Over a period of several months, we iterated and constructed an acid-resistant engineered lactose degradation bacterium, lacMAN2.0 (Figure 1). It can overexpress CAP, a non-toxic bacterial capsular polysaccharide, to protect itself from gastric acid and other microbial populations in the gut without affecting the original intestinal microenvironment, and colonize the gut for a prolonged period of time. At the same time, lacMAN2.0 can secrete the synthetic β-galactosidase (lacZ) out of the cell, instead of degrading lactose and producing gas inside the cell, which is one of the main cause of lactose intolerance: intestinal bloating.
Figure 1. Enzyme activity and acid resistance measurements of LacMAN2.0
(A) The enzymatic activity of lacMAN2.0 in the bacteria and in the medium is separately tested with the test kit
(B) Determination of lacMAN2.0 growth in acidic environments with different PH.
However, in order to make lacMAN2.0 reach the intestine more safely, we designed a protective device for LacMan 2.0 - enteric-coated capsule (FIG. 2), which is not digested by stomach acid and can release contents in the intestine. This makes lacMAN2.0 much more efficient.
Figure 2. Prepared lacMAN2.0 bacterial powder and enteric coated capsule filled with bacterial powder
Next, we tested enteric-coated capsules and bacterial powder (Figure 3). The capsules were placed in hydrochloric acid solution with PH = 1 and stood for 1.5 h. Then, the bacterial powder was removed and dissolved in PBS buffer, and a certain volume of bacterial solution was taken and evenly spread on LB AGAR plate (adding X-Gal solution), and cultured overnight at 37 ° C. The next day, it was clearly observed that the plate turned blue and X-Gal was degraded by lacZ.
Figure 3 Finalized lacMAN2.0 test
(A) Placing enteric-coated capsules containing bacterial powder in hydrochloric acid solution PH=1.0;
(B) Remove the bacterial powder in the capsule and dissolve it in PBS buffer;
(C) Apply bacterial solution to LB plate containing X-Gal;
(D) Observe the color of the plate after overnight culture.
At this point, we have completed finished lacMAN2.0 experiments and demonstrated that our engineered bacteria can survive in gastric acid and degrade lactose analogues. However, due to the limitation of time and experimental conditions, many test experiments have not been completed, such as intestinal degradation of enteric-coated clothing, whether the function of CAP can still play a role in the complex intestinal environment, etc. In future experiments, we will solve these problems one by one and introduce our products on the market.