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 )

Procedure:

1. Grow the cells with pT7 Lysis cassette overnight in the LB broth with the right antibiotics.

2. Make 10X stock solution of IPTG by adding 10µL of the 1000X IPTG in 990 µL of LB with the right antibiotics.

3. Make 1/50 times dilutions of the over night grown culture. Wait till the OD at 600nm reaches 0.4. Note the exponential phase of the cells and induce with IPTG to activate the pT7 promoter.
4. Then make the below combinations into the 96 well plate for the plate reader to take readings.
   
   

   	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	-



5. Readings were taken for BL21 as control, pT7 in BL21 plysS. (in Duplicates).