Designing a Kill-Switch
We initially attempted to create engineered bacteria that break down nitrogenous waste effectively in wastewater treatment plant. Since our E. coli can survive in saltwater, creating a kill switch for our engineered bacteria will, in theory, prevent our bacteria from thriving outside wastewater treatment plants.
We decided to vary from previous iGEM team efforts in three important ways:
1) We appended the ssrA degradation tag onto our toxin of choice, MazF. This should lead any MazF expressed due to leaky control of the promoter to be destroyed, and not damage the cell.
2) We introduced, in some of our constructs, a supplmentary copy of the cI repressor gene, in cause the light-controlled copy on pDawn was insufficient to control MazF.
3) For both MazF constructs we created, we had counterparts that contained the alpha fragment of LacZ instead of the toxin. This way, we can monitor expression levels without worrying about the cell death that high levels of MazF might cause.
Control of Gene Expression by pDawn alone
3.1 (K2268003): When light is present, c1 WILL NOT be produced. In theory, the expression of LacZ should be maximal. This is intended to be the non-lethal version of our kill-switch.
4.1 (K2268004): When light is present, c1 WILL NOT be produced. In theory, the expression of MazF should be maximal.
Control of Gene Expression by pDawn and supplementary cI Repressor
3.2(K2268005): When light is present with no ITPG, c1 WILL NOT be produced. In theory, maximal expression of LacZ should be produced. This is a non-lethal version of our kill switch.
4.2(K2268006): When light is present with no ITPG, c1 WILL NOT be produced. In theory, maximal expression of MazF should be produced.
Light control by pDawn
In order for our design to work, the light induction module present on pDawn must be operational. We tested pDawn alone, in the dark and in light, to verify that expression of the RFP reporter only occurs in the light (see petri dish below). We also observed this red fluorescence when pDawn was used in conjunction with our constructs (data not shown).