Implementation
1. Background
Functional dyspepsia (FD) is a common digestive system disease. The prevalence of FD is about 16% in the general
population and up to 18–45% in China. Characteristic symptoms of FD include epigastric pain, epigastric burning,
postprandial fullness, or early satiety that persists for at least 6 months. The prevalence of dyspepsia was 23.5%
among people (15-75 years old) who received health checkups in my country. According to Lan Yu, director of the
Gastroenterology Department of Beijing Jishuitan Hospital, the incidence of dyspepsia among Chinese people is as
high as 38%, and more than 30% of Chinese have experienced dyspepsia symptoms such as flatulence, hiccups, and
abdominal discomfort.
Clinically, dyspepsia is mainly divided into two categories: chemical dyspepsia and mechanical dyspepsia. Chemical
indigestion is actually caused by eating too much, resulting in the "supply" of a series of digestive enzymes such
as pepsin, lipase, and amylase cannot keep up, which in turn causes indigestion symptoms such as bloating and loss
of appetite. Mechanical indigestion refers to indigestion symptoms such as abdominal distension caused by
"insufficient gastric motility", which is more common in the elderly, children, people with underlying
gastrointestinal diseases, or eating a lot of indigestible food.
Fats， as an intake in everyday diet, regardless of their nutritional value, must be properly used, stored, or
discharged from the human body. Dietary fats can be difficult to break down properly, and when the digestive system
does not produce or secrete enough lipase to accommodate dietary fat intake, bloating, cramping, flatulence,
unexpected weight gain, and sometimes Symptoms such as steatorrhea. If the pancreas is not functioning properly and
the secretion of lipase is insufficient, it may lead to maldigestion and malabsorption of fat, which can lead to
malabsorption of nutrients.
Above all, for this part of the population, additional supplementation of lipase is required. Therefore, we are here
to produce a live probiotic drink, functional in releasing a due amount of lipase in the intestinal tract to help
eliminate common stomach and intestine symptoms and other health issues.
2. Target users
Specifically, our target customers are those people who are suffering from fat indigestion, especially those who
have difficulty digesting greasy food, those patients with chronic pancreatitis, and pancreatic insufficiency. At
the same time, because the product also contains probiotic ingredients which is beneficial to human health, this
also makes some people who are concerned about their health also potential customers of this product.
Our target users will be mainly focused on the Chinese region because of the large population size and special
eating habits, which provides us with a larger market. In Chinese cuisine, fried, fried and other methods are often
used to make food. Such a diet will undoubtedly increase the Chinese public's intake of fat. Besides, in the global
market, the Asia-Pacific region has a high consumption of probiotics, with a market share of about 50%.
Therefore, we take mainland China as the main product target sales area. With the improvement of people's living
standards and the change of consumption concept, their requirements for food no longer simply stay on the problem of
solving food and clothing, but more in the pursuit of safety, health and nutrition. It is believed that as long as
the publicity and consumption guidance of probiotic products are increased, our product will have a broad
development prospect in China.
3. Product
Our product will be a live probiotic drink, functional in releasing a due amount of lipase in the intestinal tract.
For its package as shown below, it will be in the form of a light blue bottle (17*6 cm and 30 mL) with a twist cap.
For its taste, it will be sweet and sour to stimulate appetite. Since our product contains live probiotics, it must
be stored under 2-10oC for maintaining activity.
Concept drawing of our live probotic drink
We will select Escherichia coli Nissle 1917 (EcN) as the host strain for our engineered plasmid in our final
product. Since 1917, E. coli Nissle 1917 (EcN), its probiotic properties, and biosafety have been verified. In
addition, the EcN strain also shows immunomodulatory and anti-inflammatory activities (Ukena et al. 2007; Zyrek et
al. 2007).
After drinking our live probiotic drink, our engineered E. coli Nissle 1917 (EcN) will pass through the esophagus
into the intestines and stomach, and then release lipase, the core functional ingredient to help fat digestion. In
order to provide time for the lipase expression process, we recommend drinking our live probiotic drink before the
meal. It is also recommended to drink after meals especially those fatty ones so as to help digest fat to avoid fat
accumulation. One tip for drinking our product, slightly shake the bottle before drink since the probiotics might
settle at the bottom.
