Team:XHD-Wuhan-B-China/Model
Model
At the beginning of our project, we discussed with team HZAU-China about two-component system since our projects were both based on TCSs. As a result, we decided to change CcaR expression level to optimize the fold-change between green/red light induction. A series of promoters: J23109, J23117, J23114, J23115 were used to control the transcription rate of ccaR mRNA. An eGFP gene is used to report the expression level under the regulation of PcpcG2-172/68 promoters.
Figure 1. (A)Circuits containing PcpcG2-68. (B)Fluorescence under red light or green light for 5 hours. (C)Circuits containing PcpcG2-172. (D)Fluorescence under red light or green light for 5 hours.
After analyzing the result, we found that as the transcription level of ccaR rises, the fold-change between green/red light induction will first rise then decline. To interpret this phenomenon, we decided to model this process by kinetic equations.
Figure 2. Fold-change of green light/red light, where 109, 117, 114 and 115 are promoters arranged in an order of increasing transcriptional intensity of ccaR.
We constructed a model based on the following schematic diagram:
Figure 3. Schematic diagram of CcaS-CcaR system.
Where Sg represents CcaS, Sa represents CcaS-P of phosphorylation state. R represents CcaR, CcaR production rate is Kr, and Rp represents phosphorylated CcaR-P. The mutual conversion rate of R and Rp is determined by Kp1 and Kp2, and β represents the maximum binding initiation of the light-controlled promoter. mRNA represents transcribed RNA, and mRNA leaks at a certain rate and is degraded at the rate of Kdacay. The protein translation rate is determined by Ktrans. GFP stands for GFP produced by translation.
Specific parameter values that we imported from a model characterizing Cph8-OmpR (developed by replacing the sensor protein of the EnvZ-OmpR osmoregulatory system, which is one of the best-characterized TCSs) [1] are shown in the table below:
Table 1. Imported parameter values.
Based on the schematic diagram, we have following equations:
Under the interference of natural light in the experimental environment[2], define the ratio of CcaS-P to total CcaS under red light conditions, y is 0.04, and define y under green light conditions as 1. By changing the rate of CcaR generation Kr in the range of 0~0.001, and simulate the ratio of green light: red light when the system under each CcaR reaches a steady state by kinetics, we have the following result:
Figure 4. Simulated fold-change curve.
Combining the results of modeling and experiment, we can see that the tendency of fold-change matches the experimental data, where 109, 117, 114 and 115 are promoters arranged in an order of increasing transcriptional intensity of ccaR.
Figure 5. Fold-change curve fitting experimental data.
After analyzing the outcomes of this model, we can predict that between the current expression level of 109 and 117, there is a peak of fold-change yet to be achieved for both 68 and 172. In this regard, we designed a series of new circuits having various RBS strengths which can provide us with subdivided output of CcaR. The result of this prediction was rather successful, we found combinations of promoters and RBSs with higher numbers of fold-change in both 68 and 172. Please view https://2021.igem.org/Team:XHD-Wuhan-B-China/Improve for more details.
With the success of finding higher fold-change, we completed the cycle of experiment-modeling-redesign-new experiment: The modeling was instructed by experimental data and then lead us to a more refined design of circuits.
References
[1] Akifumi Nishida, Ryoji Sekine, Daisuke Kiga, and Masayuki Yamamura. 2016. High-frequency Noise Attenuation of a Two-component System Responding to Short-pulse Input. In Proceedings of the 7th International Conference on Computational Systems-Biology and Bioinformatics (CSBio '16). Association for Computing Machinery, New York, NY, USA, 28–35.
[2] Olson EJ, Tzouanas CN, Tabor JJ. A photoconversion model for full spectral programming and multiplexing of optogenetic systems. Mol Syst Biol. 2017 Apr 24;13(4):926.
[1] Akifumi Nishida, Ryoji Sekine, Daisuke Kiga, and Masayuki Yamamura. 2016. High-frequency Noise Attenuation of a Two-component System Responding to Short-pulse Input. In Proceedings of the 7th International Conference on Computational Systems-Biology and Bioinformatics (CSBio '16). Association for Computing Machinery, New York, NY, USA, 28–35.
[2] Olson EJ, Tzouanas CN, Tabor JJ. A photoconversion model for full spectral programming and multiplexing of optogenetic systems. Mol Syst Biol. 2017 Apr 24;13(4):926.