Overview

There are many novel ideas and designs in our project that can be helpful for future iGEM teams. These contributions cover standard components (Parts), hardware, software, modelling. On this page, we will not only provide the final design, but also describe how the relevant construction and testing were done. We believe that our designs and our experiences can be useful to future iGEM teams and even the synthetic biology community.

2×2 transfer switch

To obtain diversified protein products by changing the environment condition conveniently, we created a 2×2 transfer switch and established a bistable switch controlled by light and two different RBS,which could work at high and low temperatures respectively. When considering how to construct the bistable switch, two classic bistable genetic circuits: l gene regulatory circuit (operating primarily by the action of two regulatory proteins CI and Cro) and functional circuits in TL phage (through operating three promoters: PRM, PR, and PL)(Atsumi S et al., 2006) were abandoned for safety and practicability reasons. Inspired by iGEM17_Toronto, we focused on regulative factors which are nontoxic and accessible such as light or temperature and got our frame of the bistable switch.

The bistable switch consists of the new-type optical integration protein LaclLOV and cI restraining factor with LVA degradation label, which were in different pathways. When exposed to blue light, the pathway with LaclLOV was restrained with the other activated. On the other hand, the restraining process of LaclLOV was restrained. Thus the proteins were expressed normally.

The other part of the conversion switch is temperature-sensitive RBS inserted to the loci downstream of the bistable switch’s genetic circuit. We have two plasmids PET28a with the bistable switch above, and two types of RBS working in different temperatures. One can work in a higher temperature (≥37℃) while demonstrating low activity when the temperature is lower. This part was based on the former search early this year, and the original version was from the team IGEM19-Rice. Also, the RBS works well in a lower temperature (27℃) and loses activity when the temperature rises. We got the idea for this part from the team iGEM20_UNILausanne, and more detailed information from the paper De novo design of heat-repressible RNA thermosensors in E. coli by Allison Hoynes-O’Connor, Kristina Hinman, Lukas Kirchner, and Tae Seok Moon. There are a group of RNA thermosensors, 24 in total, and we used the RNA thermosensor F2 in our project. We think future teams can also refer to the paper and use other types suitable to their project.

With our conversion switch, i.e., our combination with a bistable switch and temperature-controlled RBS mentioned above, this composite part can work in four different conditions to produce four nutrient substances. The four conditions are high temperature with blue light, low temperature with blue light, high temperature with no blue light, and low temperature with no blue light.

Moreover, the 2×2 transfer switch can be used in many conditions apart from our work, and we believe it will benefit future teams in the field of controlling the expression of downstream genes. The combination of light-controlling and temperature-controlling makes it feasible and easy to achieve to control the transcription and translation process. Also, this switch is very flexible as we just need to change the controlling part to change the control condition.

Flavor Substance

It is of significant importance that our team created a novel concept for the production of meal replacement that bacteria and biosynthesis methods could replace machines and chemical synthesis methods to a large extent. We believe biosynthesis technology could be taken advantage of in far more aspects than nowadays. Moreover, it's a trend that productions defined by users themselves enjoy growing popularity. Consumers could design the taste of their yogurt and enjoy a high degree of freedom with our product.

Vanillin

Vanillin(4-hydroxy-3-methoxybenzaldehyde) is one of the most widely used flavoring agents in the world. It is often used in foods, perfumes, beverages, and pharmaceuticals. It was first extracted from the seedpods of orchids, but most market demand couldn’t be met without artificial synthesis. The chemical synthesis process of vanillin is not environmentally friendly. An alternative is the use of biotechnology to produce vanillin from natural substrates.

