Team:XHD-Wuhan-Pro-China/Description
Project Inspiration and Description
Inspiration
At present, two billion people globally drink regularly, with the number increasing every year. According to the World Health Organization, ap-proximately 3 million people died from excessive drinking in 2016. Long-term or irregular drinking can lead to a variety of health problems. There are excessive examples about the damage of alcohol to one’s physical and mental state. It is proved that alcohol is related to many forms of illnesses, such as liver cirrhosis and cardiac damages.In WHO's Global Status Report on Alcohol and Health from 2018, it is stated that in China, 2016, 62.6% of the deaths in male and 41.6% of the deaths in female due to liver cirrhosis were alcohol-attributable.(Figure 1) People may be less efficient on a workday because they have drunk last night. In addition, the number of alcohol-related crimes remains high. People becomes less rational after drinking, so alcohol drinking could lead to many cases of criminal activities.
Figure 1
We hope that our product can help the people who need to work after drink to be more efficient And may help to prevent criminal cases related to excessive drinking like drunk driving.
Background
What is ADH and ALDH?
Alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) are enzymes in our liver that are important for digesting alco-hol. When we consume alcohol, our body metabolizes this chemical by 2 important enzymes: ADH and ALDH. ADH converts alcohol into acet-aldehyde, and ALDH converts acetaldehyde into a harmless chemical called acetic acid.
What is the problem about ADH and ALDH?
Although this system normally operates smoothly and prevents alcohol and acetaldehyde from damaging our liver, these enzymes do not func-tion properly in everyone’s body. Around 40% of East Asians carry a gene that results in less activities of ALDH. To make it even worse, about 80% of East Asians have mutated ADH, which converts alcohol to acetaldehyde more quickly, further preventing acetaldehyde from being metabolized, which could be harmful to our liver. Moreover, these two important enzymes cannot be easily replenished. If we could find a way to produce these enzymes in the human body, we could reduce the harm of alcohol poisoning.
What is Nissle 1917?
EscherichiacoliNissle1917 (E. coli Nissle 1917, EcN) is made by Alfred Nissle separation in 1917. It is now widely used for clinical purposes, mainly for treating diarrhea, inflammatory enteritis and constipation. This probiotic has many usefulproperties for our project, it is safe to the host, does not contain enterotoxin, hemolysis toxin, cytotoxin and oth-er pathogenic factors.
Picture: Lliriana Bisha Tagani MD, Study shows that probiotics can evolve making them highly unpredictable, 2019, gilmorehealth.com
Our Project
Our project aims to solve the problem from its root by constructing a probiotic that contains the ADH gene for producing ADH, and the ALD gene for producing ALDH. In comparison with existing products, our product deals with the issue from its base by enabling the probiotic to synthesize ADH and ALDH in the stomach and the intestines to reduce alcohol’s harm to the liver. Moreover, to ensure that energy supplied to the reactions is adequate, and the activity of enzymes remains high, we add nadE and nox genes to maintain a balance between coenzyme NAD+ and NADH. We hope that this new product can prevent alcohol poisoning and reduce liver problems for people around the globe.
Fix toxics, for ebrieties; Through probiotics, In genetics.
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.
Experimental Design
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.
Test of alcohol and acetaldehyde tolerance
Since the growth and metabolism of bacteria may be inhibited under the condition of high alcohol and acetaldehyde concentration,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.
References
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