Background

In order to offer research direction for our project, our team interviewed several professions and searched for any potential connections between cardiovascular diseases and Taiwanese society. In the course of our investigation, we came across two special phenomena that occur specifically in Taiwan, leading to one of the directions of our project - an automated probiotic platform in vivo.

First of all, as of 2022, cardiovascular diseases have been one of the top three causes of death in Taiwan, with mortality accounting for 22.5% among elderly aged 65 or above (Statista, 2022). Such a high mortality rate is definitely not a coincidence but closely related to the dietary habits of Taiwanese people.

According to a 2018 investigation, a significant population in Taiwan have high-fat diets daily, leading to the highest fat intake rate in the Asia-Pacific region (herbalife, 2018). This dietary habit is a key contributor to the high prevalence of cardiovascular diseases among Taiwanese individuals. As mentioned earlier in the description page about blood clot classification, we discussed how a sudden spike in blood pressure or disordered blood flow can lead to the rupture of atherosclerotic plaques and cause the formation of white blood clots. A high-fat diet is considered one of the contributing factors to unhealthy blood flow. Consequently, it is evident that dietary habits characterized by high-fat in Taiwan are what cause cardiovascular diseases, with fatty acids being particularly significant in blood clot formation.

In addition, since 2018, Taiwan has entered an aged society with age 65 and above growing at the rate of 5% annually. This demographic shift has brought forth various medical challenges, and one of those is the problem of medication non-compliance that happens among elders such as forgetting to take pills. This acute problem can possibly pose a critical health risk, particularly for individuals with chronic conditions like cardiovascular disease.

From the above information, our team concluded that high-fat dietary habits and the problem of medication non-compliance are the two reasons causing such a high mortality and prevalence rate of cardiovascular diseases in Taiwan. Therefore, the two problems became our team’s inspiration for developing an in-vivo probiotic platform to automatically address blood clots.

About the probiotic platform

Inspired by the high-fat dietary habits of the Taiwanese population and its influence on hypertension and disordered blood flow, our team designed a switch-like mechanism called oleate induction. This mechanism enables the in-vivo probiotic platform to produce lumbrokinase and dissolve fibrin after receiving a certain concentration of fatty acids. Therefore, the formation of white blood clots and emboli caused by the rupture of atherosclerotic plaques could be solved.

In our team’s plan, the in-vivo probiotic platform will be presented as capsules. Once the capsule is ingested, it will go through the digestive system and enter the intestines. Then, when individuals have a high-fat diet, the ingested fatty acids will induce the production of lumbrokinase. Lumbrokinase will break through the mucus layer and the epithelial cell layer of the intestine. Finally, it will begin to exert its function of producing after it enters the blood circulation system.

Our team's concept involves enabling the probiotic platform to facilitate the direct synthesis of lumbrokinase within the human intestine, ensuring its effective delivery to the specific sites where blood clot formation occurs, and prolonging each intake interval time for a longer period.

Experiments Conducted

In order to develop the probiotic platform, our team modified the pET-22b vector and transformed it into E. coli BL21 to produce lumbrokinase, thereby enabling us to confirm the production of lumbrokinase in smaller organisms. Subsequent to the transformation, we conducted several tests, including electrophoresis and western-blotting, to confirm whether E. coli BL21 successfully produced lumbrokinase when exposed to specific concentrations of fatty acids. In conclusion, the results had proven a success of the oleate-induced lumbrokinase production in our experiment.

To see more detailed information of our experiment, please visit the experimental procedures and the experiment result pages.

Thought Experiments

While the concept of a probiotic platform stands as a central pillar of our project, it becomes an obstacle that further human and animal experiments is currently not feasible. Hence, the in-vivo phase of the probiotic platform remains theoretical, and practical challenges may arise when it comes to its application within the human body. To solve this issue, our team came up with some hypothetical scenarios and thought experiments that could potentially solve the challenges that we might encounter at the in-vivo phase tests. More detailed information of the thought experiments was written in Proof of Concept.

Possible Problems and Corresponding Solution Ideas

Upon analyzing the experimental results, our team came across several issues that might cause the uncertainty of the effectiveness of the probiotic platform, and we found some possible solutions after some research.

How to prevent decomposition during the transportation process?

Within the human body, not only are proteins and peptide medicines susceptible to degradation by gastric acid in the stomach, but also bacteria and other organisms are hard to survive in the harsh environment of human stomach; therefore, conquering the obstacle of the gastric acid environment becomes imperative for the successful delivery of the probiotic platform that contains Lumbrokinase to the human intestines.

In order to overcome the challenge, our team has conceived the notion of enveloping E.coli Nissle colonies with gastro-resistant capsules. We have inspected several studies about Gastric-resistant capsules involving different protein and peptide drugs like oral insulin, and we have found out that Gastric-resistant capsules are able to avoid degradation of the active substances by the gastric juice in the human stomach.

Moreover, gastro-resistant capsules are able to reduce gastric irritation caused by the medicine. As seen in several research studies, the gastro-resistant capsules coating allows the medicine to be released only at enteric pH (Pissinati & Pereira, 2003). Since the pH in the small intestine is between 4.5 to 6.5, the pH in the large intestine is 6.0 to 8.0, and the drug covering by Gastro-resistant capsules is released at pH 6.8, it is considered that the probiotic platform can be released only when it reach the location in large intestine (Maciejewski et al., 2020). In this case, we believe that covering the probiotic platform with gastro-resistant capsules can be a possible idea to successfully carry the probiotic platform to human intestines and release lumbrokinase since there is a huge difference between the pH of stomach environment and intestine condition.

