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Team:TU-Delft/KillSwitch
From 2013.igem.org
Kill Switch
The kill switch design is based on the expression of holin and antiholin, Figure 1. Holin is a protein that forms pores in cell membranes. Anti-holin binds to holin and inhibits it's action. Once pores are formed by holin, lysozyme can access the periplasmic space and degrade the cell wall, causing cell lysis.
Kill switch constitutes a crucial part of our final system. It is activated after the production and release of the antimicrobial peptide. In that way, the E. coli bacteria are killed and the safety of the system is ensured.
Figure 1: Circuit of the kill switch
By this model we are finding the answers to the following question:
- What is the time needed for lysis to occur after induction?
Differential Equations
The kill switch circuit can be represented by the following differential equations.
Parameters
The used parameters are listed in Table 1. The dimer binding strengths are fitted on literature of Holin expression using the PcI promoter with and without antiholin. In [14] they have listed lysis times for different expression levels and from these the lethal level of Holin is estimated to be 190 molecules/cell.
| Parameter | Value | Description | Units | Reference |
| a | 1020 | Translation rate per amino acid | min-1#a-1 | [7] |
| cpconst | 0.5 | Transcription rate of Pconst | #m/min | Assumption |
| cptet | 4.16 | Maximum transcription rate of PT7 | #m/min | [15] |
| dH | 0.0348 | Degradation rate of holin | M/min | [17] |
| dH | 0.0348 | Degradation rate of Antiholin | M/min | [17] |
| dmRNA | 0.231 | Degradation rate of mRNA | min-1 | [8] |
| kb,HAH | 0.3*10-4 | Backward rate | [17] | |
| kf,HAH | 11.7*10-4 | Forward rate | [17] | |
| lT7 | 0.002 | Leakage factor of PT7 | - | Assumption |
| sH | 219 | Length of Holin | amino acids | |
| sAH | 103 | Length of Antiholin | amino acids |
Variables
| Variable | Description |
| Hm | concentration of translated holin |
| AHm | concentration of translated antiholin |
| HAHm | concentration of translated dimer formed between holin-antiholin |
| H | concentration of transcribed holin |
| AH | concentration of transcribed antiholin |
| HAH | concentration of transcribed dimer formed between holin-antiholin |
Results
The model is simulated upon activating. The lethal dosis of Holin is a 190 molecules, which is shown in the graph as the horizontal line.
Figure 2: Simulation Results
Conclusion/Discussion
In the graph of Figure 2 the behavior of the circuit can be seen and the answers to the question can be given:
The time for cell lysis to occur is approximately 12 minutes as this is the time point which the holin level passes the 1900 molecules(threshold for cell lysis to occur).
As observed the dimer is formed, reducing the amount of antiholin. After around 12 minutes the Holin level passes the 190 molecules, so the cell lysis occurs fast. From the experimental results around 30 minutes was estimated. This is significantly slower, which is mainly due to the simplification of some processes in this model. Mainly, the induction of IPTG is assumed to happen instantly, while in reality this takes time. This would explain much of the time difference.
