The real condition in the human gut is so complicated: a. Nutrition environment is hard to simulate because of the circadian rhythm of food intake, the food eaten and the enzymes released by intestine. b. The effect of gut microbiome. c. the peristalsis process in the intestine. d. the oxygen environment. To evaluate whether our project can work in the real condition, we decided to do the animal experiment.
To prove the function of the bacteria in the real condition, we conducted the animal experiment.
We tried to establish the HUA mouse model in the first stage in our animal experiment. We divided 10 male 5-week-aged balb/c mice into two groups randomly: Group 1 (n=5) and Group 2 (n=5). Set one week’s time without special handling to let them adapt to the new environment. Feed Group 2 with special food containing 0.1% adenine since week 2 while feeding Group 1 with normal food. All the feeding condition is the same expect for food.The blood of all the mice from caudal vein was taken after 3 weeks’ feeding. Isolate the serum and demonstrate the concentration of blood uric acid with ELISA. The result showed that, we can use the method to establish the HUA model successfully (Figure 11a.).
Then, the engineered bacteria was used in the animal experiment. We divided 25 male 5-week-aged balb/c mice into five groups randomly: Group Control (Group A, n=5), Group HUA (Group B, n=5), Group Allopurinol (Group C, n=5), Group engineered E. coli Nissle 1917 (transformed with BBa_K2334001) (Group D,n=5) and Group E. coli Nissle 1917 (Group E, n=5) [Figure 11b]. Set one week’s time without special handling to let them adapt to the new environment. Establish hyperuricemia animal models with all the groups except Group Control.Performe oval gavage as protocol from week 2, last for 3 weeks Isolate the serum and demonstrate the concentration of blood uric acid with ELISA.
Allopurinol is used in clinics to reduce the serum urate concentration, whose group is the negative control. Group B and C indicated that we established the HUA model successfully. Group D is the experiment group, which is of distinctive lower urate concentration compared to Group A. However, the group E has similar performance as Group D, which suggested E. coli Nissle 1917 itself has a ability to reduce the serum urate concentration, which is so surprising. We can't determine if our engineered pathway is working in vivo. But it's clear our engineered bacteria itself works. It may be related to the remodeling of the gut microbiome if a lot of bacteria were given by oral gavage (Figure 11c). Though no group diarrhea was found.