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Designed Protein Degradation Method Based on

Trim21 And Nanobody              -- Deduction

Deduction

Phenotype

Having completed parameters simulation,we can now use our model to make assessments about GFP and ErbB3 with the confidence that we have maximized our potential to obtain insights that will be directly relevant to the use of our trim21-antibody system.

To investigate the relation between antibody phenotype and time as well as the concentration of plasmid dosage, we respectively introduced equations between the phenotype and the corresponding protein concentration into our model.

For gfp, the relationship between the general fluorescence intensity and the concentration of the fluorescent substance is300x200 . In the wetlab,as the concentration of gfp is counted in nanomole and the content is very low,it can be considered that the relationship at this time is in accordance with F=2.3ρIφξlc .At this time, the fluorescence intensity is proportional to the concentration of the fluorescent substance.Simultaneously, we obtained a large number of images of gfp fluorescence intensity at different time points through wetlab. These images were expressed(by imagej)to quantify their fluorescence intensity. Then, we use Office-Excel to process the acquired data. For example, reduce the noise that brought about by background brightness and contrast Finally, we used the previous construction to obtain the relationship between plasmid concentration and fluorescence intensity decay.Our data is uploaded here.
From the previous modeling we got the relationship between concentration of ErbB3 and protein degradation time. In an ideal situation, the cell proliferation rate is exponentially related to time y=a^kt+c. By taking the data we obtained from wetlab into the equation for fitting, we can get the relationship between cell proliferation rate and protein degradation time, and thus integrate.The final relationship between cell proliferation rate and concentration of ErbB3 was obtained.Finally,we got the equation is y=0.2949e^0.0715x
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This is an image of the relation between absorbance which can quantify cell proliferation and time

Sensitivity analysis

Sensitivity analysis is one of the commonly used methods for analyzing uncertainty in models,which identifies the sensitive factors that have great influences on the calculation results of the concentration of reactant in the mechanism from the multiple uncertain factors. By analyzing and measuring the degree of influence and sensitivity of the calculation results, we can offer some advice to wetlab.. If a small change in variable can cause a large change in the calculation result, the variable is called a sensitivity factor, otherwise it is called a non-sensitive factor. For our system, we used MATLAB for sensitivity analysis to get the influence of different factors on the calculation results. Then combined with our design, we draw some conclusions for our experiment:

1、The effect of each step on the whole is determined by the parameters. For example,a number of reaction rate is described by the law of mass action(r=kc),the reaction rate r is proportional to the reactant concentration c, the size of the parameter k determines the effect of the reactant on the speed of the step, thereby ascertaining the effect of the reaction on the whole)[Fig.1][Fig.2].

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[Fig.1]

[Fig.1]Full dedimentionalization

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[Fig.2]

[Fig.2]Half normalization relative to numerator--species quantify

2、The concentration of a certain reactant(such as enzyme or ub )will affect the overall reaction rate. According to the model, when the concentration of the reactant is very low, the reaction rate increases as the concentration of the reactant increases.[Fig.3] However, when the initial concentration of reactant exceeds a certain level, their overall impact will be reduced Our analysis believes that there is a constraint relationship between each step of the reaction. It is limited to increase the concentration of a certain reactant alone to accelerate the overall response , and other restrictive factors need to be considered.[Fig.4]

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[Fig.3]

Fig.3 This plot sets to observe the effect of an isocratic concentration changes on the degradation rate.

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[Fig.4]

Fig.4 Parameter sweeps at an isocratic concentration.

3、Factors affecting the sensitivity of various reactants.The reactions that the reactants actually participate also play a role,which determine the weight of the effect of the reactants in the whole process.For instance,Ub is involved in the whole process of ubiquitination of antigen-antibody complexes from being activated to being connected to the compound, while E1,E2 enzyme only participates in a certain step(E1 only acts on the activation of ub molecules and E2 only transfers the ub)[Fig.5]Therefore, the reaction rate is more sensitive to ub than to E1,E2.

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[Fig.5]

Fig.5 The picture shows how E1 acts on the activation of ub molecules and E2 transfers the ub

Application

From the model, we have obtained the relationship between protein degradation rate and time as well as the effect of different concentration of plasmid introduction. Further more, we use these relationships to determine the specific ratio of protein decline to the corresponding time, select representative state (e.g. the interval where the concentration changes dramatically , the point proteins degrade to a special ratio)as our checkpoint, to make sure that the system works as expected. In addition, the model also shows the effect of the introduced plasmid concentration on the rate of protein degradation and the equilibrium concentration of the final protein. For future experiments and practices, we can give recommendations for plasmid introduction by computer simulation, combined with experiments or patient requirements for protein degradation time and its equilibrium concentration.

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[Fig.1]

[Fig.1]Select checkpoints

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[Fig.2]

[Fig.2]Select The Import Quantity

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