Positive feedback loop switch

We designed an upgraded bistable genetic toggle switch based on orthogonal TAL repressors and activators which is composed of a pair of mutual repressors and a pair of activators,upgradingthe classical toggle switch with positive feedback loop. Simulations of the positive feedback loop switch demonstrated bistability even at low or no cooperativity. Stochastic and deterministic simulations indicate higher robustness in comparison to the mutual repressor switch. We experimentally tested the switch by monitoring production of two fluorescent protein reporters, confirming a clear bimodal distribution of reporter fluorescence and demonstrated adoption of stable states by induction with corresponding inducer molecules. The switch persisted in a stable state after the removal of inducer molecules, which confirmed the epigenetic bistability of our system. NEW Cells could switch from one state to the other by the addition of the second inducer, which was confirmed by three methods.

Bistable genetic switch based on non-cooperative elements

Mathematical analysis of genetic switches from the literature indicates that cooperativity, which introduces a nonlinear response, is required for a functional bistable switch, consisting of two mutual repressors (Cherry et al. 2000). Macia et al. (2009) and Widder et al. (2009) proposed that bistability could be introduced by non-cooperative elements, when nonlinearity is introduced for exampleif protein A is able to repress the transcription of protein B and at the same time activate its own transcription and vice versa (Figure 1).

Figure 1. Macia et al. (2009) and Widder et al. (2009) proposed a mathematical solution for creation of a bistable switch that would not require the cooperative binding of transcriptional regulators. According to the proposed model, transcription factor A should activate its own transcription and repress the transcription of gene B and vice versa.

We, as molecular biologists, werenot aware of a transcriptional effector or effector pair that could act simultaneously as a repressor and activator, therefore we were not surprised that, to our knowledge, this type of bistable switch has not yet been experimentally implemented.

The crucial moment in our project was when we realized that the ability to design TAL repressors and activators directed against the same binding site could offer a solution to this problem and provide a unique opportunity to construct orthogonal bistable switches based on noncooperative elements (Figure 2).

Figure 2. A repressor (left) and an activator (right) competing for the same binding site.

Results

Design

We designed an upgraded mutual repressor switch, introducing two additional positive feedback loops (Figure 3), consisting of two TAL activators targeted against the same binding sites as apair of mutual TAL repressors. In other words, rather than having the same protein function as an activator and repressor, we used an activator and repressor pair, competing for the same operator. The same binding sequence for the activator and repressor introduced nonlinearity required for the bistability based oncompetition for the binding site. For the purpose of our project, we designed a bistable switch with a positive feedback loop, capable of switching between the two states through regulation by inducer molecules (Figure 4).

Figure 3. Scheme of the genetic circuit for the bistable toggle switch composed of a pair of mutual repressors and a pair of autoactivators under the control of two operators.

Figure 4. Designed states of the bistable toggle switch with a positive feedback loop. (A) Addition of inducer 1 triggers dissociation of its cognate repressor from its DNA-binding site. As a consequence, repressor A and activator B are transcribed. Activator B further activates transcription of repressor A and itself, which forms the positive feedback loop of the switch state one. Meanwhile repressor A inhibits the other state of the switch. (B) When the inducer is removed, the inducible repressor binds back to its DNA-binding site, but since activator B is still present, autoactivation is achieved, resulting in a stable state. (C) and (D) depict the switch switched to state two and the function of its other positive feedback loop.

Modeling

Before proceeding with experimental verification, we performed a thorough modeling analysis of the designed switch. We incorporated parameters obtained from the repression and activation experimental results into our simulations. We also implemented a new modular, hybrid modeling algorithm that introduced stochasticity into an otherwise deterministic approach and enabled us to explicitly model competitive transcription factor binding and a limited number of binding site repeats. Modeling is described in details in the modeling section. Both deterministic and stochastic simulations demonstrate that the positive feedback loop switch is significantly more stable than the mutual repressor switch. Most importantly, it can exhibit bistability even without cooperativity (Figure 5). This switch is also more robust than the simple mutual repressor switch in regard to leaky expression.

Figure 5. Robustness of the positive feedback loop switch. The positive feedback loop switch exhibited bistability even in the absence of cooperativity and for the minimal promoter's leaky transcription rate of 10% or constitutive promoter leaky transcription rate of 5%. In comparison, the same leaky transcription rate (with equal values of all other parameters) of 5% caused the mutual repressor switch (without the positive feedback loops) to exhibit no bistability unless high cooperativity (2 or more) was introduced into the model.

