Team:UCAS-China/Poster

Poster: UCAS-China

MEET OUR SHEEP!

Background

Helicobacter pylori (H. pylori) is a culprit of many digestive system diseases. However, the existing therapies emphasising on cleansing H. pylori, have many drawbacks such as lack of drugs and high recurrence rate. This year, inspired by the idea of H. pylori-human co-evolution, our team developed a therapy called SHEEP, which is capable of controlling the number of H. pylori in a reasonable range, instead of just wreaking havoc.

Design

The engineering bacteria is important for our therapy as the targeted killer of H. pylori. We loaded drug produce module and ammonia sensor to ensure that he can destroy the enemy precisely and efficiently. Meanwhile, we used the cold-inducible on-switch designed by UCAS-China last year and improve it to avoid biocontainment.

Here we decided to send Lactobacillus acidophilus to complete this mission. This kind of probiotics can live in our stomach and inhibit H. pylori naturally. Through literature research we got the protocol of synthetic biology manipulation, and the final demonstration will be implemented in it.

The weapon we gave our warrior is cathelicidin LL-37, which has great inhibitory effect on H. pylori. The paragraph is from the work done by Koji Hase in 2003, it shows that the antimicrobial activity of LL-37 is significantly high. And from Gu Yinxia’s work we confirmed it can be characterized in prokaryotic expression system.

Ammonia sensor is assembled by ammonia-sensitive promoter glnAp2, “NOT gate” transcription factor TetR, and lysis gene. When the ammonia concentration is high outside the cell, glnAp2 is off, so that tetR/tetA promoters will not be turned off by TetR and the lysis gene work.

Cold-inducible on-switch is a wonderful idea for biosafety. Last year, our team developed this system and tested it successfully in E. coli. This year, we improve this switch by optimize the Doc gene. Doc with TEV site sequence and degradation tag SsrA can achieve lower escape rate by decreasing leaky expression.

Modeling!

What is Hp? What is the symbiosis between human and Hp? What would happen if the fragile balance is broken? How can we make the balance more robust? How should we engineer a bacterium to realize such goal? How to ensure the biosafety of our project? These what and how questions were raised and already perfectly answered in previous parts. “Whats” inspired us, and “hows” led us to the initial concept of SHEEP. But there are still important questions left—Why? Why there is a symbiosis between human and Hp and why it is so easy to be broken? Why SHEEP can make the balance robust and why SHEEP is biosafe? Only by answering these whys can we understand the ongoing process in the human stomach, and further, can we demonstrate the efficiency and biosafety of SHEEP. These whys have been all answered by our modeling.

Our model is more than just answering these “whys”. In fact, as our modeling go deeper, we discover numerous unpredicted behaviors of the system. Therefore, more questions are raised and new methods are involved, making our model capable to reach the true nature of the system. Here we would like to remark two firsts of our model. As far as we know, our model is the first to associate the asymptomatic infection of H. pylori with the semi-stabile state of the Hp-Human system and the first to apply the information theory of individuality (ITI) to a concrete system.

Model for Design

We model the biosafety module of our design. This module is designed to trigger suicide of engineered bacteria if and only if they leave human bodies. We demonstrate that our design can form a bistable system triggered by temperature, which implies that the suicide process would irreversibly starts once the bacterium is exposed in room temperature and even if it later returns to a warm environment. The result can be concluded with the static state solution of the model.

Fig.1 Concentration of CI434 and TEV when temperature changes. It shows that the suicide process would irreversibly starts once the bacterium is exposed in room temperature and even if it later returns to a warm environment.

A concrete example of the functioning of the module is showed below. The temperature starts form and ends in the body temperature, but the switch is triggered thus the initial states and final states differs.

Fig.2 A concrete example of fig.1, showing that the initial states and final states differs even if the temperature ends in the body temperature.

Theoretically, the switch can already ensure the biosafety of SHEEP. However, in practice, the temperature in human stomach is not a constant, especially if patients wanted to have an ice-cream. An “ice-cream analysis” is done to estimate what kind of fluctuation would trigger the switch.

Fig.3 The "ice-cream analysis" showing how temperature affects in human stomach.

To make our module more robust, we have designed an “and” gate for toxin release. With simulation, we further calculate the leakage of bacteria to be 10^-5, safe enough for practical usage. This result perfectly explains the experimental statistics.

the Dynamic of Hp-SHEEP-Human System

To fully understand the relationship between Hp and human, and moreover the relation between Hp, SHEEP, and human, we examine the complicated biosystem and finally work out a dynamic that illustrates its primary characteristics. It is a dynamic that includes the interaction between Hp, nutrition, immune response, inflammation, drugs, and moreover, our engineered bacteria, with it we can simulate any question we are interested in.

