Engineering

Step1 Designing

The main purpose of our project is to reduce the content of alcohol in the human body after drinking so as to help people's livers and reduce the damage of alcohol to the human liver. The design of our project originally followed the steps below. When people drink alcohol, the alcohol passes through the cell membrane, where NAD+ promotes ethanol dehydrogenase (ethanol dehydrogenase) to become acetaldehyde (Aldehyde), and then NAD+ promotes aldehyde dehydrogenase (aldehyde dehydrogenase) to convert acetaldehyde to acetic acid (acetic acid), then these substances become acetyl coenzyme (acetv-CoA), and finally, they join the TCA cycle. The NAD+ we mentioned earlier is the conversion of NADH through NOX, which helps in the conversion between ethanol and acetate. In these cells, alcohol dehydrogenase prevents people from flushing when they drink alcohol or some of the larger bodily reactions, and aldehyde dehydrogenase helps people sober up more quickly.

Step 2 Building

1. The first is to prepare the experimental equipment we need and prepare the culture medium. We need to activate E. coliDH5α/pSB1A3-mRFP, E. coli DH5α/pMD18T insert.

2. Extract plasmid, PCR amplification, double enzyme digestion, and get recovery experiment.

3. Prepare competent cells, perform ligation reaction, and transform ligation products.

4. Obtain positive clones.

5. Transform the engineering vector into the expression host E. coli BL21, and verify the experiment.

Step 3: Test

We confirmed that the constructed EcN recombinant strain pSB-AN did carry ADH, ALD2, nox and nadE genes, while the engineered strain pSB-AA carried ADH and ALDH genes.

First, the tolerance of the engineered strains to ethanol and acetaldehyde was tested. Two engineered strains, E. coli pSB-AA and E. coli pSB-AN, were constructed, which showed that the tolerance of E. coli pSB-AN to ethanol and acetaldehyde was significantly improved.

We also tested the ability of the engineered strains to degrade ethanol and acetaldehyde. We measured the change of ethanol or acetaldehyde content in the medium after 14 h of growth of different strains in different concentrations of ethanol and acetaldehyde. The highest degradation efficiency was observed for the engineered strain E.coli pSB-AN. However, with the increase of ethanol and acetaldehyde concentration, the growth and metabolism of the bacteria were inhibited, and therefore the degradation ability of the bacteria to ethanol and acetaldehyde was gradually reduced.

Then the enzymatic activities of ethanol and acetaldehyde dehydrogenases and the content of coenzyme NAD+ were detected. To reflect more visually the functional gene expression of the engineered strains E.coli pSB-AA and E.c coli pSB-AN, different strains were tested. The enzymatic activities of various exogenous enzymes and the content of coenzyme NAD+ under in vitro culture conditions. The expression of NAD synthase gene nadE and NADH oxidase gene nox contributed to increase the content of dehydrogenase coenzyme NAD in bacterial cells, thus improving the degradation of alcohol dehydrogenase and aldehyde dehydrogenase.

Step4: Learning

Alcoholism triggers oxidative stress, lipid peroxidation and hepatic inflammation, as well as intestinal barrier damage, ultimately leading to abnormal liver function and liver damage. The recombinant EcN strain pSB-AN expressing ADH, ALDH, nadE and nox genes was shown to reverse the deleterious effects of alcoholism, promote ethanol and acetaldehyde degradation, and effectively repair the intestinal barrier. In addition to previous studies on alcoholic liver disease, the current work emphasizes the importance of protecting the function of the intestinal barrier and the gut microbial community to prevent liver injury. Our genetically engineered EcN conferring strong restorative capacity to intestinal and hepatic abnormalities is a promising drug delivery tool for these patients. However, several technical issues have hindered its development for human applications. Future studies could focus on risk assessment of the main enzymes of alcohol metabolism provided by transgenic probiotics, which would have an impact on patients with alcoholic liver injury. In the future, we need to implant a cellular autophagy program in genetically reproducible recombinant probiotics. Thus, preventing bacteria from entering the environment. This is a question that we should explore. Cellular autophagy is an evolutionarily conserved and important process in eukaryotes for the turnover of intracellular material.