Team:FZU-China/Model

Modeling

Because of the impact of the COVID-19 pandemic, we did not have time to implement the full biological circuit that we designed (we were not allowed to leave the dorms during the surge of COVID-19 cases in Fujian Province and all our classes were switched to online format). In order to show that our design could work in theory, we created a simple model to simulate the results of our full circuit.

Model description

In our biological circuit design, we use an optogenetic switch Cre-VVD to control the transcription of genes gadB and ϕX174e, which then can be translated into proteins GadB and ϕX174E. GadB catalyzes the conversion of glutamate to GABA and ϕX174E induces cell lysis. Before we start to construct the whole circuit, we would like to first test whether this design could work as expected, that is, when light is present, GABA will be constantly produced intracellularly and then be released into the extracellular environment in a cyclical fashion.

There are multiple factors that require careful consideration (proportion of cells that are remained after each lysis, the level of GABA released after each lysis, etc.), which is why we utilize a mathematical model to help us understand the dynamics of our circuit.

Note that in our complete circuit design, we have a light-generating module to provide blue light for the optogenetic switch. However, we do not put this module in our model for the following two reasons: 1) simplicity; 2) in our future implementation, we would like to have sunlight to turn on our optogenetic switch instead of using an internal light source.

We used SimBiology of Matlab to build our model. In our simplified model, DNA can generate protein in a single reaction at a constant rate. DNA-nCre-VVD and DNA-cCre-VVD make Protein-nCre-VVD and Protein-cCre-VVD, respectively. Then when light is present (light is viewed as an input as well), Protein-nCre-VVD and Protein-cCre-VVD plus light can generate Protein-Cre-VVD. This can then bind with DNA-gadB-phiX174e to generate protein GadB and protein phiX174E. GadB then binds glutamate and this reaction generates GadB and intraGABA. When [phiX174E] reaches a certain threshold, some of the cells lyse and produce extraGABA from intraGABA.

Figure 1. A schematic view of our simplified model.

When we run our simulations, we make a few assumptions:
1. DNAs do not get diluted as we assume the plasmid copy numbers remain constant in cells.
2. Light is abundant in the system.
3. Enzymes such as Cre-VVD and GadB do not get consumed, but their concentrations will decrease after cell lysis.
4. Glutamate is abundant in the system.
5. When [phiX174E] >= a threshold concentration "L", "P" percent of cells lyse.
6. There are abundant resources for growth, and cells can keep growing and multiplying forever.

Based on the above assumptions, we created a SimBiology model with five reactions.
1. [DNA-cCre-VVD] -> [Protein-cCre-VVD]
2. [DNA-nCre-VVD] -> [Protein-nCre-VVD]
3. [Protein-nCre-VVD] + [Protein-cCre-VVD] + Light -> [Protein-Cre-VVD]
4. [DNA-gadB-phiX174e] + [Protein-Cre-VVD] -> [Protein-Cre-VVD] + [GadB] + [phiX174E]
5. [GadB] + [Glutamate] -> [intraGABA] + [GadB]
And when [phiX174E] >= L,
[extraGABA] = [extraGABA] + [intraGABA],
[intraGABA] = [intraGABA]*P,
[phiX174E] = [phiX174E]*P,
[GadB] = [GadB]*P,
[Protein-Cre-VVD] = [Protein-Cre-VVD]*P.

We then ran a simulation to track the changes of [extraGABA] along with other related [molecules].

Figure 1. Simulated results of the full circuit.

As we can see in the simulation, the concentration of extraGABA increases over time as cells keep producing intraGABA and get lysed when [phiX174E] hits the threshold. After hitting the threshold, protein concentrations decrease, and [extraGABA] remains constant until [phiX174E] hits the threshold again. Note that [GadB] is exactly the same as [phiX174E] in our simulation as they are bicistronically expressed. This cycle just keeps repeating in our ideal cell growth setting while [extraGABA] keeps increasing. This simple simulated result suggests that our current biological circuit design should work in theory and the cells will keep pumping out GABA to the extracellular environment when the light is present.

However, when light is limited, [extraGABA] will increase initially; but after [Protein-Cre-VVD] decreases over time (no light, no more new Protein-Cre-VVD) because of cell division, [extraGABA] will remain constant while concentrations of all the other related molecules decrease (no Protein-Cre-VVD, no more new GadB and phiX174E).

Figure 2. Simulated results of the full circuit when light only exists for a short time.

In summary, we demonstrate that our biological circuit can work as expected in theory and release GABA into the extracellular environment when light is present. Our future plan includes characterizing the modules individually to measure the parameters that we use in this model in order to get more realistic simulation results; we also plan to test the whole circuit in cells to see whether the actual results are consistent with the simulations.

References

［1］Din, M.O., T. Danino, A. Prindle, et al., Synchronized cycles of bacterial lysis for in vivo delivery. Nature, 2016. 536(7614): p. 81-85.