Team:OUC-China
Systems biology modeling
Weak promoter
In fermentation industry, the reduction of plasmid copy number is a headache. Once the engineering bacteria loses the plasmid, it can not only lead to a decrease in production but mutate into wild type. As the wild type have more energy to support growth and production, they will become the dominant species in the fermentation tank and replace the engineering bacteria.
This is also verified when we communicate with [the company]. After that we come up with an idea that knocking out PFK in the genome of Aureobasidium melanogenum P16 as it is an essential gene to support the growth and placing it on a plasmid with a weak promoter inserted into its front end. Utilizing this, theoretically, the engineering bacteria can only survive when the plasmid copy number reaches a certain value, otherwise it will die. Therefore we decide to simulate this by systems modeling[1].
Hypothesis:
1.The transcription factor content is sufficient, it will not happen to the competition problems due to the increased promoter number.
2. Ignore the promoter leakage.
3. If the promoter is an inducible promoter, ignore the problem of the inducer content and consider it is sufficient.
A_10957 stands for the weak promtoter
Through simulations, it is founded that when the copy number reaches 4(fig1), the engineering bacteria can survive, otherwise it will die, which is enough to prove that our conjecture is correct.
Given that, there are a lot of weak promoter in genome, some others are also tested to present that not all the promoter is suitable for this part. Therefore, select two another promoters to simulate this process again. If the strength of the promoter is relatively large, its sensitivity is not good, on the contrary, its sensitivity is very high, but it is easy to cause manslaughter(fig2).
As in fig2, it is concluded that, depending on the need, promoters of different expression strengths can be selected as weak expression promoters.
Quorum sensing system
The expression of the SSRE promoter did change after adding different concentrations of IP, it can be concluded that this quorum sensing system presented in Arabidopsis thaliana is presented not only in yeast but also in Aureobasidium melanogenum P16(fig3).
Therefore, the changes and relationship between the components in the system is necessary, and systems modeling is utilized to simulation this process.
Hypothesis:
1. The engineered bacteria grow according to the normal growth curve in the fermentation tank.
2. The three proteins that play a role are highly utilized
3. In the quorum sensing system, another circuit affected by the Skn7 protein has a small and negligible effect on this circuit.
Interpretation of result
Firstly, SSRE promoter we are using is analyzed, as shown in the fig(4) is a graph of the changes in the proteins required by the system.
Because the plasmid copy number remains very high in the system, the copy number of SSRE promoter also increase. Increasing the number of promoter and analyzing again, it is found that only the final acting Skn7_P protein always decrease, while the others would basically remain at the original level(fig5).
Given that there are other subtypes of SSRE promoters that have a lower or higher range for expression scale, their cases are also analyzed. The subtypes of SSRE promoter can be regarded as mutants of the SSRE promoter, which can be regarded as one promoter changing some parameters on the original SSRE promoter. Therefore, changing the different parameters of the SSRE promoter and simulate the intracellular action of different SSRE promoter subtypes.
1.Changing the binding ability with Skn7_P protein
First, alter the ability of the SSRE promoter binding to Skn7_P proteins and the result is that GFP expression is almost unchanged(fig6). It is concluded that the subtypes of the SSRE promoter is not changing in the binding capacity.
2.Changing the dissociation rate
Based on Figure 6, there is no effect on changing the binding capacity, so adjust the dissociation rate between SSRE promoter and Skn7_P protein to analyze again. When the dissociation rate increases, the expression decreases significantly(fig7_A), the dissociation rate decreases, and the expression also increases(fig7_B).
It can be seen here that the subtypes of SSRE reduce the dissociation rate to bind more Skn7_P protein, thus activate the promoter for transcription more efficiently.
3.Changing the ability of transcription
Of course, it may also be caused by the transcription capacity of different SSRE promoters, so adjust the transcription capacity of the SSRE promoter to simulate the system(fig8).
It is the expression of the promoter with different transcription capacity. It is obvious that, the stronger capacity, the stronger expression strength.
