Team:William and Mary/Dynamic Control
Abstract
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.
Design
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).
Figure 1: Schematic of an Incoherent Feedforward Loop (IFFL) circuit. X, Y, and Z represent arbitrary transcription factors. At t=0, the production of X is activated. In region (1), the molecules of X activate the production of both Z and Y. However, the concentration of Y is insufficient to significantly repress the production of Z, so its concentration increases. In region (2), the concentration of Y has grown sufficiently large that it now exerts a significant repressive effect on the production of Z. This causes the concentration of Z to decrease. In region (3), the repressive effect from Y and the activating effect from X have balanced and the circuit has reached its steady state.
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.
Figure 2: Tuning dynamical properties of the pulsatile response of the IFFL circuit. (Red Arrow) Increasing the strength of the repression of Z by Y causes the width of the pulse to decrease, as the speed of the transition from the pulse peak to the steady state is set by Y’s repression of Z. (Green Arrow) Increasing the speed of the activation of Z by X causes the circuit to rise faster to its peak. The result of these effects is a sharpening of the pulse.
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..
Figure 3: Schematic of the Lon-based IFFL. The simultaneous induction of the circuit with ATC and IPTG serves as a proxy for the activation of the single protein X in the canonical IFFL circuit. mf-Lon is under basal repression by lacI and mScarlet is under basal respression by tetR, so neither protein is produced until the inducers are present. As long as ATC and IPTG are introduced into the cell at the same time, the fundamental structure of the IFFL is preserved.
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.
Figure 4: Some example timecourses from our ODE model of the Lon-based IFFL circuit. Our circuit is able to generate pulses of various sharpness, as well as more standard relaxation responses, all of which correspond to the behavior of a canonical IFFL. The simulations were performed by taking our protease complexing model [see Modeling section] and setting the initial conditions so that Lon and the reporter are activated simultaneously.
Results
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).
Figure 5: The Lon-based IFFL circuit can exhibit a pulsatile response. The pulsatile response should only be present in the simultaneous induction condition, as otherwise the circuit does not satisfy the structure of an IFFL. The circuit was constructed using ATC-activatable mScarlet tagged with pdt A (BBa_K2333428) on pSB1C3 and IPTG-activatable mf-Lon (BBa_K2333434) on pSB3K3. NEB 10-beta cells in M9 media with 0.4% glucose and 0.1% casamino acids were induced with 50 ng/mL ATC at t=0, along with 0.1 mM IPTG at t=0 (simultaneous Lon induction) or t= -4 hrs (pre-induced Lon) and their absolute fluorescence levels were measured with FACS at 20 minute intervals. These fluorescence values were then normalized by their maxima. Shaded regions represent one geometric SD factor above and below the geometric mean of 3 biological replicates.
Figure 6: The increased speed from the pdt system is preserved in the simultaneous induction case. (A) Pre-inducing Lon to steady state before the activation of mScarlet allows one to observe a faster speed to steady state, as expected. (B) The pdt-tagged circuit is faster than the untagged circuit even in the case of simultaneous Lon induction. Experimental conditions and data processing procedures are as in Figure 5.
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).
Fig 7: The IFFL’s pulse sharpens with increasing pdt strength. All experimental and data processing conditions are as in Fig 5.
We have demonstrated that by using the pdt system’s control over a single gene’s expression speed, we can exert predictable control over an emergent dynamical property of a genetic circuit simply through the modular swapping of a single genetic part. We anticipate that additional dynamical properties such as the period and amplitude of oscillations, the threshold and duration of temporal buffering, the properties of a signal integrator, and many more will all be amenable to predictable control through the pdt system.