Team:TU-Delft/Timer-Sumo-KillSwitch
From 2013.igem.org
Timer-SUMO-KillSwitch
The separate modules: Timer plus SUMO and Kill Switch are combined to form the complete model of the system: Timer - SUMO - Kill Switch. For the final model, the kill switch module is converted in such a way so as the holin and antiholin to be activated by the Pci promoter.
Figure 1: Circuit of the kill switch
Differential Equations
Parameters
Parameter | Value | Description | Units | Reference |
c_{a} | 1020 | Translation rate per amino acid | min^{-1}#_{a}^{-1} | [7] |
c_{T7} | 4.16 | Maximum transcription rate of T7 | #_{m}/min | [2] |
c_{ptet} | 2.79 | Maximum transcription rate of Ptet | #_{m}/min | [4] |
c_{pconst} | 0.5 | Transcription rate of Pconst | #m/min | Assumption |
c_{ci} | 1.79 | Maximum transcription rate of Pci | #_{m}/min | [3] |
d_{mRNA} | 0.231 | Degradation rate of mRNA | min^{-1} | [8] |
d_{H} | 0.0348 | Degradation rate of holin / Antiholin | M/min | [17] |
d_{TET} | 0.1386 | Degradation rate of TET | min^{-1} | [9] |
d_{CI} | 0.042 | Degradation rate of CI | min^{-1} | [9] |
k_{b,HAH} | 0.3*10^{-4} | Backward rate | [17] | |
k_{f,HAH} | 11.7*10^{-4} | Forward rate | [17] | |
d_{PEP} | 2.1*10^{-3} | Degradation rate of the peptide | min^{-1} | Assumed three times slower same as GFP |
d_{PSU} | 6.3*10^{-3} | Degradation rate of the peptide plus SUMO | min^{-1} | Assumed the same as GFP |
d_{Ulp} | 1.263*10^{-2} | Degradation rate of Ulp | min^{-1} | Assumed twice the rate of GFP |
l_{t7} | 0.002 | Leakage factor of T7 | - | Assumption |
l_{ptet} | 0.002 | Leakage factor of Ptet | - | Assumption |
l_{ci} | 0.002 | Leakage factor of Pci | - | Assumption |
k_{tet} | 6 | Dissociation constant of Ptet | #m | [10] |
k_{ci} | 20 | Dissociation constant of Pci | #m | [10] |
k_{cUlp} | 3 | Turnover rate of Ulp | min^{-1} | [6] |
n_{ci} | 3 | Hills coefficient | - | [11] |
n_{tet} | 3 | Hills coefficient | - | [11] |
s | 0 or 1 | Activation/Inactivation of T7 promoter | Binary | Assumption |
s_{ci} | 228 | Length of CI | amino acids | [12] |
s_{PSU} | 18 + 110 | Length of peptide plus SUMO | amino acids | [12] |
s_{TET} | 206 | Length of TET | amino acids | [13] |
s_{Ulp} | 233 | Length of Ulp1 | amino acids | [13] |
s_{H} | 219 | Length of Holin | amino acids | |
s_{AH} | 103 | Length of Antiholin | amino acids |
Results
In Figure 1 the results of the simulation are shown.Figure 1: Simulation Results
Conclusion/Discussion
In Figure 1 the behavior of the total circuit is seen, and the answers to the questions can be given:- How much peptides are produced by the circuit?
The total concentration is 18000 peptides per cell. - How much peptides are still uncleaved by the SUMO at the point of cell lysis?
The models shows that there is almost no SUMO-peptide uncleaved. - How many minutes does the cell lysis take from the point of induction?
After 165 minutes the Holin concentration passes 190 molecules per cell and cell lysis occurs.
Sensitivity analysis
To asses the validity of the found answers a sensitivity analysis is performed. In this case the numerical derivative of the solution is taken with respect to the parameters of the model. This derivative represents the change of the solution upon changing the specific parameter. If this change is large, the solution is very sensitive for this parameter. Also, since the solution is highly non-linear this numerical derivative is taken in both directions, as they differ greatly in this model. To compare the found values of the derivative, they are normalized by the nominal value (the solution, e.g. the lyse time). In this way a percentual change is found. Mathematically this can be expressed as:
So, for the three answers the sensitivity analysis is done, yielding the follow results and conclusions:
- The lysis time is not significantly affected by most parameters, only two are important.