When thinking about our genetic system and the controlling/producing design, one of the most important variables of our project is how long our chassis can live in the intestine and how it could behave when coexisting with a type 1 diabetic’s microbiome. Thinking of that, our team decided to simulate a in vitro intestinal environment inoculated with type 1 diabetic’s microbiome, based on the SHIME system, described by DUQUE and collaborators [1]. We chose to simulate the intestine first part, the ascendent colon, by making a 24 hour batch in a 1L stirred tank reactor at 500 mL Carbohydrate-based medium (3 g/L starch, 2 g/L pectin, 4 g/L type III mucin from porcine stomach, 1 g/L xylan , 1 g/L peptone , 1 g/L arabinogalactan, 0.4 g/L of glucose, 3 g/L yeast extract and 0.5 g/L L-cysteine) with 16 hours stabilized microbiota from a person who suffers from type 1 diabetes. The sample collection and fecal inoculum was prepared by one of our advisors, Prof. Katia Savieri, who has expertise in in vitro microbiota simulation. Stabilization of the microbiota in the bioreactor was achieved by pH control in the range 6.5 to 6.7 over a period of 24h.
Once the simulated microbiome environment was stabilized, we added a genetically modified Bacillus subtilis producing GFP in the bioreactor. The ability of Bacillus subtilis to compete with the established microbiota and to colonize this environment was analysed by following the GFP fluorescence. To make sure we would just take fluorescence from GFP produced by our probiotic, we also measured the microbiota fluorescence background and subtracted it from our measurements. As shown in figure 1, we first detected a high fluorescence signal (0-2 hours) caused by cell inoculum high concentration. Then, B. subtilis went through an adapting phase, suffering with competition (2-6 hours).
Figure 1. Bacillus subtilis ΔFluorescence (A.U.) values in the first 6 hours (left) and after 6 hours (right) in a 12 hour batch at carbohydrate-based medium (pH 6,5 - 6,8).
But finally, after 6 hours, B. subtilis grew again, showing a srfA promoter controlled GFP production over time similar to that observed in the pure culture (see figure 6 in results section). By the GFP production pattern we can assume our probiotic chassis Bacillus subtilis is capable of surviving and adapting to the intestine environment of a type 1 diabetic person for at least 12 hours.
References
[1] DUQUE, Ana Luiza Rocha Faria et al. An exploratory study on the influence of orange juice on gut microbiota using a dynamic colonic model. Food Research International, v. 84, p. 160-169, 2016.
