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Team:TU-Delft/Killswitch
From 2013.igem.org
Kill switch
To the circuit a kill switch is added for two reasons: bio-safety and secretion. The bio-safety concern is that the E.coli is in essence a treat to the humans and thus it is killed after it has done what it is supposed to. The second reason is the efficient secretion: if the peptides are not secreted naturally it is very difficult to force them. The solution is cell lysis, then all the peptides will surely be secreted.
For lysis cassettes several options are already in the part registry, these are listed in the part registry. From these options the holin with endolysin kill switch, BBa_K112808, has the most experience and good experience. Furthermore, it is easy to combine with the other parts of the circuit.
In Figure 1 the kill switch is shown in the circuit. The kill switch gets activated after the timer, the final promoter is the pcI promoter. So, at the same time as the Ulp-1 is produced the kill switch is activated.
The kill switch design is based on the expression of holin and antiholin; Holin is a protein that forms pores in cell membranes. Anti-holin forms a dimer with holin, which is not active. Once pores are formed by holin, lysozyme can access the periplasmic space and degrade the cell wall, causing cell lysis.
Figure 1: Circuit of the kill switch, Part BBa_K112808
Experiments
Aim: To check for the time taken to lyse the cells by inducing the pT7 promoter with IPTG and triggering the lysis cassette. (BBa_K1022114)
Part: The construct is made by ligating the pT7 promoter(BBa_I712074) in front of the lysis device from the biobrick BBa_K112808. This is to analyze how the lysis device can be controlled if needed.
Figure 2: Part BBa_K1022114
Description
The E.coli cells transformed with pT7 lysis cassette (BBa_K1022114) is grown on a plate reader which is capable of shaking and heating to 37˚C to take readings of the cells in exponential phase at every 10 minutes. Different range of IPTG concentration is used to characterize the bio – brick (BBa_K1022114). At time point, 160 minutes, IPTG is added according to the table below.
| No cells + 1mM | 0.1mM | 0.2m | 0.3mM | 0.4mM | 0.5mM | 0.6mM | 0.7mM | 0.8mM | 0.9mM | 1mM | Cell + No IPTG | |
| LB(µL) | 90 | 94 | 93 | 92 | 91 | 90 | 89 | 88 | 87 | 86 | 85 | 95 |
| Cells(µL) | - | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 |
| 10X IPTG(µL) | 10 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | - |
For a detailed set up of the experiment see here.
Results
The graph clearly shows the lysis of the cells after IPTG induction. 0.1mM and 1.0mM are representative for the other measurements taken.
Figure 3: Lysis of the cell after IPTG induction
Discussion
The Lysis cassette(BBa_K112808) is improved by adding a pT7 promoter (BBa_I712074) in front of it in the part BBa_K1022114. We showed the lysis device is capable of lysing E.coli efficiently and quick.
The functionality of the lysis device is improved as the promoter provides more controllability to the existing part BBa_K112808.
From the experiment, it can be clearly noted that the cells are growing exponentially and when induced by IPTG, within minutes the lysis device starts to kill the cells. Whereas the control E.coli cells with or without the plasmid used in the experiment, are growing even after IPTG is added to them. This proves that the cells do not die due to the IPTG chemical, but due to the lysis device which is activated by the pT7 promoter.
From the graph the lysis time can be estimated, it is between 30 minutes. In the modeling results, around 12 minutes was estimated. This is significantly faster, which is mainly due to the simplification of some processes as explained in the discussion on the modeling page: the induction of IPTG is assumed to happen instantly, while in reality this takes time. This would explain much of the time difference.
