Constructs

Killswitch

During the construction of a killswitch, we stumbled upon a very concerning fact. A normal killswitch inducible by a promoter can be rendered useless in the case of frameshift mutations (and any other mutation, that affects the gene-product). Therefore we chose to construct killswitches with a less likely chance of being frameshifted out of order. We have come up with two solutions, where one of them, is new to us. We have named the two concepts: The risky concept and the safe concept. This is simply because our knowledge about the risky concept is limited and only theoretically plausible. The safe concept is seen before, at least bits of it. Both of the constructs are supposed to be integrated into the genome of the bacteria in order to avoid selection pressure. You can read about the idea of our killswitches below.

The risky concept

Fmet1b= frameshifted met codon (1 basepair)
F1bp stop= Frameshifted 1 basepair stopcodon
Fmet2b= frameshifted met codon (2 basepair)
F2bp stop= Frameshifted 2 basepair stopcodon

In this risky concept we have chosen to introduce frameshifted versions of our death gene, ccdB, thereby allowing the genes to be activated upon frameshift, keeping the killswitch functional. This is, as mentioned earlier, only theoretically possible and would need testing before introducing it into our product. The promoter is activated by the presence of the synthetic sugar, L-rhamnose, which is indigestible to humans. This allows the consumer to drink a glass of water with dissolved L-rhamnose in order to kill the GMO bacteria introduced by the yoghurt.

The safe concept

In the safe concept, we made 3 separate sections of the same L-rhamnose promoter and ccdB gene, as in the risky concept, but without the frameshifted genes, allowing larger chances for at least one of them to work when you drink the L-rhamnose sugar.