Overview

The goal of our project is to arrive at a system capable of producing nutritious, delicious, and highly user-free meal replacement foods and the system should consist of a meal replacement machine, "FitYo" mobile app, and lactic acid bacteria modified by synthetic biology technology.

We choose Lactobacillus bulgaricus as our engineered bacterium, which is a probiotic that has a certain research basis in the field of synthetic biology and has good application prospects. According to the gene lines we design, under 4 different light-temperature combinations, our engineered bacteria can synthesize different flavor substances (such as vanillin) or functional substances (such as ovalbumin) to achieve adjustability in substance production. In addition, we design a suicide switch controlled by oxygen concentration to ensure that engineered bacteria will die when they escaped from the manufacturing machine, so there will be no biological pollution and environmental pollution.

For the production of meal replacements, we improve the original fermentation tank device and design meal replacement manufacturing machines suitable for sports, home, and office use. Users can use machine buttons or "FitYo" mobile app to choose desired meal replacement functions such as weight loss or muscle gain and desired flavors such as nutty flavor or vanilla flavor, then the meal replacement manufacturing machines can be adjusted to have specific light and temperature conditions, in which the engineered bacteria can take milk as fermentation substrate and produce target functional substances and flavor factor combinations, so as to create meal replacement milkshake that meets the users' requirements.

In addition, we also hope to guide the public to realize the importance of gastrointestinal homeostasis to human health through edutainment, so we design a single-player game “Oh My Gut!”, which is based on the digestive process and interspersed with the introduction of our meal replacement products, so as to further promote our products and concepts.

Experiment

Abstract

Taking into account the safety factors and the ease of gene editing, we chose the chassis bacterium for our FitYo project - Lactobacillus delbroeckii subsp. bulgaricus (L. bulgaricus) which is widely used in the fermentation process of food products. It is relatively easy to be genetically manipulated and is not harmful to human health.

A quality meal replacement product should be fat-reducing, nutritious, and delicious. Based on this concept, we plan to enable the engineered bacteria to synthesize functional substances and flavor factors. After much literature research and discussion, we selected three proteins with specific functions - RuBisCO, Amuc_1100 (outer-membrane protein of Akkermansia muciniphila), and ovalbumin. We also chose three flavor factors - 2-phenylethanol, , and vanillin.

We hope our project to be customizable. So we designed a 2×2 transfer switch with a light-controlled bistable switch as the backbone, combined with a temperature-controlled RBS element, which can switch between four states. It allows the users to produce meal replacement milkshakes with different ingredients by changing the temperature and light conditions.

In addition, biosafety is an issue that we must consider. To prevent leakage of the engineered bacteria into the environment, we designed an oxygen-controlled suicide switch based on the HIP-1 promoter with the ccdA/ccdB toxin-antitoxin system. In this way, when our engineered bacteria leak into the atmosphere with normal oxygen concentration, the engineered bacteria will initiate the suicide program and prevent the occurrence of genetic contamination.

Project description

Meal replacement has been developing rapidly these years. While its market is in fast expansion, the problems of these products remain, such as high costs, nutritional imbalance, undesirable flavor, and lack of choice for customers, etc. In this project, we manage to design a system through synthetic biology techniques to manufacture meal replacements that can meet the individual needs of potential customers with high cost-performance as well as satisfactory efficacy.

The meaning of synthetic biology is to use biotechnology to obtain artificial biological systems that do not exist in nature and to allow such systems to operate in an orderly manner under regulation. In our envisioned "FitYo" microbial meal replacement production system, the microorganism as the core component should have the following characteristics:

1. Non-toxic:

the microorganism itself does not produce toxic substances that are harmful to human health.

2. Able to act as a food producer:

the metabolites of the microorganism can be used as nutrients to provide energy or perform other beneficial functions for human health; in addition, the microorganism should be able to produce substances with specific flavors to optimize the dining experience.

3. Artificially controllable material production:

To further diversify the functions and flavors of our meal replacement, the microorganism should have specific regulatory elements that allow us to determine the combination of functional substances and flavor factors it produces according to our needs.

4. Biosafety:

Although the microorganism we use is non-toxic to the human body, we still should take several measures to prevent them from escaping from the meal replacement production system. Therefore, the microorganism should contain a suicide switch that can spontaneously kill itself after escaping to the external environment.

In real life, several kinds of microorganisms in nature can meet some of the above requirements (e.g., lactic acid bacteria are non-toxic to humans; Akkermansia Muciniphila can produce membrane proteins with weight loss functions), but we cannot find microorganism that meets all of the above requirements at the same time. Therefore, we need to find the parts that meet our requirements in nature and build specific genetic circuits, then assemble the parts into an organic whole by means of synthetic biology. With the help of synthetic biology principles and methods, our engineered bacteria can combine the above four features and produce safe, delicious, and diversified meal replacement products efficiently and conveniently.

Inspirations

2×2 Bistable Switch

We decided to construct a 2×2 bistable switch as a basic frame because this kind of switch is efficiently manipulated and the boundaries between each state of these switches are clear. Thus, we choose natural light and temperature to make sure our bistable switch is regulated in a nontoxic and accessible way. We were inspired by the light-sensitive switch design created by Team Toronto 2017. This switch mechanism is under the control of the LacI-LOV, in which it is dimerized and bound to DNA in darkness, and monomerizes in light.