Distance Between Colon and Intestine

While designing the probiotic platform, a serious problem was issued. The ideal position where E.coli Nissle lives and produces lumbrokinase would be the colon; however, the organ that absorbs nutrients and amino acids would be at the small intestine. This might lead to a problem that after lumbrokinase is produced, it would be transported to the small intestine without any protection from being nonfunctional or degraded. It may result in the final amount of absorption of lumbrokinase by small intestines and also further affect the effectiveness of the dissolve of thrombus though lumbrokinase. Currently, we still haven't figured out any solution that can improve this condition.

The Absorption Mechanism of Lumbrokinase

In most of the cases, protein is considered to be degraded into smaller molecules, which is amino acid, then being absorbed by intestine cell through different kind of mechanism, such as phagocytosis, pinocytosis, receptor-mediated endocytosis, osmosis, and channel protein. The destruction of protein structure can make it lose its functions.

However, Lumbrokinase proved that the protein structure can be completely kept during the absorption, making the protein well function when it enters the circulation system.

The Safety Concerns: Toxins Produced by E.coli

The safety of E.coli within the human body might be a concern since bacteria can be associated with health problems. In our team's project, we do not utilize E. coli for ingestion purposes. Instead, we administer probiotics because our project revolves around applying the oleate induction, which has been validated as viable in the laboratory, to probiotics. As for the issue of bacterial toxins, considering that a majority of bacteria contain endotoxins, we plan to conduct some experiments in future stages to ensure that the endotoxin will not affect the human body and only lumbrokinase is successfully absorbed by the intestine cell.

The Amount of Fatty Acid Triggering the Production

Considering the possibility that even a small quantity of fatty acid might induce the excessive production of lumbrokinase, resulting in the excessive breakdown of fibrin in blood clots and potential bleeding, we have formulated a plan to solve the problem. In our team’s idea, we planned to incorporate multiple fadR induction sites or the supplementary induction sites such as fadB with our pET-22b-LK vector. By implementing these measures, we aim to render the regulation of protein expression more stringent and less prone to unintended activation.

This prevention approach stems from our commitment to ensuring the safety and efficacy of lumbrokinase-based therapies. By introducing additional layers of control into the protein expression system, we aim to strike a delicate balance between enabling the therapeutic benefits of lumbrokinase and minimizing the potential risks associated with its overproduction. Through these considerations and design choices, we are working diligently to optimize the production of lumbrokinase for safe and effective applications in medical research and treatments.

Nutrients in Elders for E.coli Survival

It may be possible that the nutrients within elders might not be suitable or sufficient to let E. coli probiotics survive within the body. However, we believe this can also be tested and verified if we are able to conduct human experiments in further stages to assess the conditions favorable for E. coli survival in the human body, including factors like pH, temperature, nutrient levels, and other relevant factors. Also, in consideration that there are other probiotics in elder’s body, our team wished to add the probiotic platform into some kinds of bacteria that are more suitable for adapting the environment in the elders’ intestine in future stages.

Contribution

Even though our team hasn’t conducted any human or animal experiments to verify the effectiveness of the probiotic platform in vivo, we believe that the concept of probiotic platform is an innovative idea that might inspire other iGEM groups and scientists to apply similar concepts to other proteins and peptide medicine, and we believe that it would definitely contribute more to society. For instance, if the combination of probiotic platform and insulin can successfully conquer the obstacles we mentioned above, it can not only avoid the disadvantages of the current injection method, but also be considered as a competitor of the current ideal oral insulin which many scientists are concerned about.

References

CAO, Y., ZHANG , X., WANG, W., ZHAI, W., QIAN , J., WANG , J., CHEN, J., YOU, N., ZHAO , Z., WU, Q., Yuan，, xu, YUAN, L., LI , R., & LIU , C. (2013). Oral fibrinogen--depleting stroke prevention：results agent lumbrokinase for secondary ischemic from a multicenter,randomized，parallelgroup and controlled clinical trial. 4060–4065.

Pissinati, R., & Pereira, W. (2003). Enteric coating of soft gelatin capsules by spouted bed: effect of operating conditions on coating efficiency and on product quality. European Journal of Pharmaceutics and Biopharmaceutics, 55(3), 313–321. https://doi.org/10.1016/s0939-6411(03)00002-x

Wang, K. Y., Tull, L., Cooper, E., Wang, N., & Liu, D. (2013). Recombinant Protein Production of Earthworm Lumbrokinase for Potential Antithrombotic Application.

Baron, C. (2023, February). Distribution of the population in Taiwan from 2012 to 2022, by broad age group. Statista. https://www.statista.com/statistics/264727/median-age-of-the-population-in-selected-countries/

Baron, C. (2022, March). Countries with the highest median age 2021. Statista. https://www.statista.com/statistics/264727/median-age-of-the-population-in-selected-countries/

A survey on breakfast habits of generations in the Asia-Pacific region. Is your breakfast nutritious and balanced enough?. Herbalife Nutrition Taiwan. (2021). https://www.herbalife.com.tw/about-us/news-release/a-survey-on-breakfast-habits-of-generations-in-the-asia-pacific-region/