Team:UCAS-China/Description

Description

Background

Helicobacter pylori (H. pylori) is the only known pathogen that can settle in the stomach for a long time. It is one of the main culprits of gastritis, peptic ulcer, gastric lymphoma and other gastric malignant tumors. At the same time, this kind of dangerous pathogenic bacteria is also highly infectious, which can cause family aggregation infection by sharing tableware and food together.

The defects of existing treatment strategies are in a dilemma. The lack of drugs is mainly reflected in the resistance of H. pylori to triple therapy. The Meta analysis of China in 2017 showed that the average success rate of triple therapy was reduced to 71.1%, far below the acceptable rate of 80%. H. pylori also maintains a very rapid evolution rate, even after thorough cure, it has a recurrence rate of more than 3% and stronger drug resistance. In addition, the side effects of antibiotics and antacids used in triple therapy and quadruple therapy are serious, and long-term medication has great damage to human liver and kidney.

Besides, the co-evolution history of H. pylori and human beings is as long as 60,000 years, so the relationship between H. pylori and us is not a simple parasitic relationship, but symbiosis. According to the review of Blaser, the eradication of H. pylori has significantly increased the incidence rate of esophageal cancer and gastric acid regurgitation. Therefore, in the treatment of related diseases caused by H. pylori, the best strategy should be to control the number of it in a reasonable range.

SHEEP

On the asteroid B612, the little prince, who was tired of pulling up the baobab seedlings, got a sheep, which would chew off the sprouts on the ground in time, thus forming a ternary equilibrium ecosystem (or system) with the little prince and the baobabs. So for the treatment of H. pylori related diseases, we can also design a "SHEEP", so that it can eliminate the extra H. pylori at any time, and maintain the homeostasis of gastric micro-environment.

The full name of our “SHEEP” is Stomach Homeostasis Establishment for Eluding the Plethora of H. pylori. The therapy consists of three parts: the engineered bacteria that can produce drugs and target to kill H. pylori; an electronic capsule that can move freely in the stomach to take samples and photos to obtain the information of H. pylori infection; and a model that can simulate the balance process of engineering bacteria, pathogens and immune system, and predict for individualizing the treatment plan according to the infection situation.

Engineering Bacteria

Lactobacillus acidophilus is a common bacteria that can colonize in the stomach, and has already been used to treat gastrointestinal flora disorders. In addition, as probiotics, it can also effectively inhibit H. pylori.Our project chose Lactobacillus acidophilus as classic organism and constructed three modules in it to target and kill H. pylori: drug production module, ammonia sensor and biosafety module controlled by cold-induction on-switch.

LL-37 is a human cathelicidin with a good killing effect on H. pylori and can be expressed in prokaryotic cells. we put its gene under an anaerobic induced promoter so that we can primarily lower the off-target effect.

Figure 1: Gene of LL-37 under the anaerobic induced promoter, PfnrS

The function of ammonia sensor is to target H. pylori which can decompose urea into ammonia to neutralize gastric acid. When the engineering bacteria sense the increase of ammonia concentration, the activity of glnAp2 will be inhibited, then lysis protein after the inhibitory transcription factor (TetR-tetR/tetA promoters) can be expressed , so as to release drugs to kill H. pylori.

Fig.2 Engineering bacteria track Helicobacter pylori by monitoring ammonia concentrations in the environment

By combining our high-performance cold-inducible on-switch with the toxin system, we optimized the “kill switch” from its foundation last year. This year we chose to reduce the leaky expression of Doc toxin by fusing a hybrid tevS/ssrA tag to the C-terminal domain of Doc toxin to improve the performance of this switch.

Fig.3 Cold-inducible On-switch and DOC gene with SsrA

Hardware

We designed a capsule device for detecting engineered bacteria and verfied some modules design. It consists of a microcamera, float part of magnetic levitation system, sampling device and am external magnetic field control system. Microcamera connected with external device, which works in the dark, enables us take real-time images of stomach. Magnetic levitation system can locate precisely the capusule in the place we want, such as the lesion. Then sampling device can take gastric sample s and store it inside, waiting for analysis. Currently capsule robot can hardly complete the same work in stomach. Our design meets our needs and fills this vacancy. We expect to expand its functions further in the future.

Model

We look deep into the dynamics of Hp infection and further build a model with space dimension for Hp-SHEEP-Human System, in which we achieve integrated results and give detailed and rational guidance to our therapy. We build models for our biosafety module, mainly a cold-induced suicide trigger, and give a bistable solution. In our therapy modeling, we aim at an advanced and practical treatment and design a dose regimen for SHEEP therapy. What worth more mentioning is that by applying ITI(the information theory of individuality) to our model, we do the estimation for individuality in stable states in a more fundamental way.

Parts

This year, we made some improvement based on the cold-induced on-switch by changing the “Doc 1.0” gene with a “Doc 2.0”. Doc is a bacterial toxin protein, the cold-inducible kill switch utilizes host temperature as an indicator of bacterial escape, and turns on the expression of Doc at the temperature below 34 to achieve the biosafety. However, the leakage of DOC toxin protein cause a high escape rate. To solve this problem, we we chose to reduce the leaky expression of Doc toxin by fusing a hybrid tevS/ssrA tag, which is used for proteasome-dependent degradation, to the C-terminal domain of Doc toxin. When the temperature is lower than 34°C, TEVts, as a component of cold-inducible switch, will be highly expressed, and cleave tevS to release ssrA tag from Doc protein. Through experimental measurement, the escape rate of engineering bacteria has decreased from 10^(-3) to 10^(-6).

Human Practice

No man is an island even the waves roan in foamy anger. Although our ability of communication is restricted due to the pandemic, we managed to stay in touch with the world. By interviewing multiple entities like companies and doctors, interacting with publics through a questionnaire, inspiring future academics and communicating with our iGEM communities, we obtained a better idea of how our technology can be applied in society.

We always believe that it is of great importance to view our project from as many different aspects as possible, which can give us the general idea about what the world think about our work and what can we improve to better serve the world. This year, we refreshed our HP organizing methodology by a “problem -solution” loop: Not only seek answers with certain questions, but also discover problems behind our expectations.

Reference

[1]Scott MG. et al., 2002. The human antimicrobial peptide LL-37 is a multifunctional modulator of innate immune responses. J Immunol. 169(7):3883-91.

[2]Higashikuni, Y.; Chen, W. C.; Lu, T. K., Advancing therapeutic applications of synthetic gene circuits. Curr Opin Biotechnol 2017, 47, 133-141.