Team:Gaston Day/Basic Part

Kill Switch

We have designed a passive kill switch that will significantly reduce the risk of engineered bacteria surviving in the event of accidental release. In our design, arabinose acts as an environmental cue to the pBAD promoter. Colicin is a toxin produced by E.coli that can destroy the cell, but is safe to use in the event of human exposure. When the arabinose level is high, the pBAD promoter is active and drives the production of the Tet repressor to prevent the production of colicin that kills the E. coli. On the other hand, when the arabinose level drops, the pBAD promoter activity is reduced, allowing the concentration of the repressor to decrease. This will allow the expression of colicin and result in the death of the bacteria. The construct shown in Figure 1 is used to test which concentrations of arabinose induce the pBAD promoter. As concentrations of arabinose increase, the production of GFP should also increase. The optimal concentration determined in this experiment will be used for all other pBAD constructs.

Our second and third constructs (Figures 2 and 3) are designed to determine the effectiveness of the Tet repressor system by measuring the reduction in GFP production. We combined the Tet repressible promoter with GFP (Figure 2) and combined the pBAD promoter with the Tet repressor coding region (Figure 3). Because the concentration of the Tet repressor and the production of GFP are inversely related, when the level of Tet repressor is high, we should observe low concentrations of GFP.

When both the pBAD and Tet repressor systems are working, we will pair colicin with the Tet repressible promoter (Figure 4). In order to build this construct, we must already have production of the Tet repressor so that colicin production will be repressed in the transformed cells. Otherwise, if colicin is active, it is possible that any cell transformed by the intended plasmid would die. We can test the effectiveness of the kill switch by reducing the amount of arabinose in the growth medium and measuring the change in the number of viable cells as colony forming units.

The final step will be combining the pBAD promoter and Tet repressor coding region in the same plasmid with the Tet repressible promoter and colicin construct (Figure 5). It is important to have an active stop signal between the two constructs because we don’t want run through transcription of colicin causing the cell to die.

Construct 1:

Test the function of the arabinose promoter and determine the most effective concentration of arabinose for induction.

Construct 2:

To be used in combination with construct 3 to test the function of the Tet repressor. the complete test plasmid is construct 4.

Construct 3:

To be used in combination with construct 2 to test the function of the Tet repressor. The complete test plasmid is construct 4.

Construct 4:

Test the function of the arabinose inducible promoter linked to the Tet repressor system. Under arabinose induction, GFP production should fall.

Construct 5:

Intermediate construct containing the lethal gene behind the Tet repressible protein.

Construct 6:

The final kill switch design. Under arabinose induction, the clicin will be suppressed using the Tet repressor. If the level of arabinose falls, as would happen in an accidental release, the Tet repression will stop, causing production of colicin and cell death.