Team:BNU-China/Model3
Selection of Degradation Tags
Introduction
We want to create new DNA barcodes automatically by coupling CRISPR/Cas9 system with cell cycle. In order to degrade Cas9 timely, we planned to create a fusion protein of Cas9 and Clb2 protein, so that Cas9 can be degraded together with Clb2. Then we found in literature that the first 124 amino acids of Clb2 also has degradation ability. In order to evaluate their efficiency in promoting the degradation of Cas9 and choose the better one, we constructed models before experiments.
Models and Results
1.The full-length Clb2 protein
For the full-length Clb2 protein, we used model to evaluate its efficiency in degrading Cas9 based on the relevant data obtained from the literature review. We referred to the model of 2019 Team: Fudan-TSI. First, we simplified the model and carried out numerical calculation through dimensionless analysis to simulate the change of concentration with time in the protein degradation process.
It is known from the literature that the binding and dissociation rates of the degradation tags and binding sites differ by about an order of magnitude. Therefore, we set the relative rate to v_r=10. On this basis, we set ten parameters related to the degradation reaction process:
(PS: The symbols shown in the equations below represent the relative concentration of its corresponding substance in the table.)
Considering the combination and dissociation between the components, the following ordinary differential equations are given:
Through the qualitative simulation with initial given conditions, the images of all the following concentration of components with time are obtained. It should be noted that the unit of horizontal axis time in the figure is 20min, to facilitate calculation in the process of solving the equation.
Figure 1. Curve of relative concentration of each component over time
It can be seen that Cas9 fused to the full-length Clb2 degrades quickly before the components stabilize, which can not ensure the function of it. So we turn to the first 124 amino acids of clb2.
2.the First 124 Amino Acids of Clb2
For the first 124 amino acids of Clb2, as the relevant data can not be obtained from the literature, we chose to carry out experiments to calculate and verify the degradation rate first.
We combined the first 124 amino acids of Clb2 with GFP, getting them expressed by the inducible promoter GAL1, and induced their expression by galactose. The basic flow chart is shown below:
Figure 2. Flow chart of this model
The protein degradation rate is measured by observing the changing rate of fluorescence intensity. Our experiment provides a feasible idea for the prediction of the degradation rate of the first 124 amino acids of Clb2.
The detailed information of the parameter names are as follows:
The ODE equations describing this process are as follows:
We used Flow Cytometer to test the fluorescent intensity of protein by time and get the following curve.
Figure 3. Experimental fluorescence intensity with the change of time
After inducing the protein expression, the data from the experimental group showed that during degradation, the fluorescence intensity of the target protein has a decreasing trend. We fitted the following data by Matlab which the image trend conforms to the exponential function and obtains the following relation:
Figure 4. Fitting curve of the relationship between fluorescence intensity and time
The fitting of 
is close to 0.99 which indicates a good fitting result. To further get the relationship between protein degradation rate and time, we need to explore the relationship between fluorescence intensity and protein concentration first. Regarding the search results from BBa_E0040, we know that the relationship between fluorescence intensity (FT)(au) and protein concentration (
) (nM) is a direct proportion.
Finally, we obtained the function of the protein concentration ()(nM) by time(t) (h):
Figure 5. Fitting curve of the relationship between protein concentration and time
Due to the expression of the protein was suppressed completely in the data of experiment group, we think that:
Then, we obtained the relationship of protein degradation rate by time and got the constant of the average protein degradation rate:
That is to say the degradation rate of GFP fused to the first 124 amino acids of Clb2 is 0.298h-1.
Discussion
Comparing the degradation rate of full-length Clb2 with its first 124 amino acids, we think the degradation rate of the full-length protein is too fast. That is to say, when the concentration of each component is not stable, the relative concentration of Cas9 has been lower than 5%. In this case, Cas9 doesn't work well, which means using the full-length Clb2 is not suitable for our project.
Although the first 124 amino acids of Clb2 is relatively slow, it can maintain the working concentration of Cas9 in the desired time and accelerate its degradation to a certain extent when compared with full-length Clb2, so as to achieve the purpose of coupling its function with the cell cycle.
Therefore, we chose the first 124 amino acids of Clb2 as our degradation tag.
References
[1] Ringrose, L., Lounnas, V., Ehrlich, L., Buchholz, F., Wade, R., & Stewart, A. F. (1998). Comparative kinetic analysis of FLP and cre recombinases: mathematical models for DNA binding and recombination. Journal of molecular biology, 284(2), 363–384.
Journal of Molecular Biology, 1998, 284(2):0-384.
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