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Team:SDU-Denmark/Project/Future
From 2012.igem.org
Future Applications
Our idea to battle obesity gives rise to a lot of different aspects of the future. In this section we will try to describe some of the ideas we had for future applications. When devising these future ideas, we have always had in mind, the safety considerations needed for GMO. Therefore this section will begin with our killswitch.
Killswitch
During the construction of 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.
Bacterial chassi
The next thing we want to discuss is the transfer of the construct to a probiotic bacteria. For this part we want to use a lactobacillus, since it is the normal type of bacteria used in probiotic cultured yoghurts. The lactobacillus is a normal inhabitant of the gut, and several strains of the specie are already characterized as probiotic. We found a strain (Lactobacillus johnsonii) that have the ability to produce inulin polymers (the bacterial version with a degree of polymerization (DP) up to 10.000 units), and also happened to be in the group of probiotic lactobacillus strains. This might have some unknown features that handles the inulin production (fx. transport out of the cell) and therefore has the potential to be a very useful chassi.
Promoter
As we have mentioned, the end product should be a cultured yoghurt. This suggests to us, that we might use a heat shock promoter, allowing the construct to remain untranscribed, as it is held in the refrigerator and then transcribed at a quick pace when eaten (at 37°C).
Anti-colonization compartment
We found a part made by the Chinese team, XMU-china (2011). The part enables bacterial density limitations by synthesizing a signaling molecule capable of activating a death gene, if it has a high concentration. This idea allows us to avoid the chance of our bacteria inappropriately colonizing our gut. Below we have posted a link to the part. http://partsregistry.org/Part:BBa_K658001
Cellulose compartment
Because the waste product of inulin production is a glucose unit (per fructose added to the polymer), we also thought to add another part to our construct. This part should be the cellulose compartment, which would allow us to convert glucose into cellulose. Cellulose is, unlike inulin, a non-soluble fiber that is found in almost any plant organism. Cellulose is indigestible to humans, as well as inulin, but has a lot of beneficial health attributes (Helps to prevent cancer, healthier feces etc.). Of course you might consider the further decrease of survivability as an opposing factor in the decision of adding the cellulose compartment. The excess glucose could be used for energy supply, keeping the bacteria alive. In order to decide whether the cell has the capacity for the cellulose compartment, we suggest empiric testing to measure survival rates of the bacteria. Keep in mind, though, that the bacteria are only supposed to be able to survive for some hours in the gut, and produce what is necessary (mostly for reasons of biosafety).
Delivery System
In order to provide the best circumstances, when delivering our probiotic bacteria, we think microencapsulation methods to be of great use. We want to encapsulate our bacteria in a mixture of some coating polysaccharides that will shield the bacteria through the gastrointestinal tract (GI). These coating polysaccharides should be a base of alginate mixed with some prebiotic starches such as pectin, according to literature.[1][2]
