iGEM 2022 — Team lzu-hs-china-b

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# Notebook

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# 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.

![](https://static.igem.wiki/teams/4331/wiki/engineering-1.png)

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

![](https://static.igem.wiki/teams/4331/wiki/engineering-2.png)

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

![](https://static.igem.wiki/teams/4331/wiki/engineering-3.png)

4. Obtain positive clones.

![](https://static.igem.wiki/teams/4331/wiki/engineering-4.png)

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

![](https://static.igem.wiki/teams/4331/wiki/engineering-5.png)

## 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.

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## What should this page contain?

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Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

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* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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# Results

## 1. Test results of tolerance of engineered strains to ethanol and acetaldehyde

In this study, two engineering strains E.coli pSB-AA and E.coli pSB-AN were constructed to test their performance in degrading ethanol and acetaldehyde.

## 2. The activity of ethanol and acetaldehyde dehydrogenase and the content of coenzyme NAD+

Fig.1 Engineering strains and control strains express the activity of related enzymes. (A) The enzyme activity of alcohol dehydrogenase and acetaldehyde dehydrogenase; (B) NAD+ content in different strains; (C) The enzyme activity of NADH oxidase.

In order to reflect the expression of functional genes of engineered strains E.coli pSB-AA and E.coli pSB-AN more directly, we detected the activities of various exogenous enzymes and the content of coenzyme NAD+ in different strains cultured in vitro. Results as shown in Figure 1, E.coli 1917 showed almost no activities of alcohol dehydrogenase, acetaldehyde dehydrogenase and NADH oxidase in the crude enzyme extracts of each bacterium, and the content of coenzyme NAD+ was very low. The ADH activity of E.coli pSB-AA was 47.91±3.12 U/mL, and the ALDH activity was 33.57±2.59 U/mL. No NADH oxidase activity was detected. However, the ADH activity of E.coli PSP-AN was 81.41±3.64 U/mL, the ALDH activity was 57.56±1.48 U/mL, and the nox activity was 14.4±2.29 U/mL. The content of coenzyme NAD+ was also significantly higher than that of E.coli pSB-AA. These results indicated that the expression of NAD synthetase gene nadE and NADH oxidase gene nox could contribute to the increase of the content of dehydrogenase coenzyme NAD in bacterial cells, thus improving the degradation ability of alcohol dehydrogenase and acetaldehyde dehydrogenase.

## 3. Detection results of the tolerance of engineering strains to ethanol and acetaldehyde

Fig.2 The tolerance of different strains to different concentrations of ethanol. (A) The growth curve of E. coli1917; (B) The growth curve of E. coli 1917 with pSB-AA; (C) The growth curve of E. coli 1917 with pSB-AN.

Fig.3 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.

The growth curves of engineered strains E.coli pSB-AA and E.coli pSB-AN in the medium containing different concentrations of ethanol and acetaldehyde were drawn to show the tolerance of engineered strains to ethanol and acetaldehyde. As shown in Figure 2 and Figure 3, when ethanol concentration> 6%, acetaldehyde concentration> 0.3% would seriously inhibit the growth of E.coli 1917; When ethanol concentration> 8%, acetaldehyde concentration> The growth of E.coli pSB-AA was inhibited at 0.4%, and the tolerance of E.coli pSB-AA to ethanol and acetaldehyde was improved to a certain extent. And when the concentration of ethanol> 10%, acetaldehyde concentration> At 0.5%, E.coli pSB-AN began to inhibit the growth of engineered strain E.coli pSB-AN, indicating that the tolerance of E.coli pSB-AN to ethanol and acetaldehyde was significantly improved.​

## 4. Test results of the ability of engineering strain to degrade ethanol and acetaldehyde

Fig.4 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.

In order to alleviate the damage caused by excessive drinking, the ability of the engineered strain to degrade ethanol and acetaldehyde is crucial. Here, we detected the content changes of ethanol or acetaldehyde in the culture medium of different strains after growing in different concentrations of ethanol and acetaldehyde for 14 h, so as to measure the degradation ability of the strain to ethanol and acetaldehyde. As shown in Figure 3.3, the degradation efficiency was the highest when the ethanol content was 2% and the acetaldehyde content was 0.1%. The degradation rates of ethanol and acetaldehyde of the engineered strain E.coli pSB-AA were 54.3% and 41.4%, respectively. The degradation rates of ethanol and acetaldehyde of E.coli pSB-AN were 61.5% and 53.5% respectively. However, with the increase of ethanol and acetaldehyde concentration, the growth and metabolism of bacteria are also inhibited, so the degradation ability of bacteria to ethanol and acetaldehyde is gradually reduced.​

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