Team:William and Mary/Heat
Temperature Controlled Systems
Background
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.
Design
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.
Figure 1: Schematic of BBa_K2680051
Initial Testing of ts-CI System
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).
Figure 2: Relative fluorescence (fluorescence/max) measurements of the temperature activatable circuit BBa_K2680051 when grown at 30C and then activated by exposure to 37C for the entirety of the experiment (A) or transiently (B). Dots represent the geometric mean of 3 (B) or 6 (A) distinct biological replicates (colonies) and the blue shaded region represents one geometric standard deviation above and below the mean. The grey shaded region in (B) represents the period in which the temperature was 37C. Normalized fluorescence (relative to max) was calculated by dividing each colony relative to it's maximal expression.
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).
Figure 3: Optical density (absorbance) measurements of the temperature activatable circuit BBa_K2680051 on psb1C3 + BBa_K2680050 psb3K3 when grown at 30C and then activated by exposure to 37C for the entirety of the experiment (A) or transiently (B). Dots represent the geometric mean of 3 (B) distinct biological replicates (colonies) and the blue shaded region represents one geometric standard deviation above and below the mean. The grey shaded region represents the period in which the temperature was 37C.
Refinement of ts-CI System
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. We reasoned that one way to resolve this would be to express all transcriptional units together on a single plasmid on the low-medium copy backbone psb3K3. However, when we tested this single plasmid construct we found that while cell growth was normal, there was no appreciable gain in fluorescence upon induction (Figure 4a). As this construct was sequenced confirmed (see WM18_016 3K3 in sequencing on Data and Methods), we deemed it likely that there was simply too much mf-Lon expressed relative to mScarlet-I-pdt. This matches with previous where the amount of ratio of mScarlet-I to mf-Lon needed to be far higher than what our construct would likely achieve in order to observe a pulse. We reasoned that one potential