Team:Stockholm/Model
As a part of our collaboration, NUS 2017 offered their modelling expertise about our toxin-antitoxin system and they kindly simulated our two gene switches, tryptophan and cumate expressing the Im2 and Colicin E2, respectively. The main objective is to provide insight into the genetic circuits and specifically to roughly simulate how the addition of chemicals ( tryptophan and cumate) would affect the expression of Im2 and Colicin E2. Moreover, they conducted a sensitivity analysis to determine how we can improve our system by choosing other regulatory units.
Certain kinetic values about the system were found from literature whereas the rest kinetic data and timing were arbitrary. Figure 1 demonstrates the functional Im2 Response of tryptophan system (0.001 Km). Upon addition of tryptophan at t = 2160 sec, there is a sudden drop in Im2 production as all the accumulated TrpR at steady state, readily binds to IM2 to repress the Trp operator. This sudden drop in IM2 production is caused by this rapid repression of the Trp Operator. However, TrpR must still be produced to maintain repression of the Trp Operator and because of the delay it takes to produce TrpR, we observe a small bulge that is indicative of time taken for TrpR to be produced, bind to TrpR Tryptophan and reach the necessary quantity to repress the Trp Operator.
Based on the sensitivity analysis, they decided to examine how varying Km of the Trp impact the response of the circuit. Figure 2 summarises the effect of varying Km in the genetic circuit. Their conclusion is to employ Trp Operator of Km 0.0001 or lower for this circuit because it will repress undesired IM2 production to a level where the quantities are extremely low and potentially insignificant. Moreover, they propose to vary the strength of the Trp Operator and RBS to gain more insight into the circuit.
All the kinetic data and timing were arbitrary in the cumate system since there was a lack of relevant information in the literature. Figure 3 shows the E2 response over time in absence or presence of cumate. Cumate is added again at t = 2160 sec. At first, we observe a delay of E2 repression since the cymR is not expressed in adequate quantities to repress the Cumate operator. However, CymR quantities manage to repress expression of E2 after a while. It should be noted that in practice, the delay of E2 repression is significantly more detrimental than the delay of IM2 repression. This is because E2 is a toxin and its undesired expression can kill the host bacteria at an unsuitable stage, for example in a stage before the bacteria has completed its primary function.
Based on the sensitivity analysis, they decided to examine how varying Km of the cumate impact the response of the circuit. Figure 4 summarises the effect of varying Km in the genetic circuit. The main finding is that decreasing the Km is successful in reducing the undesired expression of E2 to very low levels and it may prevent avoid accidental killing. However, this could affect the overall expression of E2 upon addition of cumate and it may compromise the killing mechanism. Their conclusion is that Km = 0.1 is the most suitable choice for a well - regulated system.
Our interpretation is that we cannot avoid the leaky expression of both systems, however, E2 leaky expression should be considered more crucial. Therefore, we came to realise that we should first introduce the tryptophan system with Im2 to our host and characterize the system to appreciate the level of regulation. Then, we should introduce cumate containing E2. Following this approach, any leaky expression of E2 would be neutralized by the baseline expression of Im2 until the cymR reaches adequate quantities to repress the cumate regulatory system.
Modelling of gene switches
Introduction
Results
Tryptophan system expressing Im2
Cumate system expressing Colicin E2
Conclusions
