the math modeling section. Briefly, two differential equations were created that described the rate of synthesis of each repressor as a function of the concentration of the opposite repressor, cooperativity, and repressor dilution/decay. Both of these equations were set equal to 0 to ensure that the cell was in a steady state. This model demonstrates the need to balance promoter strength with the strength of repression by tuning ribosomal binding sites strengths. It also shows that repressor cooperativity is necessary for bistability as was shown in the mathematical modeling section. Results Figure 2: Measuring the IPTG toggle-point concentration. The toggle-point of the plasmids was first investigated to determine the amount of input that must be detected by the cell for a toggle to occur. This was looked at specifically for IPTG. Figure 2 shows that a step-like toggle occurs at an IPTG concentration between 10-4 and 10-5 M. Based on this data, 100% of cells should “remember” an IPTG concentration >10-4 M, while a concentration below this will cause none or a portion of the cells to toggle. Similar measurements were done for aTc concentration and lambda temperature sensitivity. Figure 3: Experimental results for a) pTAK and b) pIKE plasmids. The pTAK and pIKE constructs were then tested for the ability to toggle between the on and off states. In Figure 2, the top row of boxes a and b represent the experimental results for the different plasmid types. The bottom two rows of boxes a and b are controls, which behave as expected. All of the cells were started in the on state though 6 hours of exposure to IPTG. All but one of the experimental constructs remained in the on state after removal of IPTG. The promoter and repressor strength were likely not well-balanced in the construct that didn’t retain memory. The cells were then exposed to their second input (42 degrees C or aTc), causing them to toggle into the off state and remain off even when the input was removed. Conclusions