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Revision as of 16:10, 30 October 2017
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Abstracts:
One part of our aim of modeling is to verify the effect of signal molecules on the promoter and to make the biological expression process clearer and more organized. What’s more, the main purpose is to verify the feasibility of the experiments of expression group and guide the progress of the experiment more smooth. However, the expression of the gene line is relatively long, if the theory is wrong, and the mistakes are found after the experiment, there will be a great harm on the experiment schedule, and moreover, the change will be very difficult. Therefore, the feasibility of modeling validation before expression experiments can provide a theoretical basis for expression groups and provide guidance for the success of their subsequent experiments. In addition, the detection of biomolecules is usually difficult to achieve, because the amount of biomolecules in the human body is too small, so we considered using the double fluorescence system to achieve the amplification by mathematical model and detect the amount of material on . Therefore, we use the support of literature , use the model to verify the linear relationship between the GFP and RFP ratio, to verify that the dual fluorescence system can be used for our project detection link, which play a guiding experimental team successfully completed the role of the experiment.
Objective:
One part of our aim of modeling is to verify the effect of signal molecules on the promoter and to make the biological expression process clearer and more organized. What’s more, the main purpose is to verify the feasibility of the experiments of expression group and guide the progress of the experiment more smooth. However, the expression of the gene line is relatively long, if the theory is wrong, and the mistakes are found after the experiment, there will be a great harm on the experiment schedule, and moreover, the change will be very difficult. Therefore, the feasibility of modeling validation before expression experiments can provide a theoretical basis for expression groups and provide guidance for the success of their subsequent experiments. In addition, the detection of biomolecules is usually difficult to achieve, because the amount of biomolecules in the human body is too small, so we considered using the double fluorescence system to achieve the amplification by mathematical model and detect the amount of material on . Therefore, we use the support of literature , use the model to verify the linear relationship between the GFP and RFP ratio, to verify that the dual fluorescence system can be used for our project detection link, which play a guiding experimental team successfully completed the role of the experiment.
Introduction:
In this model, we made the following assumptions: (1) having constructed lysine-deficient Escherichia coli and obtained accurate experimental data (2) the promoter sequence unchanged after the inhibition of protein binding to the promoter. (3) the terminator can completely terminate the promoter (4) When the inhibitory protein is absent, the promoter can stably express red fluorescent protein (RFP) (5) don’t consider the expression of the leakage of gene lines.
The first part:
1.The gene line which produce green fluorescent
BIT_Figure_The gene line which produce green fluorescent
First, according to the gene line above, we can get the expression, transcription and degradation of the following genes. Among them, k1 is the producing rate of mRNAlacI, k-1 is the degrading rate of mRNAlacI, k2 is the producing rate of proteinlacI , k-2 is the degrading rate of proteinlacI :
BIT_Figure_Model 9
The expression formula of promoter activity was constructed by Hill equation. β1 is the maximum starting ability of the promoter (generally set to 1), M1 is the amount of leakage of the promoter, SluxR is the amount of regulation factor, kd is the SluxR value when the model change trend is changed:
BIT_Figure_Model 10
Finally, according to the differential equation to express the expression, transcription and degradation process of the whole gene line:
BIT_Figure_Model 12
ModelAccording to our gene line, it can be concluded that the amount of proteinlacI can be approximated as the amount of proteinGFP (green fluorescence).
BIT_Figure_Model 13
Among them, according to the database in the official website of igem , we can know the value of some parameters: the maximum capacity of start-up promoterβ1 = 0.6; the amount of the leakage of promoter M1 = 0; n1 = 2. So we put these known parameters into the equation and get the value of kd at equal distances: 50,100,150,200, finally draw a rough image:
BIT_Figure_The relationship between regulation factor and gene expression rate
From these differential equations, combining the gene line of the whole expression group,we can get the model of green fluorescent protein expression and get the conclusion: the concentration of lysine from the upstream is higher , the rate of survival of lysine-deficient Escherichia coli is higher , the fluorescence intensity of GFP is greater . And this is also consistent with the original intention of the team members who design this line diagram. We proved the feasibility of this gene line.
2.The gene line which produce red fluorescent
BIT_Figure_The gene line which produce red fluorescent
Without considering the inhibitory effect of protein lacI on the promoter pLac, according to the gene map of the above chart, imitating the green fluorescence generation process, we can get the expression, transcription and degradation of the following genes. Among them, k3 is the producing rate of mRNARFP, k-3 is the degrading rate of mRNARFP, k4 is the producing rate of proteinRFP , k-4 is the degrading rate of proteinRFP:
BIT_Figure_equ14
The expression formula of promoter activity was constructed by Hill equation. β2 is the maximum starting ability of the promoter (generally set to 1), M2 is the amount of leakage of the promoter, S is the amount of regulation factor, kd is the S value when the model change trend is changed:
BIT_Figure_equ15
Finally, according to the differential equation to express the expression, transcription and degradation process of the whole gene line:
BIT_Figure_equ16
From these differential equations, theoretically, the RFP expression rate is approximately the same as the expression rate of GFP, regardless of the effect of protein lacI on the expression of the promoter pLac. While taking the inhibition of protein lacI on the promoter pLac into account, from these differential equations, combining the gene line of the whole expression group,we can get the model of green fluorescent protein expression and get the conclusion: the concentration of lysine from the upstream is higher , the rate of survival of lysine-deficient Escherichia coli is higher , compared with GFP ,the fluorescence intensity of GFP is lower. And this is also consistent with the original intention of the team members who design this line diagram. We proved the feasibility of this gene line.
The following was the initial value of each dependent variable in the model.
BIT_Figure_The initial amount
Through the modeling of the first part, we could verify the feasibility of the gene line of the expression group and verify that the dual fluorescence system can really play the role of mathematical amplification in theoretically.
The second part
Dual fluorescence system
We introduced a gain factor, Fg, which could be used to quantify the change in fluorescence.
BIT_Figure_equ17
GFPs and RFPs represented GFP and RFP fluorescence measurements at the level of expression of the relative fluorescence units, whereas GFP0 and RFP0 represented the fluorescence measurements of the leaked expression. From the above conditions,GFP0/RFP0=1
Considering the dual fluorescence system in the expression will have a very complex side effects, affected by many factors, beyond our current level, so we decided to assuming that the linear relationship for first, then combined with our search of the literature data, using professional literature data prove that the dual fluorescence system linear relationship.
From the experimental data and the conclusion shown in the literature, it could be seen clearly that there is an obvious mathematical linear relationship between the two fluorescence systems.
BIT_Figure_Data BIT_Figure_ the dual fluorescence
It could be proved by the literature data that the dual fluorescence detection system was feasible and had a great amplification effect. During the next experiment, we could construct a feasible dual fluorescence detection system based on the literature.
Conclusion
According to the modeling of two parts, we could guarantee that the feasibility verification of gene line and the theory of dual fluorescence system, and then determine the relationship between output and input by simulating the expression of dual fluorescent system. Based on the modeling of two parts, we can also get this conclusion: The input signal was further linear amplified by the ratio of red and green fluorescent signal. So we succeeded in modeling, providing theoretical basis for the expression group, providing theoretical support for the purpose of our team's final testing, providing the necessary guidance for the success of their subsequent experiments.
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