Constructs

The System

The above picture illustrates our idealized system the way we wanted it to be . It consists of two plasmids with FFT and SST respectively. Both plasmids have a toxin-antitoxin system introduced which works as a form of safety to prevent horizontal gene transfer (read more about it further down the page). The two enzymes, FFT and SST, produce together the non-digestible inulin from sucrose. Along with the two plasmids there is also a sigma-E gene that functions as a regulator gene in our kill-switch system.

The sigma-E is an alternative sigma factor that controls the extracytoplasmic stress response in E. coli. The idea is to have an L-Rhamnose sensitive promoter that expresses this essential gene.

The L-Rhamnose would be in the yoghurt with our bacteria and function as a limiting lifetime for the consumed bacteria. L-Rhamnose is a non-digestible sugar which passes through the system and thereby inactivates the sigma-E gene. The consumed bacteria from the yoghurt would inevitably die after a while.

Data For Our Favorite New Parts

Data For Pre-existing Parts

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.