Team:TU-Delft/KillSwitch

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.