We designed a pathway to produce vanillin after literature research. It is accessible carbon sources that act as the ingredient of this pathway. Producing vanillin from ferulic acid could be much easier, but biosynthesis of vanillin from simple carbon sources is more attractive because they are cheaper and more readily available. The production of vanillin involves nine enzymes on three plasmids. The first plasmid(BBa_K4256011) consists of four enzymes aimed at L-tyrosine overproduction. The second one(BBa_K4256012) includes three enzymes involved in the production of ferulic acid from L-tyrosine. The last plasmid(BBa_K4256013) with two enzymes has the function of converting ferulic acid to vanillin. This pathway and plasmid design come from reference literature(Ni et al., 2015; Nakagawa et al., 2011).

2-Phenylethanol

Phenylethol is a type of compound that possess the aroma of rose petals. Lactobacillus itself provides the pathway for synthesizing Shikimic acid(Cristina Serrano-Amatriain et al., 2016), which could be further converted to phenylalanine. 2-Phenylethanol could be directly synthesized from phenylalanine.2-Phenylethanol has a wide range of applications. It serves as an antimicrobial, antiseptic, disinfectant, and an aromatic essence and preservative in pharmaceutics and perfumery. In our project, we target to use it as an aromatic essence for flavoring our product.

The initial design of the genetic circuit for the synthesis of 2-Phenylethanol includes several reactions, including the synthesis of phenylalanine from a simple carbon chain and further synthesis of 2-phenylethanol.

Then, due to a chance encounter, we learned that the Shikimic acid pathway present in prokaryotes is more efficient in synthesizing aromatic amino acids. So we modified my genetic circuit and simplified it to a polycistron structure with only three genes(BBa_K4256203、BBa_K4256204 and BBa_K4256202), thus reducing the metabolic stress on the bacteria.

3-methylbutanal

Lactococcus lactis has been successfully applied as a starter to increase the level of 3-methylbutanal produced during the ripening of cheese. It means that it could very well be expressed by engineered Lactobacillus bulgaricus. The ideal concentration range of 3-methylbutanal in hard Cheddar cheese is 73 to 210 μg/kg, which is responsible for a rich nutty flavor (Afzal et al., 2017). So researchers can do more stretching experiments to find the best concentration if they want to add this flavor to their meal replacement. Moreover, to fit different tastes, researchers can find ways to achieve adjustable concentrations in the engineered chassis bacteria. In terms of the pathway to synthesize 3-methylbutanal, there is both a direct pathway (Mazé et al., 2010; Wei et al., 2013; Afzal et al., 2017) and an indirect pathway (Ward et al., 1999; Afzal et al., 2012; Luo et al., 2018). If other people want to use it, choose the proper one!

Functional Substance

Amuc_1100

Akkermansia muciniphila, or AKK for short, was discovered in 2004. AKK is an oval-shaped Gram-negative bacterium isolated from the human intestine. It is widely distributed in the human intestine as a mucus-degrading bacterium. Amuc_1100, a membrane protein synthesized by AKK bacteria, has been shown to have some weight loss and immune benefits and is not harmful to human health.

Amuc_1100 not only reduces host adiposity, insulin resistance, and dyslipidemia but also promotes 5-hydroxytryptamine (5-HT) synthesis through signaling with TLR2 (Toll-like receptor), an important neurotransmitter that regulates gastrointestinal function. Acum_1100 can speed up the synthesis of 5-HT, a slow step in 5-HT synthesis, in RIN-14B cells with the help of Tph1 enzyme, and can reduce the expression of SERT in Caco-2 cells through direct interaction with TLR2, which ultimately promotes 5-HT synthesis. 5hydroxytryptamine (5-HT), also known as serotonin, is a multifunctional bioindolamine. It serves as a key neurotransmitter of the nervous system, as well as a key signaling molecule regulating the function of the gastrointestinal tract and other organs. Its functions of regulating gastrointestinal motility are related to the realization of the AKK weight loss effect.

Ovalbumin

As the main protein component in egg whites from poultry, ovalbumin, which consists of 385 amino acid residues and an acetylated N-terminus, is a significant, comprehensive, and reliable source of daily nutrition intake for humans. It has been a common diet since ancient times and has become an ideal functional ingredient and nutritional supplement in food industries.

