© 2020 Team:Fudan/Poster
Make calcium supplement sustainable
![abstract](https://static.igem.org/mediawiki/2020/d/d2/T--Fudan--Poster_bone_appetit.png)
- Student Leader: Shizheng Tian
- Student Members: Shuojin Liang, Mingwei Li, Ziyuan Wei, Jingqi Zhang, Tianhui Xu, Zhenru Zhang, Yiyang Yao, Yiyang Lu, Yuanyuan Liu, Emily Mo, Xiaohui Jiang, Xinran Li, Gaochen Zhang
- Advisors: Liang Cai, Yijie Pan, Yuchang Lee, Jingyi Hu
- Team PI: Daru Lu
- Affiliation: School of Life Sciences, Fudan University; with funding from Top Talent Undergraduate Training Program and YF Capital
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In Asia, many old people suffer from insufficient calcium intake and related diseases such as osteoporosis. Considering that traditional calcium supplementation is not efficient and user-friendly enough, we intended to develop a kind of engineered bacteria based on E. coli that can colonize in the human intestine and secrete peptides to promote calcium absorption.
We introduced the quorum sensing system to colonize bacteria in the intestine. Once the bacteria have successfully colonized the intestinal tract, the luxPR promoter will be activated and a series of short peptides that can bind to calcium and promote calcium absorption of intestinal epithelial cells will be expressed. For safety, a kill switch is added accordingly.
Our project provides a novel delivery method to provide calcium supplements sustainably. And we are actively seeking commercialization possibilities to expand our project impact, to reach more population, for the elderly's good health and well-being.
![what we have done](https://static.igem.org/mediawiki/2020/4/43/T--Fudan--Poster_todays_special.png)
Where we came from
A large portion of the elderly Asian population suffers from insufficient calcium intake and related diseases like osteoporosis. Experts recommend that osteoporosis prevention should begin as early as middle age.
Curious about how calcium deficiency affects the elderly and the middle-aged, we conducted a survey to learn whether/how we are able to help with calcium supplementation. Our overall results indicate that the younger generation has a strong awareness of bone health and has taken action to supplement their calcium intake.
More than half of our respondents aged between 40 and 60 replied that they know more than 3 seniors with fractures from falls. We realized that health issues surrounding bone health are no longer figures on paper but tangible challenges in many families. Calcium deficiency is clearly an urgent issue that requires immediate change. However, the current calcium supplementation approaches are inefficient and not user-friendly, particularly towards the elderly. In early 2020, we initiated Bone Appetit, a project that strives to build efficient, user-friendly and sustainable calcium supplementation for the elderly.
Where we are now
In the past 10 months, we combined our work in lab, production, entrepreneurship, education and safety. We developed a thorough proof of our concept involving modeling a kill switch, designing and testing secretion peptides guided calcium absorption peptide (CaAP), designing, modeling and testing a MccB17 based quorum sensing system, and developing a robust measurement scheme (CaAsst).
Our overall engineering success could not be achieved without the help of modeling and learning from the experienced. We modeled the initiation and steady states of a quorum sensing system.
At the early stage of colonization, AHL will be rapidly produced and reach a high concentration intracellularly, leading to the generation of AHL-LuxR heterodimers and the initiation of quorum sensing.
Although the number of molecules may change, the AHL-LuxR interaction is relatively stable, suggesting a self-steady state to maintain quorum sensing. For more details, please visit /Team:Fudan/Model.
We consulted various experts to improve our project. We collaborated with BNU-China when designing our Kill Switch.
In order to deliver quality education to the elderly, we started educating the young and instruct them to educate their parents and/or grandparents. We formed partnerships with other iGEM teams to better educate the young and those from the rural areas. We actively reached out to high school students, undergraduates and all age groups with accessible educational opportunities.
Our contributions to #iGEM
To conclude, our team has contributed to the iGEM community in various aspects, from inclusive education to contributing new parts to the Registry, from improved modeling to a robust measurement and a product development plan. We hope that our project is not just limited to providing solid foundations for future research to build upon, but also contributes to achieving the sustainable development goals outlined by the United Nations.
![sdg3](https://static.igem.org/mediawiki/2020/2/21/T--Fudan--Poster_sdg3.png)
Ensure healthy lives and promote well-being for all at all ages.
Our project aims to provide a holistic solution to the problem of osteoporosis among seniors. Our online general biology course Shengyin and senior-friendly textbooks, were designed to educate the elderly on the importance of bone health.
We paid special attention to post-menopausal women who are more likely to suffer from osteoporosis. Since Chinese women favor traditional crosstalk, we combined the traditions with basic synthetic biology knowledge to deliver our education in the form they can appreciate with ease. We performed it for them on our nursing home visit. They all laughed and enjoyed our performance while learning something new.
Another thing we did is to promote a traditional Chinese sport - Mu'lan'quan to them. Many elder women liked the sport because it is both elegant and healthy. The movements of Mu'lan'quan combine dancing and Kungfu and is preferred by many females. It is convenient to practice - anybody can do it with a fan. We hope our effort could help the elderly women strengthen their bone health and lower the risks of osteoporosis.
