Due to the inherent limitations that we found to be present in our chemically induced IFFL system, a new method of gene expression control was required if we wanted to test our decoding circuit. Ideally this new method of gene expression control would be easy to turn on and off, modular enough to work in a wide variety of systems, and have the potential to be useful in applied projects. Based on these requirements we eventually settled on using an expression system whose activation was controlled by temperature. Temperature based control presents several advantages over similar systems (ex. optogenetics), namely in that temperature is easy to manipulate with non specialist lab equipment, and that temperature based systems are well characterized and could potentially be useful for in vivo systems.

Over the course of iGEM’s history, a number of temperature controlled systems have been described and characterized, prominent examples include heat sensitive repressors as well as RNA thermometers. Based upon our research, we initially chose to design our temperature controlled IFFL based using the temperature sensitive mutant of lambda cI (ts-cI). In this system, ts-CI acts as a repressor of the lambda phage promoter R0051 at low temperatures, but is irreversibly denatured by temperatures of 37C or higher. Thus our circuits based on this system contained a module that constitutively expressed a high level of ts-CI, as well as modules that placing the production of both our reporter (mScarlet-pdt) and inhibitor (mf-Lon) under the control of R0051. When grown at 30C ts-CI would be able to repress the production of both the reporter and inhibitor, but when the temperature was increased to 37C ts-CI would be denatured, allowing the production of both. If temperature was lowered again, then more ts-CI would quickly be produced and the production of both the reporter and repressor would be repressed.

We initially sought to validate that we could create constructs that were controlled by temperature. To that end we created a simple inducible circuit containing a constitutively expressed ts-CI and a mScarlet-I-pdt under the control of R0051. By growing these circuits at 30C before inducing them at 37C, we were able to determine that the system was indeed controlled by temperature (Figure 2a). After confirming that activation of the system was controlled by temperature, we next tested to determine if reducing the temperature of the activated circuit would cause the production of mScarlet to cease. We found that indeed the system does stop production of mScarlet-I-pdt after the temperature is reduced, with a short delay due to the need for some ts-CI to be produced (Figure 2b).

Next, we designed a separate temperature controlled system (BBa_K2680050) that expressed mf-Lon, which we hoped to combine with K860052 to form a complete IFFL. However, while cells co-transformed with both of these parts grew fine at 29C, when the IFFL was turned on by heating to 37C, no change in fluorescence values was apparent. Upon inspection of the optical density values, the reason became clear. Upon activation of the IFFL an immediate drop in cell density occurred, indicating that the constructs were toxic to the cells (Figure 3a). This was not overly surprising, as previous teams have reported that high expression of mf-Lon has a cytotoxic effect on E. coli. Given that the promoter (R0051) driving the expression of mf-Lon is phage derived and has extremely strong expression, it is reasonable to believe that an overly high expression of mf-Lon led to this result. Next, we determined that the expression of mf-Lon from our construct actually led to cell death and did not merely inhibit growth (Figure 3b).

We first attempted to get around this issue by using a lower temperature of 35C to activate the cells, but found that this still lead to cell death (data not shown). Based on these results, we reasoned that we might want to reduce the expression of mf-Lon as well as the expression of mScarlet-I-pdt, as in conjunction they appeared to strongly cytotoxic.