Team:BNU-China/tem
We create new DNA barcodes automatically by getting CRISPR/Cas9 system to work during the G2-G1 phase of the cell cycle. Our experiments verify that the combination of the CLB2 promoter and Cas9 could couple the Cas9 expression with the cell cycle. What’s more, we also have to consider the Cas9 degradation rate, which needs to be degraded before the early G1 phase to ensure that the parental and offspring barcodes are different.
Initially, there were two options for our degradation label. One was to select the complete CLB2 protein, and the other was a part of CLB2 (the first 124 amino acids). What changes directly in both cases is the efficiency of protein degradation, but it is not the only determinant. Therefore, we conducted simulation through the model and experimental data to select an appropriate degradation label.
1.The complete CLB2 protein
For the complete CLB2 protein, we used the model to calculate its degradation rate 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 
. 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 the half-life of CLB2 is about 30 minutes. However, the degradation rate of the whole length is fast, which has serious disadvantages. 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.
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.
To observe the degradation rate of the 124 amino acids more directly, we determined to turn to model.
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 target protein.
The detailed information of the parameter name is 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 the 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 experimental groups, we think that:
Then, we obtained the relationship of protein degradation rate by time and got the constant of the average protein degradation rate:
Comparing the total length of CLB2 with its 124 amino acids, although the degradation rate of CLB2 124 amino acids is relatively slow, it can maintain the working concentration of Cas9 in the desired time and accelerate its degradation to a certain extent, so as to achieve the purpose of coupling its function with the cell cycle. Therefore, we chose 124 amino acids of clb2 as our degradation label.
References
Ringrose L , Lounnas V , Ehrlich L , et al. Comparative kinetic analysis of FLP and cre recombinases: mathematical models for DNA binding and recombination[J].
Journal of Molecular Biology, 1998, 284(2):0-384.
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