Team:XHD-Wuhan-Pro-China/Design

Design

We understand that the process of alcohol decomposition can be divided into a process: alcohol dehydrogenation into acetaldehyde, acetaldehyde dehydrogenation into acetic acid, (Figure 1). Alcohol dehydrogenation into acetaldehyde requires the participation of alcohol dehydrogenase (ADH), acetaldehyde dehydrogenation into acetic acid needs the participation of acetaldehyde dehydrogenase (ALDH). Therefore, in order to achieve the efficiency of laminate dehydrogenase, we can start by increasing the number of ethanol dehydrogenase, acetaldehyde dehydrogenase.

Why we chose Nissle 1917 as biological chassis?

Nissle 1917 is a food-grade probiotic. Studies showed that EcN has good efficacy for common gastrointestinal diseases, and is widely used to prevent infectious diarrhea, ulcerative colitis, and other inflammatory intestinal diseases. EcN can stabilize the environment in the intestines, regulates the immune system, and plays an important role in maintaining normal physiological function. In recent years, EcN expression exogenous gene as a carrier of disease diagnosis and treatment more and more research, clinical treatment has a number of recombinant EcN gene, used to treat intestinal diseases, HIV and other diseases success stories, plasmid recombination technology has been relatively mature.

Plasmid building

Sober Up 1.0

After obtaining valuable advice from Dr.Yang of Wuhan Union Hospital, we chose adh and ald2, which are genes that encode for ADH and ALDH in Saccharomyces cerevisiae S288C. We used the lacP promoter and cloning to build a recombinant plasmid pSB-AA in order to synthesize a higher amount of ADH and ALDH to promote the metabolism of alcohol and acetaldehyde.The adh and ald2 genes form our production module.In this manner, we hope to reduce the amount of alcohol the liver needs to metabolize and gain more control over the harm of alcohol and acetaldehyde on the body.

Sober Up 2.0

After further analysis on the chain of alcohol metabolization, we found out that, when the probiotic take in excess amounts of alcohol, NAD+, a substance essential for the activation of the two enzymes, is continuously converted to NADH. When the NAD+/NADH ratio in the probiotic falls, the metabolism of alcohol and acetaldehyde is inhibited. On the advice of Professor Ma from Huazhong Agriculture Univeristy,we inserted the NAD synthase gene sequence nadE from E. coli K12 and the NADH oxidase gene sequence nox from Lactobacillus Breus to our original recombinant plasmid PSB-AA and constructed the recombinant plasmid PSB-AN.The nadE and nox genes form our acceleration module.In this way, the balance between NAD+ and NADH could be restored, contributing to a higher reaction rate.