RNA Themometers

During the suitable RBS designing process, I got help from my tutor Xiujie Wang. She helped me to build the overall idea. She advised using the antisense RNA and inserting the antisense sequence of RBS into a plasmid with a controllable promoter. When the promoter works and transcripts antisense RNA out, this RNA will combine with the RBS and therefore stop the assembly of the ribosome, restraining the process of translation.

Synthesis of 2-Phenylethanol

The initial design of the genetic circuit for the synthesis of 2-Phenylethanol includes several reactions. Then, due to a chance encounter, I learned that the manganese pathway present in prokaryotes is more efficient in synthesizing aromatic amino acids. So I modified my genetic circuit and simplified it to a multi-cistron structure with only three genes, thus reducing the metabolic stress on the bacteria.

Flavors

The tastes we favor vary from each other. Vast arrays of yogurts on the market inspired us to produce flavor substances for better tastes. Many of our members complained about fixed flavors from food production factory, thus, we realized the importance of suiting the taste of consumers as well as maintaining users’ autonomy. We must thank Professor Jiangyun Wang for encouraging us to produce flavor substances and giving us precious resources in this direction. We created this part and stepped in this direction based on his advice.

Suicide Switch

We plan to store and work with the engineered bacteria in the of the meal replacement machine, so we designed suicide switches that are sensitive to oxygen concentration to ensure biosafety.

We choose the ccd system to design an effective self-killing system. The CcdA/CcdB Type II Toxin-antitoxin system is one example of the bacterial toxin-antitoxin (TA) system. The ccd system (control of cell death) of the F plasmid encodes the CcdB protein (101 amino acids; toxin) and the CcdA antidote (72 amino acids) (Madl et al., 2006). In recombinant DNA technology, the ccdB gene is widely used as a positive selection marker (e.g. the Invitrogen's Zero Background and Gateway cloning vectors).

RuBisCO

The inspiration for selecting the functional substance Rubisco came from a survey we had seen before: Neel Ocean and his team found that a person's mental health is directly proportional to the amount of fruit and vegetables they eat each day. We conducted further research and found that Kimura et al.'s study in 2018 showed that Rubisco proteins in green leafy vegetables are digested in the intestine to produce peptides with anti-anxiety effects. Then we choose Rubisco anxiolytic-like peptides (rALPs) with anxiolytic effects as one of the functional substances.

Hardware

We designed and made a portable and extensible IoT (Internet of Things) device to provide a needed condition and produce the Fit-Yo with the help of engineering bacteria. It also served the experiment with user-defined parameters, including light and temperature. We used some modules to implement the functionalities needed to test the engineering microbe. We also proposed and designed some future improvements. Experience in safety and sources is available, aiming to help future teams create their own and make improvements.

Software

This year our software covered a variety of fields for our project, including an entertaining science game, an APP for our hardware machine, and a convenient tool for creating wiki.

To better match the hardware design and contribute to a better ecosystem for our project, we created an APP using Python language based on kivy and kivymd library. This APP allows convenient manipulation of our meal replacement machine through WiFi and enables the one-stop purchase of our products and services within an APP. The APP is highly compatible with multiple platforms, including Windows, Linux, macOS, Android, and iOS.

We have also developed a science breakout game called "OhMyGut" based on the pygame library in Python. Players can operate the game's main character called changbao through the main digestive organs of the human body to complete different challenges while gaining relative scientific knowledge. The game has a total of 8 levels, each level has a different style of play and role.

Furthermore, we've developed a python program that converts Word files to HTML files automatically. By just setting the path of CSS and javascript files and changing the constant head part of the designed wiki, it can convert the content of a Word file to an HTML according to the input design style.

Model

We modeled three modules for production, biosafety, and human-related weight management. The production part is divided into three parts modeling the bistable switch, temperature conversion, and minimum time algorithm, while the biosafety part is modeling the oxygen suicide switch. The weight management component uses statistical methods to evaluate a one-week weight loss program for people with different characteristics to predict the functionality of our product.

Challenges

COVID-19 is a sudden and continuous disaster. In China, for the health of most people, the anti-epidemic policies have never been relaxed. Due to the multiple rebounded epidemic, many difficulties come one after another.

COVID-19 has made us not enough time to communicate and cooperate.

The first difficulty came in February 2022. New cases of COVID-19 came out and closed management was implemented in our school. We have to stay in the dorm, living and taking lessons. There is no available time and space for all our members to have discussions and skill training. The greatest difficulty came in late May. Because of a Large-scale, to some extent, epidemic backlash in Beijing, many students went to their hometown to avoid outbreaks of aggregated pandemic outbreaks on campus. Therefore, though we prepared our program early in the winter of 2021, there are not enough members to conduct experiments throughout June, July, and August. In almost 4.5months, we were just able to discuss online and do some research.

COVID-19 has made it not convenient for us to do experiments as like before.

Firstly, it spent more time for us to buy tools, materials, biologicals, etc. Biotechnology companies needed time to recover from the epidemic and logistics cost more time. Secondly, also most importantly, we were not able to go to other research laboratories to use their experimental apparatus, especially anaerobic incubators, flow cytometry, mass spectrometer, HPLC, and microbial identification systems, as we expected. Because of the restrictions on travel based on the pandemic situation, we were often prohibited from leaving the campus and a significant number of group members failed to enter the campus or conduct experiments in laboratories.

COVID-19 has made our HP hard to conduct.

It is difficult for us to launch large-scale social campaigns offline, such as conducting extensive public surveys and paying a visit to famous local manufacturers. Although we think it may not be as effective as offline, we are attempting to achieve these goals online and trying our utmost to receive better effects.