Positive feedback loop switch

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.

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.

Construction and experimental testing of the bistability of the switch

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).

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.

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.