We began by comparing CFP/YFP ratios from circuits containing an aromatically tuned variant to the circuit containing the untuned EnvZ-V5 (left panels in Figure 5).
Here, for each aromatically tuned variant, a different relationship between steady-state signal output and receptor level was observed, apparent as a change in the slope of the CFP/YFP trendlines that are summarized in Table S1, suggesting that the ratio of kinase to phosphatase activities was different within each receptor and always different than wild-type EnvZ.
Those containing the WLF-4 (m = −90), WLF-3 (m = −18), and WLF-1 (m = −91) variants exhibited a sharp decrease in YFP fluorescence as increasing levels of the tuned variant were present.
Our experiments show that SATenstein solvers achieved dramatic performance improvements as compared to the previous state of the art in SLS algorithms; for many benchmark distributions, our new solvers also significantly outperformed all automatically tuned variants of previous state-of-the-art algorithms.
Data for the WYA (Trp-Tyr-Ala) series of aromatically tuned variants.
During our analysis, we assessed whether osmosensing circuits containing the aromatically tuned variants possessed normal levels of CFP fluorescence, YFP fluorescence, and CFP/YFP ratios.
We also observed that circuits containing several tuned variants appeared to be sensitive to the level of EnvZ-V5 present under the low osmolarity regime.
We also noted that circuits containing aromatically tuned variants became more sensitive to changes in the receptor concentration than their wild-type counterpart, suggesting a new way to study mechanisms underpinning receptor concentration-dependent robustness.
It is noteworthy that circuits containing the aromatically tuned variants of EnvZ-V5 did not show the expected decrease in CFP/YFP at higher levels of EnvZ-V5; therefore, all data points above EnvZ-V5 levels of 0.1 were included during calculation of subsequent trendlines.
Increased sensitivity to changes in SHK levels was seen for all osmosensing circuits containing an aromatically tuned EnvZ variant.
In addition, the majority of aromatically tuned EnvZ variants retain the ability to respond to stimulus, suggesting that their signal output is biased but not locked in either a stimulus-deprived or -saturated conformation.
