Team:TU Darmstadt/Model/Kill Switch Modeling

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Introduction Model One Model Two Model Three

Introduction

This year we had to face several challenges. Due to the coronavirus outbreak and the resulting lockdown, we were not able to work in the wet lab and had to focus our work on the dry lab. Therefore, we decided to focus our resources in an all-round modelling effort. We used homology modeling to calculate a 3D structure of our laccase to improve our enzymes. The biofilm modeling simulated the growth of the biofilm and the third modelling branch was built around a MATLAB model of a kill switch, based upon the comXQPA system [1],[2]. The latter will be described in the following.
The kill switch model consists of three independent models which build upon each other. The first one describes the quorum sensing part, by simulating five individual cells and their interaction. The second one was built after the first model and describes one cell, to test different other parameters in the cell, whilst reducing its complexity. The third model improved the second one by describing two additional parameters.
The circumstances allowed us to put more time into refining and testing our models, so we were able to build a more in-depth model of the kill switch. We also hypothesized that a good working model may theoretically prove our kill switch. With matching data from the lab, the model should be able to precisely fine-tune the kill-switch.
Due to limited lab access, we opted for a new strategy and devised the work to a conceptual model of our kill switch with MATLAB SimBiology and MATLAB SimBiology Analyzer[3]. The models are based on ordinary differential equations which are solved with a numerical analyser. We developed three iterations of our models.
In the following text, we would like to show you the process of the three models we created.

Model One

Questions

1. Is it possible to simulate different cells in the model?
2. Is it realistic to model the quorum sensing part of our kill switch?
3. What rate of change values do we need to look for?

Assumptions

1. The pathways of the quorum-sensing system can be described as rate of changes
2. The reactions are simple mass-action reactions

Model description

In the first model, we focused on the quorum sensing part. For this purpose, we simulated five individual cells that share a common extracellular space.
First, the cells are equally producing ComX from pre-ComX and release it into the extracellular space. From there it can be transported back into the cell via unspecific membrane transporters. Inside of the cells, ComX activates ComP which phosphorylates ComA to ComA-P.

Goal

The goal of the first model was to experiment with MATLAB SimBiology and produce the first searchable rate of change values. It should be a starting point to refine the model. Also, we wanted to show that it is possible to simulate the quorum sensing part of our model.

Results and future development

This initial model helped us to confine first values and to gain an impression of how the software and the modeling works. Also we extrapolated one critical problem: The problem with this model is that the ComX is distributed in a uniform manner, which is unrealistic because the cells are neither equally distributed nor in an equal development stage. Due to the number of cells, there will be some that are more efficient than others. So, we decided to break it up and only simulate one cell. With lab access, it should be possible to determine the needed values by measuring the extracellular ComX concentration of different colonies or we could try to find the minimal number of cells so that the colony survives and the kill switch is not triggered.
For the further development of this model, it is essential to know how high the intracellular concentration of ComX must be in the cell to phosphorylate ComA. This task needs to be performed in the future, for instance by another iGEM team.

GraphicalAbstract
Figure 1: Here you can see a scheme of the first model. The green ellipses represent the species while the boxes represent the enzymes that catalyze the reaction. Along the black lines the reactions take place.

Model Two

Question

1. Where do the educts for pre-ComX and ComA come from?
2. Which rate of change values do we need for the Model?

Assumptions

1. The pathways of the quorum-sensing system can be described as rate of changes.
2. The model is built around mass-action equations.
3. One cell stands for an undefined cluster of cells.
4. The educts for the pre-ComX and ComA somehow are connected to the promoter we use.

Model description

To address the problems we encountered in the first model, we built a second one. The most fundamental change was that we did not simulate individual cells but rather one cell or more precise an undefined cluster of cells. Like the first model, pre-ComX is converted into ComX and then released into the extracellular space. Then it is transported back into the cell and acts as a cosubstrate for ComP. ComA is phosphorylated and activates the promoter in a new subunit. The new subunit describes the promoter and therefore the step after the phosphorylation.
Another question arose as to where the educts for the pre-ComX and ComA come from. Therefore, we linked the promoter subunit with unknown transition units, which synthesize the educts.