Fig1. interaction between Hp, SHEEP, and human

After mathematization, we simulate the behavior of the Hp-Human system under different conditions. Our results clearly show the bistability of the Hp-Human system. With the very same set of parameters, the system has two stable states. One we name as the incubating state. It has a relatively low Hp population as well as its inflammation and immune response level. The other we name as the pathological state. It has a high Hp population, serious inflammation, and a maximum immune response level. That perfectly suites the reality—the very same person infected can either have no symptoms or be critically suffering. Deeper analysis shows that the incubating state is semi-stable, if the environment fluctuation exceeds the threshold, the system would irreversibly fall into the pathological state. Our analysis further shows that in most cases we cannot expect the incubating states to last for our entire life, since the state transition may be triggered by merely a single big feast. It is also found that poorer nutrition conditions can increase the stability of the incubating state, which explains the fact that infected people in developed areas usually have a higher risk of suffering from critical symptoms.

Fig2. Effect of nutrition on the growth of HP

Of course, things change if we apply the system with SHEEP. Engineered La could establish a new stable state together with Hp, which we name as the homeostatic state. This state has an even lower level of inflammation and immune system activation when compared to the incubating state. The homeostatic state is robust to fluctuations. Even doubling the nutrition condition would not break the balance, let alone one or two feasts. With SHEEP, we no longer need to worry about anything.

Model for Therapy

Merely knowing about what happens in our stomach is not enough, making SHEEP really an advanced, and practical treatment is our final goal.

Our model suggests traditional treatments unavoidably need high drug concentrations while they all take a very long time to eliminate all the Hp. However, with the dose regimen we design for SHEEP, less drug is needed and the total duration of the therapy is also shortened to 10% of the duration of the traditional treatment. Here is an example of how SHEEP shortens the duration of therapy.

Fig.1 A comparison between traditional treatment and SHEEP therapy. With the same duration of therapy, SHEEP cured Hp but traditional treatment failed.

For further exploration, we will apply the family evolutionary tree and machine evolution to extend our therapy method. Safe, efficient, swift, and further, intelligent, that is our SHEEP.

Estimation for Individuality in Stable States

We have found three stable states of the system. A question naturally raises—are these stable states the result of mutualism or they are induced by one-species’ dominance? The information theory of individuality (ITI) established by David Krakauer provides a general method to work on such problems. Here we will skip the detail of the theory and only introduce its idea. ITI take a system as signals evolving with time and take the interaction as information flow. With a technique called decomposition of mutual information, one can separate the information flow between particular parts of the system, which characterizes the individuality of the respective part.

Fig1.Time Evolution of the System

Fig2.Information decomposite

We run stochastic simulation around the stable states and applied the system with ITI analysis and here is what we learn. In the incubating state, Hp and humans are both of high organismal individuality and are independent of each other. Both Hp and humans adapt the situation well but as they rely on innate coding rather than encode through ongoing interaction, in the face of huge pulses the state cannot keep its balance. For the pathological state, Hp and humans are still both organismal individuals. However, their relations with the environment differs. Analysis implies that Hp is dominating the state while humans have to make extra efforts to survive the state. Finally, for the homeostatic state, all Hp, SHEEP, and humans are of high colonial individuality. They all encode through ongoing interaction, which makes the homeostatic state capable to overcome extreme fluctuation.

Hardware

Our hardware intends to design a real-time observation and sampling system in the stomach.

Hardware Design

The whole working system is composed of a capsule working in vivo and a control magnetic field working in vitro. There are mainly three parts in the capsule, the micro camera, the sampling device and the floating part of the magnetic levitation system.

Micro camera

The micro camera is designed to take pictures in real-time in the dark and transmit the images of various parts of the stomach, especially the lesions, for doctors to diagnose.

Sampling device

We designed a wheel type structure for the sampling device, which can be remotely controlled to sample and store the samples in a respective capsule.

Magnetic Levitation System

The magnetic levitation system is divided into two parts, which are a float inside the capsule and a magnetic field controller outside the capsule. Through such design, we can control the position of the capsule in the body by changing the magnetic field in vitro, and take photos and samples of various parts of the stomach.

Hardware Result

We verify some modules’ function of the device, and make the capsule move freely in the stomach in three-dimensional space. In the theory, it can accurately reach any position in the stomach to complete the detection and sampling work.

Hardware Future

Our expectations for this capsule are as follows:

1. Minimizing the volume of the capsule to make it easier to be swallowed in.

2. Make use of microfluidics to enable our capsule to detect multiple bacteria in real-time.

There has been some intestine capsule robot designs with powerful functions. Our team studied it last year. If we could combine two designs, this hardware design would be much more competent.

💡 ←→ ⚙

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.

💡What is a better H.pylori therapy?

After interviewing clinical expert, we aimed at the lack of targeting medicine and turn out to be our design of ammonia sensor that can specifically detect and target H.pylori.

💡What constrains our SHEEP?

From our SHEEP questionnaire, we heard public’s complaint over current transplanting methods, which became an inspiration and a strong motivator in our capsule robot design.

⚙Stability is your advantage over current treatments.

TreatGut’s idea of replacing “elimination” sparkled us to focus more on the homeostasis establishment, which has made the novelty of our project.

⚙A modifiable design worth a better try.

When we were struggling to cut the cost, DaXing led us a brand new path to approach cost-effectiveness by sharing the cost, rather than just lowering it.

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