A genetic bistable switch utilizing nonlinear protein degradation

Background Bistability is a fundamental property in engineered and natural systems, conferring the ability to switch and retain states. Synthetic bistable switches in prokaryotes have mainly utilized transcriptional components in their construction. Using both transcriptional and enzymatic components, creating a hybrid system, allows for wider bistable parameter ranges in a circuit. Results In this paper, we demonstrate a tunable family of hybrid bistable switches in E. coli using both transcriptional components and an enzymatic component. The design contains two linked positive feedback loops. The first loop utilizes the lambda repressor, CI, and the second positive feedback loop incorporates the Lon protease found in Mesoplasma florum (mf-Lon). We experimentally tested for bistable behavior in exponential growth phase, and found that our hybrid bistable switch was able to retain its state in the absence of an input signal throughout 40 cycles of cell division. We also tested the transient behavior of our switch and found that switching speeds can be tuned by changing the expression rate of mf-Lon. Conclusions To our knowledge, this work demonstrates the first use of dynamic expression of an orthogonal and heterologous protease to tune a nonlinear protein degradation circuit. The hybrid switch is potentially a more robust and tunable topology for use in prokaryotic systems.


Parameters and Variables
It should be noted the degradation rate of all proteins in TS is the doubling time of the cell, indicating the absence of ssrA degradation tags.

Figure S.1 -Bistable TS
The full transcriptional bistable switch consists of two promoters and three genes, and is based off of our bistable TES from figure 1 of the main paper. The repressor CI434 is used in place of mf-Lon. In order for CI434 to repress PRM, OR3 needs to be changed to a CI-434 binding site [32]. Just as in TES, the main positive feedback loop is the PRM-CI loop. The second loop consists of PLtetO-1 making CI434, repressing PRM.

Figure S.2 -Rate-balance plot of a bistable TS
Just like for the bistable TES, having only the PRM-CI positive feedback loop, bistabile behavior is not possible. Bistable behavior is conferred once the second positive feedback loop. In the degradative TES, the linear degradation of CI is changed into a nonlinear degradation to create a bistable condition (figure 1e). With the TS, bistability was created by adding more nonlinearity into the CI production rate (figure S.2, dashed line).

Figure S.5 -Flow cytometry of switch deactivation
This series of flow cytometry plots shows a time course for SW6 switching from the "on" to "off" state. GFP expression uses the PRM-GFP reporter. For all scatter plots, the vertical axis is GFP fluorescence measurement and the horizontal axis is the forward scattering measurement. For all histograms, the vertical axis represents the number of counts and the horizontal axis represents GFP fluorescence. During the "high" to "low" transition, usually we see a bimodal distribution representing both the "on" and "off" population. As time progresses, the "on" population shrinks and the "off" population grows. Instead of observing a bimodal distribution during the transition we see the whole "on" population move to an intermediate state, then move into the "off" state.

Figure S.6 -Flow cytometry with full induction by inducers
In these plots we observed the effect of adding inducers on PLtetO-1. In the plots above GFP is reported using the PLtetO-1-GFP reporter. When aTc is added we observed high expressions of GFP shing that PLtetO-1 is fully active (top two plots). When IPTG is added a bimodal population distribution was observed (bottom two plots). One population has no GFP expression, the other population shows that PLtetO-1 is partially active. This indicates that TetR is unable to completely shut off pLtetO-1.

Figure S.7 -Flow cytometry without added inducers
In these plots we observed the behavior of PLtetO-1 at steady-state. Once again the PLtetO-1-GFP reporter is used. When inducers are removed and cultures are allowed to come to steady-state a strains initially cultured overnight in aTc appear identical to strains initially cultured in IPTG.

S.4 Single Feedback Loop Bistable System
It may be possible to create a bistable system with only the PRM-CI positive feedback loop, and a constitutive expression of mf-Lon. This however would require the Michaelis constant for mf-Lon to be significantly smaller that the measured valued.