Overview

Kill switch is a safety system which is necessary to control the release of genetically modified organisms (GMOs) into the environment. Since we aimed to construct a biofilm with GMOs which will convert CO to CO2, we needed a kill switch that can prevent our system to cause any harm to the environment. In order to achieve that aim we used the kill switch designed by Berkeley 2008 iGEM Team (Bba_K112808) and also we tried to develop it.

How The System Works ?

In order to construct our kill switch system we needed a reagent that E.Coli will grow and in the absence of that reagent all the bacteria should die. For that purpose we prefered to use IPTG (Isopropyl-beta-D-thiogalactopyranoside) because it cannot be metabolised by E.Coli and that is why its concentration remains constant in the cell.[1] When IPTG is present in the cell it will bind to LacI which can be produced by the promoter J23116. Basal LacI expression is already present in E.Coli but at low levels and it is hard to control the system with that much LacI. Hence, we prefered to produce it with the help of J23116. Our kill switch system includes the expression of endolysin, holin, antiholin and their production is controlled with the initial concentration of IPTG in the environment. Actually cell lysis can occur without IPTG but the lysis rate will be high. By using IPTG we aimed to keep our system under control.

Endolysin-Holin System; Endolysin and holin are the proteins needed for the cell lysis. Endolysin is a lysozyme and its funtion is to hydrolyze the peptidoglycan bacterial cell wall but it needs holin to form pores on inner membrane, then it can reach periplasm and cell lysis occur.

The fate of the Gram-negative envelope during holin-endolysin lysis:[2]

In this figure holins are shown as clear ovals and endolysin is shown as notched circles.

• a-)Pre-Hole Configuration: Accumulation of holin is present in the inner membrane, while endolysin accumulates in the cytosol. Holin action can be prevented with the inhibitors (shown with star and black ovals).
• b-) Hole Configuration: When the holes are formed by holin in the inner membrane, endolysin can attack the murein which is important for cell lysis to occur.

 

Antiholin; It is the protein that blocks the function of holin and it is important for the regulation of cell lysis.[3]

In our system antiholin is constitutively produced by the promoter J23116 and when antiholin level is in between 1000-3000 molecules, the cell will die.[4] Actually,it is the number of free holin molecules that determines if the cell will die or not.

Modelling

KILL-SWITCH MODEL

  The aim of kill-switch model is to predict the behavior of kill-switch mechanism in different IPTG concentrations. We first tried to model mechanism by the help of ODEs. Then we used SimBiology toolbox of MATLAB to simulate system.

Introduction:

As can be seen from the diagram our circuit involves constitutive production of LacI. LacI Promoter is negatively regulated by LacI. The control mechanism of our system is inhibition of LacI by IPTG molecule. Since LacI is inhibited by IPTG its effect on LacI promoter changes with respect to IPTG concentration. Holin and Endolysin expression is controlled by cI promoter which is negatively regulated by cI. Our first assumption in the model is that when the free Holin molecules, which means number of Holin molecules minus number of Antiholin molecules, reach 3000[ref] cell lysis occurs. Because cI concentration depends on the activity of LacI promoter, we can conclude that in the presence of IPTG the concentration of cI will be high and expression of Holin and Endolysin will be lowered. Due to the constitutive production of Antiholin, we can say that free Holin molecules will be lowered and chance of cell lysis to occur will drop. However, to understand the overall behaviour of system with respect to IPTG concentration we need to model and simulate the system.

Mathematical Model:

In our model, we included all processes shown below.

Equations for mRNA transcription

     

 

 

 

 

  Equations for Protein Translation      

 

 

 

 

 

 

 

Parameter	Description
N	Plasmid copy number
Px	Promoter Strength
ax	Degradation Rate
Kx	Disassociation (Equilibrium) Constant
nx	Hill Coefficient
tx	Translation Rate
kx	Binding on rate
k-x	Binding off rate
Dx	Diffusion rate

&nsbp; &nsbp;

Simulation Results

When the IPTG concentration is 0;

Individual graphs can be accessed here. Because the critical point in kill-switch is “Holin-Antiholin” molecules, we ran simulation in different IPTG concentrations and to understand result easier we select the outputs as Holin and Antiholin. Since translation rate of endolysin is more than Holin and Antiholin we can assume that there will be enough endolysin to make cell lysis occur. Also from the graph above we can understand that endolysin will be in sufficient amount.

By the help of our model, we can conclude that when there is no IPTG molecule, cell lysis occur in 275 s. However when there is 100000 IPTG molecules, cell needs 2290 s to lyse. As can be seen from graphs until a very high IPTG concentration like in the Run14, the increase in Holin can eventually cause lysis. As a result of our model we concluded that after an IPTG concentration of 700000 molecule cell lysis will not occur. We are not considering the generation time of cell in this model since it can later be used for different organisms. If we consider e.coli with a generation time of 20 min, even in the medium with IPTG of 100000 molecules, it will divide before it can lyse.

When IPTG concentration is 700000;

Individual graphs can be accessed here.

Sensivity Analysis

Since the challenging part of modelling is choice of parameters, we conducted a sensitivity analysis to see how much result of our model depends on parameters. By the help of those, the critical values to be measured through experiments can be understood easily.

References

[1]Hansen LH, Knudsen S., Sorensen SJ. (1998). The effect of the lacY gene on the induction of IPTG inducible promoters, studied in Escherichia coli and Pseudomonas fluorescens. Current Microbiology,36 (6), 341-347. doi: 10.1007/s002849900320

[2]The fate of Gram-negative envelope during holin-endolysin lysis [Image]. (2000). Retrieved September 23, 2012, from: http://dx.doi.org/10.1016/S0966-842X(00)01705-4

[3]Tran TA, Struck DK, Young R. (2007). The T4 RI antiholin has an N-terminal signal anchor release domain that targets it for degradation by DegP. Journal Of Bacteriology, 189 (21), 7618-7625. doi: 10.1128/JB.00854-07.

[4]Christos G. Savva, Jill S. Dewey, John Deaton, Rebecca L. White, Douglas K. Struck, Andreas Holzenburg, Ry Young. (2008). The holin of bacteriophage lambda forms rings with large diameter. Molecular Microbiology, 69 (4), 784-793. doi: 10.1111/j.1365-2958.2008.06298.x