Considering one of our target groups who are patients suffering from chronic pancreatitis or digestive system
disorders, we will indeed conduct rigorous further research to determine the specific amount of lipase needed to
secrete for different categories of customers in order to adjust suitable live probiotic formulas accordingly.
4. Experimental Design and Proof
We chose a genetic engineering approach to construct an E. coli system capable of
expressing lipase. The Lactobacillus W1 and Pseudomonas SP genomes lipases were inserted into the plasmid pET28a to
construct an E. coli expression vector in E. coli DH5α and sent to the company for Sanger sequencing. Then, the
recombinant expression plasmid was transformed into E. coli BL21 (DE3), so that the lipase gene could be expressed
heterologously. Following that, we used IPTG to induce protein expression and obtained the lysate to measure the
activity of the lipases. Finally, the optimum temperature and pH of the two lipases were preliminarily tested to
explore the optimal condition for the lipase expression.
Figure：Enzyme activity of lipase (W1 and SP) at different PH and different temperature, respectively
According to the experiment results in the laboratory, we did not only confirm the successful expression of
W1-lipase and SP-lipase in our engineered E. coli BL21 (DE3) but also explored the optimal expression condition
which will be at pH=9 and around 37-42oC.
5. Safety
Safety has always been our primary criterion, and our team fully complies with the iGEM lab safety provisions, which
are thoroughly guaranteed during our experiments.
● We attach great importance to the safety of experimental procedures and experimental products. The experimental
substrate we used is Escherichia coli, which has been widely used in experiments and industrial production, showing
very good and stable properties.
● In addition to the raw materials of the experiment, we strictly abide by the laboratory's operating specifications
throughout the experimental process. Not only did the team form operational notes about the experimental steps, but
also made safety tips for every key link.
We also have considered the safety of our final product, a live probiotic drink. To meet the requirement of probioc
products in food industry, we will select Escherichia coli Nissle 1917 (EcN) as the host strain for our engineered
plasmid in our final product. Since 1917, E. coli Nissle 1917 (EcN), its probiotic properties, and biosafety have
been verified. Besides, we indeed conduct rigorous further research to determine the specific amount of lipase
needed to secrete for different categories of customers in order to adjust suitable live probiotic formulas
accordingly.
6. Challenges
After team summary and expert consultation, we believe that the challenges mainly come from the following aspects.
● The first challenge we face is the stereotype of GMO technology. Especially in China, many people are not very
familiar with synthetic biology and are cautious about some concepts including genetically modified. Therefore, the
number one difficulty we face in promoting our products is the acceptance of the technology by the general public.
● Secondly, we have not conducted any clinical trials. Although we have tested the activity of lipase at different
temperatures and pH values, due to the difference in each person's body constitution, as well as different eating
habits and living habits, it will affect the function of lipase in the human body.
● Policy and management requirements are important aspects that our team has to face. In China’s application, the
number of live active probiotics in the product is required to be higher than 106 CFU/g (ml). Besides, products
should pass the health food inspection and evaluation technical specifications as well as to pass the test of
General Standard for Health (Functional) food (GB16740-1997). Probiotics (powder) import customs clearance
procedures need to provide information such as import and export business rights, ingredient content, certificate of
origin, etc. In addition, we will seek the local medical departments for advice on the operation procedures to
permit our products for sale.
● There are also special requirements for production plants, not only in terms of hygiene but also related safety
standards. Because probiotic products require strict control of temperature and supervising the manipulation of the
genome, the production process needs to be carefully monitored and conducted in a safe environment.
7. Future Plan
● Market Expansion
Per our previous analysis, we will take more effort into the publicity, and consumption guidance of probiotic products so that we could attract more potential consumers and achieve a broader market.
Per our previous analysis, we will take more effort into the publicity, and consumption guidance of probiotic products so that we could attract more potential consumers and achieve a broader market.
