Team:GA State SW Jiaotong/DMM
A dynamic simulation model of the toxin-antitoxin (hok/sok) mechanism
Model Background
Model background introduction
To ensure the safety of the engineered bacteria in the natural environment, we need to ensure that: 1. The engineered bacteria cannot survive in the natural environment (suicide) after escaping from the working environment (scalp) due to chance factors; 2. The engineered bacteria should commit suicide quickly after finishing the scalp environment, i.e., they cannot continue to survive on the scalp.
The project designed a dual-regulated suicide switch system in the engineered bacteria used to achieve the above objectives. This suicide switch system mainly contains two exogenous genes, Sok and Hok. The Hok gene encodes the Hok virulence protein, which causes the bacteria to lyse itself and die. The Sok gene can transcribe and produce Sok mRNA, binding to Hok mRNA to form an mRNA complex. This mRNA complex will be degraded by enzymes, thus preventing further translation of Hok mRNA into Hok virulence protein and thus preventing the bacteria from lyzing itself and dying. Based on this mechanism, the Hok gene is regulated by the tryptophan manipulator, and the lactose manipulator regulates the Sok gene. At this time, if the bacterium is in a survival state, it is only necessary to add tryptophan and IPTG to the bacterial solution because tryptophan can inhibit the expression of tryptophan manipulator and prevent the transcriptional expression of Hok gene. At the same time, IPTG can induce the expression of lactose manipulators and allow the regular transcriptional expression of Sok gene, thus ensuring the survival of the engineered bacterium. On the contrary, only the added tryptophan and IPTG need to be washed away with water when the engineered bacteria finish their work. At this time, the expression status of Hok and Sok will be reversed, i.e., Hok is expressed commonly, and Sok is inhibited, which in turn ensures the death of the engineered bacteria; the schematic diagram is shown in Figure 1.
In summary, this project is to introduce a plasmid containing a toxin-antitoxin (hok/sok) mechanism into the engineered bacterium to accomplish the task of surviving while working and dying by suicide after the work is completed. And this mechanism ensures the safety of the engineered bacteria in the natural environment.
Fig 1. Schematic diagram of toxin-antitoxin (Hok/Sok) mechanism.
Model introduction
In the bacterial growth competition model chapter, the model results strongly argued the stability of the engineered bacterium Bacillus Subtilis WB600 to the scalp environmental homeostasis. This model is mainly used to theoretically simulate the dynamic expression of the suicide switch plasmid in vivo during drug administration in the engineered bacterium Bacillus Subtilis WB600 and investigate the theoretical feasibility of this mechanism.
Dynamic simulation of suicide mechanism
2.1 Simulated dynamics of the common regulated toxin-antitoxin (hok/sok) mechanism
In order to investigate the toxin-antitoxin (hok/sok) mechanism more clearly, its dynamic expression under normal regulation was first investigated. The normal regulation of the toxin-antitoxin (hok/sok) mechanism represents the dynamic process of transcriptional translation under the normal expression of both Hok and Sok genes. The dynamic process of Hok gene is shown in Figure 2, where:
Fig 2. Simplified drawing of the common regulated toxin-antitoxin mechanism for plasmid maintenance.
For the dynamic mechanism of Fig1. the changes in the concentrations of Hok-mRNA, Sok-mRNA, Hok-protein and Complex can be described by the following equation:
Num is the plasmid copy number per cell volume.
According to the actual situation and relevant reference, the plasmid copy number was set to 6 [1]. The above equation was solved directly by Matlab (see Table 1 for the parameter values). The results were obtained as shown in Figure 3. The results show that in the common, regulated toxin-antitoxin mechanism, Sok-mRNA, Complex, and Hok-protein all show a growth trend before 20 min; between 20 min and 60 min, the growth trend gradually slows down; and at about 60 min, it basically steady state was reached. At the steady-state, the concentration of Hok-mRNA was maintained at 0.1, Sok-mRNA at about 30, Complex at about 60, and Hok-protein at about 14. In summary, the concentration of Hok-protein under the common, regulated toxin-antitoxin mechanism showed a low concentration at the initial stage (before 20 min) and a high concentration at the later stage (after 20 min).
Fig 3. Simulated dynamics of the common regulated toxin-antitoxin mechanism.
Table 1. The value and meaning of parameters.
Simulated dynamics of the dual-modulated toxin-antitoxin (hok/sok) mechanism
After understanding the dynamic trend of Hok-protein concentration of the toxin-antitoxin mechanism under the common mechanism, the dynamic simulation of the dual-modulated toxin-antitoxin mechanism was started. In contrast to the common regulated toxin-antitoxin mechanism, the dual-modulated toxin-antitoxin mechanism was working from the beginning, i.e., in the environment of IPTG and tryptophan, when Sok gene was expressed normally and A fold repressed hok gene. gene is repressed. After the steady-state work is completed, the bacterium is in an environment without IPTG and tryptophan, where Sok gene is inhibited and Hok gene is expressed normally. The following equation can describe the overall dynamic mechanism:
Where, indicates the working time threshold of the engineering bacteria, because the system can reach the steady-state very quickly, so the time threshold is selected without affecting the subsequent system changes, considering the actual use, it is set to 15min here. The suppression effect is set to 1/60 of the common, that is, being suppressed 60 times. The Matlab solution was applied to it, and the results were obtained as shown in Fig. 4 and Fig. 5. The results showed that the engineered bacteria reached a steady state at 2.3 min during the initial working period, and at the steady-state, Hok-mRNA, Complex and Hok-protein were all 0, and Sok-mRNA was stable at about 33, while the expression of Sok-mRNA had almost no interference to the host [8], and this state ensured the stability of the work of the host. At the end of the working time, i.e., t, Sok-mRNA decreases to 0 within 2 min, Complex and Hok-mRNA start to increase and stabilize around 8 and 21, respectively.Hok-protein starts to increase exponentially when Sok-mRNA decreases to 0. It rises to 500 at 25 min, and its reaches at 200 min steady state with concentrations up to 3858, ensuring the cellular suicide effect.
Conclusion
In summary, the present model theoretically simulated Hok-protein dynamics under the common, regulated toxin-antitoxin mechanism and the dual-modulated toxin-antitoxin mechanism. The results showed that both mechanisms are characterized by low Hok-protein concentrations in the initial phase and high concentrations in the final step. However, the dual-modulated toxin-antitoxin mechanism has a poor concentration of Hok-protein at the initial stage (0) and a very high concentration at the end-stage (3858) compared with the common, regulated toxin-antitoxin mechanism. It is more suitable for the functional characteristics of this project. Moreover, the dual-modulated toxin-antitoxin mechanism is more controllable, i.e., the time threshold is manipulated by the addition and removal of tryptophan and IPTG, which provides a more significant advantage for the application of the project product. Finally, the dual-modulated toxin-antitoxin mechanism was chosen to design the suicide switch for the project-engineered bacteria.
Code appendix
Reference
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