Team:CAU China/Project2

In order to develop a novel beverage, which helps us avoid getting drunk or intoxication when consumption of large amount of alcohol is unavoidable, we firstly specify our project by define three functional modules --- alcoholic oxidation unit (AOU), principle unit to provide the power to eliminate alcohol by enzymatic reaction; accessory unit (AU), indispensable unit to support the normal work of AOP, primarily contains acidic resistance part and time elongation part; biosafety unit (BU), ensure the biosafety of our design beverage, safe to consumers and friendly to environment.

To mimic the normal metabolism of ethanol in human liver, we tried to present a recombinant strain occupying active Alcohol dehydrogenase (ADH)and Aldehyde dehydrogenases (ALDH), which can oxidize alcohol to aldehyde and finally to acetic acid.Acetic acid turns out to be subject of beta-oxidation.

To ensure the best performance of our protein and strain, accessory unit have to tackle serial problems, such as how to survive and work in gastric acid, how to avoid the effect of gastric empting, and so on. Because the core of our beverage’s function is two successively enzymatic reactions (AOU), in which, the reaction condition (pH and Temp.) and reaction time count most. Thus, we primarily focus on these two points.

On account of the gastric acid, we thought the resistance of both our strain and our enzymes affect the performance. For the strain, we domesticated our E. coli to grow in acidified LB medium, and tested the possibility of using lactic bacteria, simultaneously. As to the enzymes, we adopted both random mutation and rational reformation as strategies to make a high enzymatic activity in relative low pH.

In case of reaction time, we introduced a new part into our strain, enabling it secreting Cholecystokinin, a peptide hormone secreted in the duodenum, mediating digestion in the small intestine by inhibiting gastric emptying and gastric acid secretion, which will promise our enzymes a longer reaction time in stomach and our strain a weakened acid secretion stomach.

To eliminate the unwanted effect of our product to consumers and environment, biosafety unit was designed to prevent such influences.

1, Ourfunctional strain cannot grow in the out-lab nature.

2, the only place to survive after drinking is our stomach, thus once the strain escaped, the alkaline surrounding in duodenum would kill the bacteria, naturally.

3, to ensure the extinction of our bacteria in digestiontrack correspond to the low ethanol concentration, a promoter which is able to sense the alcohol concentration was intended to promote the expression of killer protein.