Team:XHD-Wuhan-Pro-China/Proof Of Concept
The construction and cultivation of the engineered strain
We have successfully constructed plasmid pSB-AA and pSB-AN, which were transformed into Escherichia coli Nissle1917, then grown in LB broth containing Ampicillin antibiotics respectively at 37 ℃, 200 rpm overnight.
The result of cultivation
The result of colony PCR
Therefore, our construction of the engineered strain is successful.
Test of alcohol and acetaldehyde degradation ability
We designed and tested the ability of our probiotics to degrade alcohol and acetaldehyde, which is important for achieving our final goal: to achieve maximum degradation of alcohol and acetaldehyde. Here, we detected changes in the level of alcohol and acetaldehyde in the medium after different strains grew in alcohol and acetaldehyde of different concentrations for 14 hours, so as to measure the ability of the strains to degrade alcohol and acetaldehyde. As shown in Figure 3.3, when the alcohol content is 2% and the acetaldehyde content is 0.1%, the degradation efficiency is the highest, in which the degradation rates of alcohol and acetaldehyde for E. coli PSB AA are 54.3% and 41.4% respectively; The degradation rates for E. coli pSB-AN were 61.5% and 53.5% respectively. It can be seen that the engineering bacteria with plasmid PSB AN have stronger ability to degrade alcohol and acetaldehyde. This proves that our idea of enhancing the metabolic capability of alcohol and aldehyde dehydrogenase by adding nadE and nox genes is tenable. However, with the increase in the concentration of alcohol and acetaldehyde, the growth and metabolism of bacteria are also inhibited, so the ability of our bacteria to degrade alcohol and acetaldehyde is also gradually reduced.
Fig.3.3 The degradation ability of different strains to different concentrations of ethanol and acetaldehyde. (A) Ethanol content of different strains after 14 h; (B) Acetaldehyde content of different strains after 14 h.
Test of alcohol and acetaldehyde tolerance
According to the test results above, our bacteria’s ability to degrade alcohol and acetaldehyde gradually decreases with the increase of alcohol and acetaldehyde concentration. This is because the growth and metabolism of bacteria will be inhibited under the condition of high alcohol and acetaldehyde concentration. Therefore, we designed and carried out an experiment to test the alcohol and acetaldehyde tolerance of the bacteria by simulating environments with different alcohol and acetaldehyde concentrations that our probiotics may encounter in the small intestines. Firstly, the growth curves of E. coli PSB AA and E. coli PSB AN in the media that contains alcohol and acetaldehyde of different concentrations were drawn to show the tolerance of the strains to alcohol and acetaldehyde. As shown in Figure 3.1 and Figure 3.2, when the concentration of alcohol was larger than 6%, and the concentration of acetaldehyde was larger than 0.3%, the growth of bacteria E. coli 1917 would be seriously inhibited; When alcohol concentration was larger than 8%, and acetaldehyde concentration was larger than 0.4%, the growth of E. coli PSB AA was inhibited. Thus, the tolerance of E. coli PSB AA to ethanol and acetaldehyde was improved to some extent. When the concentration of alcohol was larger than 10%, and the concentration of acetaldehyde was larger than 0.5%, the growth of E. coli PSB AN was inhibited. Hence, the tolerance of E. coli PSB AN to alcohol and acetaldehyde was significantly improved. It can be seen that E. coli PSB AN with significantly greater tolerance to alcohol and acetaldehyde is more suitable for being our engineered bacteria. This can well solve the potential problem that excessive alcohol intake will directly kill our probiotics in real application.
Fig.3.1 The tolerance of different strains to different concentrations of ethanol. (A) The growth curve of E. coli 1917; (B) The growth curve of E. coli 1917 with pSB-AA; (C) The growth curve of E. coli 1917 with pSB-AN.
Fig.3.2 The tolerance of different strains to different concentrations of acetaldehyde. (A) The growth curve of E. coli 1917; (B) The growth curve of E. coli 1917 with pSB-AA; (C) The growth curve of E. coli 1917 with pSB-AN.
In conclusion, it is shown through our in vitro experiments that our bacteria after engineering may be able to function normally.