● Product Industrialization
The pharmaceutical company is aimed to establish a factory in Beijing, which takes advantage of the infrastructure support and technical assistance of the region. The comprehensive network in logistics and the pool of talents from research centers would facilitate our supply chain and also provide needed assistance in innovation and quality control.
The pharmaceutical company is aimed to establish a factory in Beijing, which takes advantage of the infrastructure support and technical assistance of the region. The comprehensive network in logistics and the pool of talents from research centers would facilitate our supply chain and also provide needed assistance in innovation and quality control.
● Research and Development
So far we have successfully constructed the lipase expression system in E. coli BL21 (DE3). In the near future, we will further extract the recombinant plasmid and transfer it into E. coli Nissle 1917 to test the expression performance. Besides we will conduct more investigation to explore the probiotic drink formula for gradually implementing our final product.
So far we have successfully constructed the lipase expression system in E. coli BL21 (DE3). In the near future, we will further extract the recombinant plasmid and transfer it into E. coli Nissle 1917 to test the expression performance. Besides we will conduct more investigation to explore the probiotic drink formula for gradually implementing our final product.
8. Reference
[1]Luo X, Wang L, Fang S, Qing X, Jiang T, Yang Y, Su X, Wei W. Chinese Herbal Medicine for Functional Dyspepsia
With Psychological Disorders: A Systematic Review and Meta-Analysis. Front Neurosci. 2022 Jul 14;16:933290. doi:
10.3389/fnins.2022.933290. PMID: 35911981; PMCID: PMC9330302.
[2]Saxelin, Maija. “Probiotic Formulations and Applications, the Current Probiotics Market, and Changes in the Marketplace: A European Perspective.” Clinical Infectious Diseases, vol. 46, 2008, pp. S76–79. JSTOR, http://www.jstor.org/stable/40307429. Accessed 24 Sep. 2022.
[3]Ukena, S. N., Singh, A., Dringenberg, U., Engelhardt, R., Seidler, U., Hansen, W., Bleich, A., Bruder, D., Franzke, A., Rogler, G., Suerbaum, S., Buer, J., Gunzer, F., & Westendorf, A. M. (2007). Probiotic Escherichia coli Nissle 1917 inhibits leaky gut by enhancing mucosal integrity. PloS one, 2(12), e1308. https://doi.org/10.1371/journal.pone.0001308
[4]Zyrek, A. A., Cichon, C., Helms, S., Enders, C., Sonnenborn, U., & Schmidt, M. A. (2007). Molecular mechanisms underlying the probiotic effects of Escherichia coli Nissle 1917 involve ZO-2 and PKCzeta redistribution resulting in tight junction and epithelial barrier repair. Cellular microbiology, 9(3), 804–816. https://doi.org/10.1111/j.1462-5822.2006.00836.x
[2]Saxelin, Maija. “Probiotic Formulations and Applications, the Current Probiotics Market, and Changes in the Marketplace: A European Perspective.” Clinical Infectious Diseases, vol. 46, 2008, pp. S76–79. JSTOR, http://www.jstor.org/stable/40307429. Accessed 24 Sep. 2022.
[3]Ukena, S. N., Singh, A., Dringenberg, U., Engelhardt, R., Seidler, U., Hansen, W., Bleich, A., Bruder, D., Franzke, A., Rogler, G., Suerbaum, S., Buer, J., Gunzer, F., & Westendorf, A. M. (2007). Probiotic Escherichia coli Nissle 1917 inhibits leaky gut by enhancing mucosal integrity. PloS one, 2(12), e1308. https://doi.org/10.1371/journal.pone.0001308
[4]Zyrek, A. A., Cichon, C., Helms, S., Enders, C., Sonnenborn, U., & Schmidt, M. A. (2007). Molecular mechanisms underlying the probiotic effects of Escherichia coli Nissle 1917 involve ZO-2 and PKCzeta redistribution resulting in tight junction and epithelial barrier repair. Cellular microbiology, 9(3), 804–816. https://doi.org/10.1111/j.1462-5822.2006.00836.x