Due to its commonness, ovalbumin has been studied in detail by researchers during the past decades. And it is involved in a wide range of subjects for structural or immunological analysis. Researches indicate that ovalbumin also shows anti-bacterial and anti-hypertensive activities, which are of non-negligible advantages to human health.

Retrieved from the NCBI database initially and codon-optimized subsequently, the chicken ovalbumin sequence (Gallus gallus) was expressed in E. Coli (pET-28a(+) plasmid) and synthesized by professional institutions.

RuBisCO

RuBisCO (ribulose-1,5-bisphosphate carboxylase), with a molecular weight of about 530 kD and generally composed of 8 large and 8 small subunits, is a critical enzyme both in plant photorespiration and photosynthesis that determines the rate of carbon assimilation. It is also known as the most abundant protein on earth.

Articles reported that the oral digestion of spinach with pepsin-trypsin sequentially treating the large subunit of RuBisCO can achieve certain anxiolytic effects in mice. Some peptides in the digestion products can activate the central δ-opioid receptor, which can play an anxiolytic effect through the gut-brain axis.

Considering that the RuBisCO large subunit has about 80% homology among different plants and is not toxic to humans, we introduced the RuBisCO protein large subunit gene from Cucumis sativus into our engineered bacteria. Under the regulation of the gene element, the RuBisCO protein large subunit could be expressed in the engineered bacteria and added to the meal replacement products as a functional substance.

Suicide Switch

We designed an oxygen-sensitive suicide switch based on a ccdA-ccdB toxin-antitoxin system.

Most prokaryotes contain many toxin-antitoxin modules. These modules generally consist of a pair of genomes encoding two components, one of which is a stable toxin protein, and the other is an unstable homologous antitoxin. The ccdA-ccdB toxin-antitoxin system we use is a relatively non-toxic killing switch for human and other mammalian cells. The mechanism of action of the toxin ccdB is to prevent DNA spinase from functioning, which leads to double-strand breaks in DNA; ccdA prevents the toxicity of ccdB by forming a compact ccdA-ccdB complex. In our suicide switch, the toxin ccdB coding region is inserted into the loci downstream of the T7 promoter, and the antitoxin ccdA coding region is placed under the HIP-1 promoter regulated by FNR (a transcriptional regulator that contains [Fe-S] clusters of sensing proteins), the activity of which is controlled by the concentration of oxygen present in the environment. Thus, when engineered bacteria are exposed to atmospheric concentrations of oxygen, the switch is activated and the amount of toxin proteins in the cells increases dramatically, effectively killing escaped engineered bacteria.

Visit our design page to know about our genetic circuits.

Visit our parts page to know about our part numbers and details.

Hardware

We designed, made, and open-sourced the FitYo-maker. It can provide user-defined parameters, including light and temperature. The two-layer structure is extensible.

We left the machine running for a long time to test its stability. With the feedback from users, we improved the temperature-controlling speed and safety. We documented some experiences while choosing heaters and wires, which may help future teams avoid fire hazards.

We added an automatic data recording program. It runs on a user device connected to the FitYo-maker. The IoT FitYo-maker is designed to take advantage of JS libraries in the future, allowing future teams easy to implement more functions based on the device we made and present IoT technology.

All the designs, including the 3D model, the main list of standard components, wiring, and the source codes are open-source by the link on our wiki page, which may help reproduction by other teams.

We also design some methods to improve the function of this extensible machine. We hope the concept of open-source equipment will benefit the whole of society.

Software

Free Science breakout game: Oh My Gut

We have designed a science breakout game to players learn the science of the human digestive system in a pleasant game experience. It contains a variety of ways to play, there are solving puzzle levels like a maze, as well as adventure parts that are similar to Super Mario. All ages and kinds of people can have fun through our game. Moreover, the design of each level is based on the characteristics of the corresponding digestive organ, while every character’s attributes setting also takes sufficient scientific basis into consideration. Everyone can download our game and learn more about our project through the web page of our game.