However, it is also possible that the two parameters change at the same time, so the two parameters are adjusted at the same time(fig10).From fig10, it is clearly analyzed that transcriptional capacity has a greater impact on the amount of expression
Then simulate the changes of the three phosphorylated proteins after changing different promoters(fig11).
From fig11, it can be seen that the more SSRE promoter production increases, the more phosphorylated SKn7 protein are utilize.
Throughout the system, the plasmid copy number remains relatively high, so it is also necessary to simulate the performance of different SSRE promoters at different plasmid copy numbers(fig12).
From Fig(12), the expression of different subtypes of SSRE promoters do not increase significantly when the copy number reaches two. This presents that when the number of engineering bacteria in the fermentation tank has not reached a certain level, the expression of foreign genes will be very low. This ensures that engineering bacteria are able to compete with wild type in fermentation tank.
RNA transporter, riboswitch, hardware
Due to the epidemic situation, our partner NJU-China did not have enough time verify the performance of RNA transporter by exosome. But we want to know the interaction between our system and our hardware. Systems modelling is utilized to simulate the interaction between them.
Hypothesis:
1.Engineering bacteria in fermentation tanks grow according to the normal fermentation curve.
2.RNA transporters and hardware performance are stable
The reaction can be described as follows.
As the three elements affect each other so much, to a certain extent, they can be treated as a small system, so they should be analyzed together. But it is still necessary to analyze the performance of each component
1.RNA transporter
To analyze the performance of RNA transporter, it is to analyze the binding alibility of RNA transporter. Changing the binding ability of the RNA transporter to visualize the content of the product at different locations in the system(fig13).
According to fig13, it is concluded that as the binding capacity increases, the content of products transported out of the cell also increases. However the content in cell is always higher than that out cell.
2.Riboswitch
There are only two factors that affect the action of the riboswitch, one is the concentration of intracellular products, and the other is the binding capacity of riboswitch to the product. As in the fermentation process, it can be approximated that the products in the engineering bacteria are accumulated from scratch, and the changes in the amount of products in the cell are similar without considering the addition. Therefore, during the simulation analysis, changing the binding capacity to visualize the changes of the YopE protein content and riboswitch-ligand complex(fig14).
From Figure 14, it is presented that sensitive riboswitch can greatly reduce the possibility of manslaughter as it can function even if the concentration of the product is extremely low.
The original intention of adding riboswitch elements is to eliminate low-yield engineered bacteria, so it is also necessary to simulate the role of riboswitch switches in engineering bacteria at different production levels(fig15).
It is a strong support that with the level of fermentation increasing, the expression of YopE protein receives a significant inhibition which means that more riboswitch-ligand complex generates.
As the copy number of plasmids remains high in CHIP system, it is vital to simulate YopE protein expression at different copy numbers(fig16).
It is shown in fig16, with the increase of the copy number, the YopE protein expression increases a lot which may cause manslaughter.
As the main reason for this process is that in the early stage of fermentation, the concentration of intracellular products is very low, so it leads to the accumulation of YopE protein. To deal with this problem, we intend to artificially add a certain concentration of the product to the fermentation tank at the beginning of fermentation, so that the product can be transported to the cell by the RNA transporter, thereby inhibiting the expression of YopE protein to a certain extent and reducing the risk of manslaughter(fig17).
From fig17, it is concluded that, adding products into fermentation tank can inhibit the expression of YopE(K_bind = 0,it stands that the engeneeing bacteria without the riboswitch, the YopE protein can expression freely.K_bind=0.3, it stands the result of the normal riboswitch)
In summary, it can be seen that there are many factors that affect CHIP components, and different combinations of components will change more. Through the above simulation results, the optimal circuit can be analyzed to meet the fermentation requirements, and the performance of a certain component can be changed to control the circuit to a certain extent. Our software was developed based on this to give the optimal solution by simulating the situation of the CHIP component under different component combinations.
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References arrow_downward
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- Hilborn, R.C., Brookshire, B., Mattingly, J., Purushotham, A. and Sharma, A. (2012). The Transition between Stochastic and Deterministic Behavior in an Excitable Gene Circuit. PLoS ONE, 7(4), p.e34536.
