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.colis are killed and the safety of the system is ensured.

Figure 1: Circuit of the kill switch

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 [14].

Parameter	Value	Description	Units	Reference
a	1020	Translation rate per amino acid	min^-1#_a^-1	[7]
c_pconst	0.85	Transcription rate of Pconst	#m/min	Assumption
c_ptet	2.79	Maximum transcription rate of Ptet	#m/min	[15]
d_H	0.0348	Degradation rate of holin	M/min	[17]
d_H	0.0348	Degradation rate of Antiholin	M/min	[17]
d_mRNA	0.231	Degradation rate of mRNA	min^-1	[8]
k_Atc,in	0.9			Assumption [11]
k_Atc,out	0.05			Assumption [11]
k_b,HAH	0.3*10^-4	Backward rate		[17]
k_f,HAH	1.2*10^-4	Forward rate		[17]
l_ptet	0.002	Leakage factor of Ptet	-	Assumption
n_tet	3	Hills coefficient	-	[11]
s_H	219	Length of Holin	amino acids
s_AH	103	Length of Antiholin	amino acids

Results

The model is simulated upon activating. The lethal dosis of Holin is a 1000 molecules [17], which is shown in the graph as the horizontal line.

Figure 2: Simulation Results

Discussion

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.