Team:TU-Delft/Killswitch

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 )

Description

The BL21(DE3) 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. BL21 is used as a control.