Team:Tianjin/Demonstrate
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DEMONSTRATE
Overview
On this page, you will see the figures of our experimental results. We used the Hitachi F-2500 Flourescence Spectrophotometer and the Leica DMi8 inverted microscope to detect Fluorescent proteins and used the Thermoscientific Luminoskan Microplate Reader to detect Luciferases. Through a series of experiments, we obtained a large amount of experimental data, and obtained the oscillation curves through data processing. The experimental results show that our reconstructed KaiABC system runs successfully.
Result analysis
1. Experiments using the interaction of KaiC and SasA, and between KaiC and CikA
By timing sampling and testing, we have obtained a series of final results, which included the experimental group, the control group, and the experiment minus the control group. The result charts of the experiment minus the control group and corresponding result analysis are shown below. (For other complete result charts, please click on the measurement page).
Specifically, for instance, the sample named as (pABaC+pCiRbS+p1F)-(pCiRbS+p1F) means that the data is derived from the results of subtracting corresponding strains as control group containing two plasmids, pCiRbS and p1F, from strains as experimental group containing three kinds of plasmids, pABaC, pCiRbS (to reconstruct KaiABC system) and p1F (to report). The same is true for others.
Figure1. interaction of KaiC and SasA or KaiC and CikaA with p1F
Oscillation analysis with 1F(Gal1 promoter +Fluc)
1. In the measured 3500 min, the oscillations in 700-2000 min are extremely unstable and nearly absent, while the oscillations for the rest of the time are relatively stable.
2. The cycle is about 500min (about 8.3h).
3. The amplitude changes greatly, and there is no fixed value. In comparison, the yeast cell strain containing pbCiRS has a slightly smaller amplitude change than the strain containing pCiRbS.
Figure2. interaction of KaiC and SasA/CikaA with p1N
Oscillation analysis with 1N(Gal1 promoter +Nanoluc)
1. In the measured 4200min, the overall oscillation trend is relatively good. However, to be specific, the oscillation of yeast cell strain containing pCiRbS is somewhat unstable during the period of 1600-2200min and 3000-3500min, while the strain containing pbCiRS is unstable in the 3000-3500min.
2. The period of oscillation of the yeast cell strains containing pCiRbS is about 550 min, which is slightly floating. The variation of the oscillation period of yeast cell strains containing pbCiRS is comparatively large, swinging between 400min and 700min.
3. The amplitude changes greatly, and there is no fixed value. In comparison, the yeast cell strain containing pbCiRS has a slightly smaller amplitude change than the strain containing pCiRbS.
Figure3. interaction of KaiC and SasA/CikaA with p2N
Oscillation analysis with 2N(Gal1 promoter +Nanoluc reporter gene)
1. The overall oscillation trend is good and it has been oscillating persistently for the measured time.
2. On the whole, the oscillation period of the yeast cell strains, which are introduced with plasmid containing Gal2 promoter, is a little less stable compared to the oscillation period of the yeast cell strains containing Gal1 promoter. Talking of the strain containing pCiRbS, the period of oscillation in 0-1500 min is about 400 min, while the period of oscillation in 1500-3200 min is about 700 min and the period of oscillation after 3200 min is reduced to about 500 min. In the sametime, the period of oscillation of yeast cell strain containing pbCiRS also changes greatly, varying between 400-600 min, but the period becomes stable when the measurement has passed 2900 min.
3. The amplitudes of both strains are unstable and vary greatly, while the oscillation of yeast cell strain containing pCiRbS is slightly more stable in comparison with that of strain containing pbCiRS.
2. Experiment using the interaction between KaiC and KaiB
In this section, since the magnitude of the change in the experimental group data is not significantly different from the magnitude of the change in the control group data, we did not treat the data in this group similarly to the above results.
Figure4. (paBAS+pbCRCi+p1F) and (pbCRCi+p1F)
Oscillation analysis
1.There was no significant difference between the experimental group and the control group, indicating that the reporter gene was not stably expressed and the oscillation system was not successfully reconstructed.
Analysis of results based on experiments
1. Experiments using the interaction of KaiC and SasA, or KaiC and CikA
(1)Comparing the strains controlled by Gal1 promoter, the yeast cell strains containing pbCiRS are more stable than the strains with pCiRbS, indicating that the recombination of AD-KaiC (KaiC fused with the activation domain of Gal4 ) and BD-CikA (CikA fused with the DNA binding domain of Gal4)is better than that of AD-KaiC and BD-SasA (SasA fused with the DNA binding domain of Gal4)from the oscillation amplitude perspective. Possible causes are analyzed as follows:
● The TEF1P used is more stable than the TDH3P.
