One of our major goals in developing the pdt speed-control system was to allow future teams to obtain control over the dynamical properties of their circuits through modifications that they make at the level of a single genetic part. As a proof-of-concept demonstration of this capability, we construct an Incoherent Feedforward Loop (IFFL) circuit whose dynamical properties are controlled by Lon activity. We demonstrate that we can predictably tune the sharpness of the circuit’s pulsatile response simply by swapping the choice of pdt.
A simple example of a dynamical circuit is the incoherent feed forward loop (IFFL), which consists of three proteins X, Y, and Z which regulate each other such that X activates Y and Z, and Y represses Z. This circuit architecture can generate a pulsatile response upon activation of X (Figure 1).
By tuning the properties of the different interactions within the circuit, one can control the overall dynamics of its response. For example, there are several ways to control the sharpness of the pulse in the circuit’s temporal response. One approach is to increase the strength of Y’s repression of Z (Figure 2, red arrow). This will cause the circuit’s relaxation to steady state to occur more quickly, narrowing the width of the pulse. One could also increase the speed of X’s activation of Z (Figure 2, green arrow), which increases the slope of the circuit’s initial rise to its peak.
Using the Lon-pdt system, we constructed a minimal IFFL circuit which relies on Lon’s proteolytic degradation of a pdt-tagged reporter as the circuit’s inhibition step (Figure 3). By choosing to use Lon as the middle inhibitor (Y) protein in the IFFL, we place both the Y-Z inhibition strength and the X-Z activation strengths under the control of the same property— the strength of the pdt on Z. Because the sharpness of the pulse is now driven by the strength of Lon’s activity, we predict that by swapping out different choices of pdt on the tagged reporter we will be able to control the sharpness of the circuit’s pulse..
However, we were concerned that our Lon-based circuit might be too unorthodox to function effectively as a pulse-generating IFFL. Although we can diagram our circuit in a way that takes the form of an IFFL’s structure, both the fact that we are including small-molecule inducers as a node in the circuit and the fact that our Y-Z inhibition step is realized by post-translational degradation rather than transcriptional repression, are unconventional choices which are not often seen in the canonical descriptions of IFFL circuits. Therefore, to confirm our intuition that our Lon-based circuit would function as a valid IFFL, we simulated an ODE model of our circuit over a wide range of parameters to investigate the types of responses which the circuit can generate (Figure 4). We found that our circuit can exhibit a wide range of responses, all of which are characteristic of an IFFL circuit’s behavior. With this result, we were confident that the design of our circuit was sound, and we moved on to experimentally measure its behavior.
We proceeded to experimentally validate whether our Lon-based IFFL can generate measurable pulses in experimental conditions. We constructed our circuit using IPTG-activatable mf-Lon and ATC-activatable mScarlet tagged with pdt A and measured its activation response over 200 minutes. As a negative control, we also measured the same circuit when Lon was activated 4 hours before the mScarlet, to ensure that a pulsatile response is a property of the IFFL design rather than of the Lon-pdt system itself. We found that the circuit was indeed able to generate a pulse, and that the pulse was a result of the IFFL design (Figure 5). We also compared the circuit’s response to an untagged version of the circuit in both Lon-induction conditions, to validate that the speed change induced by Lon is still present in the simultaneous induction case (Figure 6).
Based on our theoretical understanding of the IFFL curve, we predict that as we decrease the strength of the pdt, the observed pulse should widen. In the no-pdt condition we should not see a pulse, as there is no Y -| Z reaction and hence the circuit is no longer an IFFL. We performed measurements of our circuit’s response to six choices of pdt, where all other parameters and conditions were held constant. We confirmed our prediction that the circuit’s pulse sharpens with increasing pdt strength (Figure 7).