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.

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

We analyzed the performance of the switch by using two fluorescent proteins with a sufficientspectral distance (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 has a unique ability to determine 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 transfected noninduced cells expressed only one of the two fluorescent proteins.The expression of one or the other reporter proteins is most likely a result of stochastic events or possibly a slight imbalance of the amount of transfected plasmids (Figure 7A).Importantly, a very low number of cells expressed both reporters. This bimodal distribution of fluorescence clearly demonstrates the intrinsic bistability 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 7B and 7C).

Confocal microscopy confirmed high expression of the expected and no expression of the opposite fluorescent reporter protein in both induced states (Figure 8). This means the addition of an inducer shifts cells to a corresponding state which is preserved even after the inducer has been removed (Figure 9). 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.

Switching between the two states NEW

The ultimate test of the bistable switch with a positive feedback loop functionality is to switch the system between the two states by the addition of the second inducer. This is a longer experiment as it requires the system to settle first into the initialselected state by the addition of the first inducer and then switch into the second stateafter the addition of the second inducer. Nevertheless we managed to demonstrate this functionality of our switch during the time between the regional and world jamboree using a secretory luciferase assay and fluorescence microscopy.

Results

After the induction of state I with pristinamycin we observed expression of reporter I, blue fluorescent protein BFP (Figure 10). Two days after the addition of pristinamycin it was removed and exchanged with erythromycin. We observed expression of the reporter mCitrine, an indicator of state II of the switch only one day after inducer exchange. We can still see some BFP emission in the next few days, due to a relatively long half-life, but it is clear that mCitrine expression prevails eventually.

We also demonstrated flip-flop using a cytosolic firefly luciferase, fLuc and a secreted alkaline phosphatas ( SEAP) as state I and state II reporters. A secretory Renilla luciferase was used for normalization. Cells were induced with either erythromycin (2 mg/L) to trigger expression of fLuc or with pristinamycin (2 mg/L) to trigger expression of SEAP. Inducer I was removed after one day and replaced with inducer II. Expression of reporters was analyzed 3 and 6 days after inducer exchange. We observed reduced expression of firefly luciferase and elevated expression of SEAP when pristinamycinwas replaced by erythromycin and vice versa when the cells were first stimulated with erythromycin and then withpristinamycin (Figure 11).

Our system has considerable inertia as first the transcriptional factors must be degraded as well as the reporter proteins.The degradation could be accelerated by the addition of PEST degradation signals, which we already anticipated and prepared parts with TAL regulators that include pest signals. These regulators exhibit increased fold induction but have not yet been tested within the switch context.

References

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

Cong, L., Zhou, R., Kuo, Y.C., Cunniff, M., Zhang, F.(2012) Comprehensive interrogation of natural TALE DNA-binding modules and transcriptional repressor domains.Nat Commun.3,968.

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.