Goal

The goal of the model was to determine the missing values for the model and to prove that it is working, as well as to get values to compare with real lab data.

Results and future development


Although the results of the first simulations looked promising, another problem arised as we were not able to determine the values just by looking into the literature.
If we would set up the model on simple mass-action equations, we would only need the so-called "rate of change" values, which are called kf values and describe the rate with which educts are converted to products. The model may work well with the right rate of change values and would help us to fine-tune our kill switch by defining various levels of proteins inside the cell. Unfortunately, our literature research did not come up with rate of change values we could use in our model, so we considered making the model a concept and help future iGEM teams move their project forward. Some starting points for fine-tuning the model are:

• Integrate an enhancer upstream of the promoter
• Simulate additional interference factors within the cell
• Add a second security layer, e.g. another kills switch-like system
This ideas are for future iGEM teams to discuss.


Figure 2: Here you can see a scheme of the second model. The green ellipses represent the species while the boxes represent the enzymes that catalyze the reactions. Along the black lines the reactions take place.

Model Three

Question

1. Is it possible to describe the necessary ComX Threshold in the extracellular space, by using the SimBiology Event functions?

Assumptions

1. The pathways of the quorum-sensing system can be described as rate of changes.
2. The model is built around mass-action equations.
3. One cell stands for an undefined cluster of cells.
4. The extracellular ComX concentration need to be above a certain threshold to be used by comP to phosphorylate ComA.
5. The educts for pre-ComX and ComA are produced continuously.

Model description

The third model built on the second one and refined some aspects of it. On the one hand, a transcription and translation subunit was introduced, which simulate the RNA/DNA synthesis and the associated degradation. On the other hand, event rules were introduced, which simulate a dynamic concentration limit of ComX. That means, only if the ComX concentration rises above a specific threshold, the cells can absorb it back.
Translation and transcription unit:
The added transcription and translation subunit simulate the degradation of DNA, RNA, and proteins. For example, not all mRNA reaches the ribosomes and a certain part of mRNA is removed on the way.
ComX Concentration limit:
The cells need a certain concentration of ComX to activate ComP. If the concentration in the extracellular space rises above a concentration of 0.4 nM, the kf value of ComP increases a hundredfold. If the value of ComX is below 0.4 nM, the kf value is set to 0.01 and the reaction does not take place. The 0.4 nM value is an example to demonstrate the function of the event rules and does not relate to real-world data.

Goal

The goal was to conceptualize the model so that future research into the characterization of the kill switch could be used to build a working model.

Results and future development

The model works in terms of describing the basic function of the kill switch. At this point, the model needs characterization in the lab to be validated. In the future it could be refined by increasing the complexity or by using the results from the lab, thereby making it more plausible.
figure
Figure 3: Here you see a scheme of our kill switch model three. The green ellipses represent the species while the boxes represent the enzymes that catalyzes the reaction. Along the black lines the reactions take place. Also here you see a transcription and translation subunit and a tunnel protein which transports ComX into the extracellular room.

Model download

We, from iGEM Technical University Darmstadt, want to share our model files with you. We used the Matlab SimBiology version 2020a for our project, but it may work with older versions of Matlab. If you want to download the model, you may click here to download it.

References

[1] MATLAB and SimBiology Toolbox 2020a, The MathWorks, Inc., Natick, Massachusetts, United States. [2] ŠPACAPAN,LINKS BETWEEN EXTRACELLULAR PROTEASES, MATRIX COMPONENTS PRODUCTION AND CELL-CELL COMMUNICATION IN A Bacillus subtilis BIOFILM ,DOCTORAL DISSERTATION,2019 [3] Vipul Singhal, Zoltan A. Tuza, Zachary Z. Sun, Richard M. Murray bioRxiv 2020.08.05.237990; doi: https://doi.org/10.1101/2020.08.05.237990