Team:TU-Delft/KillSwitch

From 2013.igem.org

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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 <i>E.coli</i> bacteria are killed and the safety of the system is ensured.
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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 <i>E. coli</i> bacteria are killed and the safety of the system is ensured.
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<p>Figure 1: Circuit of the kill switch</p></div>
<p>Figure 1: Circuit of the kill switch</p></div>
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<p>By this model we are finding the answers to the following question:
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<li>What is the time needed for lysis to occur after induction?</li>
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<h2 align="center">Differential Equations</h2>
<h2 align="center">Differential Equations</h2>
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<img src="https://static.igem.org/mediawiki/2013/5/5b/Kill_switch.png" width="600" >
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<img src="https://static.igem.org/mediawiki/2013/7/7c/Killswitch_equationsDelft.png" width="600" >
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<h2 align="center">Parameters</h2>
<h2 align="center">Parameters</h2>
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The used parameters are listed in Table 1. The dimer binding
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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 [[Team:TUDelft/Modeling_References|[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.  
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strengths are fitted on literature of Holin expression using the PcI promoter with and without antiholin
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[[Team:TUDelft/Modeling_References|[14]]].
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<h2 align="center">Variables</h2>
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{| align="center" border="1"
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|'''Variable'''
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|'''Description'''
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|H<sub>m</sub>
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|concentration of translated holin
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|AH<sub>m</sub>
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|concentration of translated antiholin
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|HAH<sub>m</sub>
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|concentration of translated dimer formed between holin-antiholin
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|H
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|concentration of transcribed holin
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|AH
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|concentration of transcribed antiholin
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|HAH
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|concentration of transcribed dimer formed between holin-antiholin
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<h2 align="center">Results</h2>
<h2 align="center">Results</h2>
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The model is simulated upon activating. The lethal dosis of Holin is a 1000 molecules [[Team:TUDelft/Modeling_References|[17]]], which is shown in the graph as the horizontal line.
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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.
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<img src="https://static.igem.org/mediawiki/2013/0/00/Killswitch_results.png" width="700">
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<img src="https://static.igem.org/mediawiki/2013/f/f5/Modeling_results_kill_switch.jpg" width="650">
<p>Figure 2: Simulation Results</p></div>
<p>Figure 2: Simulation Results</p></div>
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<h2 align="center">Discussion</h2>
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<h2 align="center">Conclusion/Discussion</h2>
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As observed the dimer is formed, reducing the amount of antiholin. After 55 min the Holin level passes the 1000 molecules, so cell lysis would occur within an hour.  
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In the graph of Figure 2 the behavior of the circuit can be seen and the answers to the question can be given:
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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).
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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 <a href="https://2013.igem.org/Team:TU-Delft/Killswitch" target="blank">experimental results</a> 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.  
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Latest revision as of 15:43, 4 October 2013

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:

  1. 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.

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