● The combination of KaiC and CikA is more stable than the combination of KaiC and SasA.
(2)In the comparison of the two kind promoters, the periods of oscillation of the yeast cell strains containing the Gal1 promoter are more stable than those controlled by Gal2 promoter. Possible causes are analyzed as follows:
● The two-hybrid system has different start-up efficiencies for the two Gal promoters, resulting in periodic fluctuations in oscillation.
(3)The resulting oscillation periods in our experiment are much less than the 24 hours of cyanobacterial oscillation as we expected before. Possible causes are analyzed as follows:
● The system we constructed in yeast cells is different from the oscillating system in cyanobacteria, resulting in the variation of proteins expression ratio and expression level. More specifically, the concentration ratio of these three core proteins, KaiA, KaiB and KaiC, is 1:1:4 (by weight) according to previous studies, but what strength ratio of constitutive promoters we have respectively used in the three cassettes of KaiA, KaiB and KaiC, is about 1:1.4:1(by amount of substance).
● The physicochemical environment in yeast cells differs from that in cyanobacteria, affecting the physiological function of KaiC protein, thus the period of oscillation changes.
● The fusion of KaiC and AD affects the original structure and function of KaiC, which may affect the oscillation period.
● The KaiC gene might be mutated during the process of reconstituting the KaiABC system, resulting in the formation of a mutant of the KaiC protein, thereby affecting the oscillation cycle.
● Due to the diversity of the redox environment and the metabolic pathway inside the cells, the content and change trend of ATP in yeast are different from those in cyanobacteria, which affects the oscillation period.
(4)The period and amplitude of our oscillating system are unstable, especially the amplitude.Possible causes are analyzed as follows:
● For one thing, no TTFL(transcriptional/translational feedback loop) is introduced when reconstituting the oscillating system in yeast, for another the environment of yeast oscillation is also different from the stable condition in vitro. As a result, the proteins in living cells are continuously degraded and formed, thus the content and proportion of KaiA, KaiB and KaiC proteins change with the variation of cell state, resulting in its’ oscillation period and amplitude change.
● The effect of protein content and ratio on the amplitude of the oscillation is greater than the effect on the period, so the amplitude change is more significant in this experiment.
(5)In the comparison of the oscillations of yeast cell strains containing different reporter genes, the oscillation description of strains containing 1N is best.
2. Experiment using the interaction between KaiC and KaiB
The reporter gene of the strain containing the paBAS, pbCRCi and p1F was not successfully activated. Possible causes are analyzed as follows:
● KaiB needs to undergo an allosteric process before binding to KaiC. The construction of the fusion protein may affect the variant of KaiB, which affects the binding of KaiB to KaiC, resulting in failure of the downstream promoter.[7]
Analysis of results based on modeling
We propose some assumptions in combination with the model based on our experimental results (If you want to see detailed modeling information, click on the modeling page.)
(1)Temperature: We suspect that there is a large temperature difference between yeast and cyanobacteria, so temperature becomes an influencing factor. According to the model, we know that an increase in temperature will result in a significant period reduction and will cause the oscillation attenuation. According to previous studies, we know that there is temperature compensation in the cyanobacteria, and the period of cyanobacteria oscillation does not change with temperature.[8] Here we suspect that there are some proteins or other components in cyanobacteria that correct the temperature change. In the course of the experiment, we did not synchronously transduce them into the yeast, so the above experimental results were formed. The existence of this mechanism is the direction of our further discussion.
(2)Phosphorylation: We have demonstrated through model tests that the oscillations decay rapidly with accelerated phosphorylation. Therefore, we have two conjectures: one is that phosphorylase in yeast plays a better role in promoting phosphorylation, and the other is that yeast provides sufficient or excess ATP/ADP to accelerate the rate of phosphorylation. This direction is also the focus of our further research.
(3)Concentrations of KaiA, KaiB and KaiC: Unlike the envisaged results, the concentrations of KaiA, KaiB and KaiC did not have much effect on our model in the testing of mathematical models.
Summary
We reconstructed the KaiABC circadian clock system derived from Synechococcus elongatus in yeast, and used the yeast two-hybrid system to initiate the reporter gene to characterize the oscillation results of the core proteins. We utilized a variety of core protein pairs to construct the yeast two-hybrid system. The yeast two-hybrid system driven by the combination of KaiC-SasA and KaiC-CikA successfully characterized the results of periodic oscillations. The above results indicate that the KaiABC oscillating system we constructed in eukaryotic yeast has successfully run. On this basis, we simulated and analyzed the reconstructed KaiABC oscillation through the model, and we inferred the possible factors affecting the oscillation period and oscillation attenuation, and explained the cause of the shape of the experimental curve successfully.