Construction and experimental testing of the bistability of the switch

For experimental implementation of the positive feedback loop toggle switch we introduced the following components into cells (Figure 5): a pair of TAL:KRAB repressors (TALA and TALB), controlled by the opposite TAL (TALA controls the transcription of TALB and TALB controls the transcription of TALA), exactly as in the mutual repressor switch a pair of TAL:VP16 activators (TALA and TALB), each activating its own transcription and transcription of the opposite TAL repressor two of the constructs were tagged with fluorescent reporter proteins (BFP and mCitrine) via a t2A sequence, which ensured the equimolar production of the fluorescent reporter and TAL regulator Both TAL repressor and activator pairs (A and B), controlled by inducible repressors Constitutively expressed inducible repressor constructs Inducer molecules (pristinamycin and erythromycin)

Figure 5. Functional components (operons and inducers) of the positive feedback loop switch. .

We analyzed the performance of the switch by using two fluorescent proteins with good spectral separation (BFP and mCitrine), which enabled easy detection and quantification by confocal microscopy and flow cytometry.

To analyze the bistability we first used flow cytometry, a technique which allowed us to detemine the number of cells expressing either one or both of the fluorescent reporter proteins. Although cells were transfected with the complete switch device including both reporters, the analysis of cells demonstrated clear bimodal distribution - the majority of the analyzed noninduced cells expressed only one of the two fluorescent proteins, their selection probably resulting from stochastic events or due to a slight imbalance of the amount of transfected plasmids (Figure 6A). This bimodal distribution of fluorescence clearly demonstrates the intrinsic bistabilty of our system in comparison to the mutual repressor switch (classical toggle) topology, where a large fraction of cells expressed both fluorescent reporters. The addition of either one of the inducers switched the reporter production towards the corresponding fluorescent protein (Figures 6B and 6C).

Figure 6. The bistable switch with a positive feedback loop exhibits a bimodal distribution of fluorescence. (A) Non-induced HEK293 cells transfected with plasmids forming the switch (see Figure 5) transcribe either BFP or mCitrine as determined by flow cytometry. (B) HEK293T with the switch plasmids induced with erythromycin (2 mg/L) express mainly mCitrine and (C) cells induced with pristinamycin (2 mg/L) express BFP. Samples were analysed five days after induction.

Confocal microscopy confirmed high expression of the expected and no expression of the opposite fluorescent reporter protein in both induced states (Figure 7). This means the addition of an inducer shifts cells to a corresponding state which is preserved even when the inducer is removed (Figure 8). The system remained in a stable state several days after the removal of the signal, which further confirms the epigenetic bistability of our positive feedback loop switch.

Figure 7. Positive feedback loop switch exhibiting two different states at induction with pristinamycine (PI) and erythromycine (ER) inducer molecules. HEK293T cells were cotransfected with the following plasmids : PCMV_mCherry transfection control (20 ng), [A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, [B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 5 ng), PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16, PCMV_[ETR]_TALA:KRAB, PCMV_[ETR]_TALB:VP16, (all 10 ng), PCMV_PIP:KRAB, PCMV_E:KRAB (both 200 ng). Pristinamycine and erythromycine were added to final concentration of 2 µg/ml. Fluorescence was measured 3 days after induction.

Figure 8. Positive feedback loop switch exhibiting stable states at removal of inducer molecules. HEK293T cells were cotransfected with the following plasmids: PCMV_mCherry (20 ng), [A]_PMIN_TALB:KRAB, [A]_PMIN_TALA:VP16_t2A_BFP, [B]_PMIN_TALA:KRAB_t2A_mCitrine, [B]_PMIN_TALB:VP16, (all 5 ng), PCMV_[PIR]_TALB:KRAB, PCMV_[PIR]_TALA:VP16, PCMV_[ETR]_TALA:KRAB, PCMV_[ETR]_TALB:VP16, (all 10 ng), PCMV_PIP:KRAB, PCMV_E:KRAB (both 200 ng). Pristinamycine and erythromycine were added to final concentration of 2 µg/ml. Media was replaced 3 days after induction and fluorescence was measured 3 days after removal of inducers.

References

Cherry, J., L., Adler, F., R. (2000) How to make a Biological Switch. J. Theor. Biol. 203, 117-133.

Macía, J., Widder, S., Solé, R. (2009) Why are cellular switches Boolean? General conditions for multistable genetic circuits. J. Theor. Biol. 261, 126-135.

Widder, S., Macía, J., and Solé, R. (2009) Monomeric Bistability and the Role of Autoloops in Gene Regulation. PLoS ONE 4, e5399.

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