Figure X: Oh My Gut Game.

As the game is based on the pygame library, we have also open-sourced our game’s python codes in the GitLab.] Other teams can download the exe files to play our games and make changes and innovations to the game.

Compatible Application based on Python

This year the development of our APP was carried out on the Kivy and KivyMD libraries. Different from the general team choice of APP development platform - Android studio, we innovatively choose a more scalable and flexible python language to design our app. What’s more, based on the Kivy library we can pack all the programs into executable files compatible with multiple platforms, including Windows, Linux, macOS, Android, and iOS.

Useful wiki tools: Word2html

In the process of building our wiki, since most of the team members did not know how to write web pages and are not familiar with the markdown format, they wrote relevant Word documents and our members for designing wiki then converted them into html files. This conversion process would take a lot of time and energy if all the pages were handled manually, so we developed a python-based Word2html program. By just set the path of css and javascript files and changing the constant head part of designed wiki, it can convert the content of Word file to a html according to the input design style. Hopefully, future iGEM teams can benefit from this highly-efficient tool.

References

Atsumi S, Little JW. A synthetic phage lambda regulatory circuit. Proc Natl Acad Sci USA. 2006 Dec 12;103(50):19045-50.

Ni, J. et al. Mimicking a natural pathway for de novo biosynthesis: natural vanillin production from accessible carbon sources. Sci. Rep. 5, 13670; doi: 10.1038/srep13670 (2015).

Nakagawa, A. et al. A bacterial platform for fermentative production of plant alkaloids. Nat. Commun. 2:326 doi: 10.1038/ncomms1327 (2011)

Cristina Serrano-Amatriain et al. (2016). Folic Acid Production by Engineered Ashbya gossypii. Metabolic Engineering, 38pp. 473-482.

Afzal, M. I., Boulahya, K. A., Paris, C., Delaunay, S., & Cailliez-Grimal, C. (2013). Effect of oxygen on the biosynthesis of flavor compound 3-methylbutanal from leucine catabolism during batch culture in Carnobacterium maltaromaticum LMA 28. J Dairy Sci, 96(1), 352-359.

Afzal, M. I., Delaunay, S., Paris, C., Borges, F., Revol-Junelles, A. M., & Cailliez-Grimal, C. (2012). Identification of metabolic pathways involved in the biosynthesis of flavor compound 3-methylbutanal from leucine catabolism by Carnobacterium maltaromaticum LMA 28. Int J Food Microbiol, 157(3), 332-339.

Plovier H., Everard A., Druart C., et al. A purified membrane protein from Akkermansia muciniphila or the pasteurized bacterium improves metabolism in obese and diabetic mice[J]. Nat Med, 2017, 23(1): 107-113.

Clara D, Amandine E, Céline D，et al. Supplementation with Akkermansia muciniphila in overweight and obese human volunteers: a proof-of-concept exploratory study [J]. Nature Medicine, 2019.

de RUYTER P G, KUIPERS O P, de vos WILLEM M. Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin[J]. Applied and Environmental Microbiology, 1996, 62(10): 3662-3667.

Kimura, Saeko, Tomoki Uchida, Yuki Tokuyama, Mayumi Hosokawa, Junya Nakato, Atsushi Kurabayashi, Masaru Sato, et al. "Identification of Rubisco Anxiolytic-Like Peptides (Ralps) by Comprehensive Analysis of Spinach Green Leaf Protein Digest." Biochemical and Biophysical Research Communications 505, no. 4 (2018/11/10/ 2018): 1050-56.

Mengesha A , Dubois L , Lambin P , et al. Development of a flexible and potent hypoxia-inducible promoter for tumor-targeted gene expression in attenuated Salmonella.[J]. Cancer Biology & Therapy, 2006, 5(9):1120-1128.