Members of all age groups are stakeholders of osteoporosis prevention and osteoporosis treatment. Seniors are an integral part of our society, and it is our responsibility to ensure their health and quality of life.
![sdg4](https://static.igem.org/mediawiki/2020/7/74/T--Fudan--Poster_sdg4.png)
Ensure inclusive and equitable quality education and promote lifelong learning opportunities for all.
With the rapid development of technology and society, the older generation is gradually lagging behind. The elderly is facing fraud, misinformation, and diseases. Lifelong education has become an urgent need for them. We dedicated ourselves to exploring education approaches, trying to bring high-quality education for the elderly and protect them from fraud and misinformation.
We used a previous unexplored education approach to educate the elderly - educating the younger generation and instructing them how to educate their grandparents. Compared with traditional education strategies, our approach offers emotional support from the younger generation and is comfortable for the elderly. In countries like Japan, Korea, Singapore, and China, where the bond between grandparents and grandkids is incredibly tight, our approach is undoubtedly effective.
To educate the youth, we have held online summer camps for high school students to pass on aged care, in addition to synthetic biology which we have been teaching in the past several years. We developed an online audio course on basic biology knowledge, which could be listened to by both the youth and the elderly.
Our free online audio course The Voice Of Biology runs on multiple popular platforms with easy access to all age groups. We incorporated graphics to attract younger audiences and provided large font textbooks to accommodate seniors and those with hearing impairment. 上传两个ogg格式的音频文件
![sdg9](https://static.igem.org/mediawiki/2020/e/e5/T--Fudan--Poster_sdg9.png)
Build resilient infrastructure, promote inclusive and sustainable industrialization and foster innovation.
Our project aims at converting engineered probiotic into a sustainable calcium supplement ready for the market. Our product targets seniors suffering calcium deficiency and strives to lower the risks of osteoporosis amongst seniors. Our product is highly efficient and does not require frequent intake.
Our product is a genetically engineered probiotic that can colonize the intestine and secrete a short peptide - CaAP, which increases the amount of soluble calcium by binding to calcium ions and increasing the absorption of calcium by intestinal epithelium. Compared with other calcium supplement products such as calcium pills, our product has its unique advantages.
We have outlined a five-year product development plan, and signed a letter of intent with RuiShi Inc.
![demo CaAP](https://static.igem.org/mediawiki/2020/d/d4/T--Fudan--Poster_caap.png)
CaAP stands for Calcium Absorption Peptide.
According to literature, we selected peptides GPAGPHGPVG, FDHIVY, YQEPVIAPKL, NDEELNK, DHTKE. All of these peptides can form calcium-peptide complex through their Asp, Glu and His residues, and facilitate uptake by cells. We linked these peptides through Phe-Arg (FR) junctions. When CaAP is released into the intestinal lumen, CaAP could be further cleaved into functional oligopeptides in the presence of digestive enzymes (trypsin and chymotrypsin). We contributed a basic part BBa_K3606000 to the Registry, which is RFC[10] assembly compatible.
We added a signal peptide to the N terminus of CaAP to guide its secretion into the periplasmic space of E. coli. Because it is so small, it will then leak out of the cell wall into the outside environment (intestinal lumen). There are 2 existing prokaryotic signal peptides (PelB, PhoA) in the Registry, and we contributed 3 more (NSP4, OmpA, DsbA).
Driven by a quorum sensing promoter, E. coli only express signal peptide linked CaAP after their colonization.
We demonstrated in vitro released CaAP by SDS-PAGE, in trichloroacetic acid precipitated bacterial culture media samples.
![demo mcbABCD](https://static.igem.org/mediawiki/2020/d/d8/T--Fudan--Poster_mcbefg.png)
Based on previous work, we use AHL+LuxR, TetR and the antimicrobial peptide microcin B17 (MccB17) to achieve an on/off switch-like controlling of bacterial colonization and CaAP expression.
McbA,B,C,D jointly complete the secretion of MccB17. Among them, peptide product from mcbA is the precursor of the antimicrobial peptide, and McbB,C,D is responsible for the post-translational modification of McbA, to produce MccB17. McbE,F assemble the channel to export MccB17. McbG provides the immunity against MccB17. If the engineered bacteria express only McbA,B,C,D, but neither McbE,F nor McbG, intracellular accumulated MccB17 will kill itself. When the surrounding bacteria absorb MccB17 but not McbG, they would die.
We place mcbABCD under the control of a constitutive promoter, and use PtetR drive the expression of McbEFG. When the density of engineered bacteria P2 is low (as shown below in the animation) and PtetR promoter is on, expressed McbEFG would protect P2 while MccB17 would kill other bacteria. Because P2AD does not express McbEFG, they die even before other bacteria.
After the density of engineered bacteria increases to a certain extent, the heterodimers formed by the products of luxI and luxR will accumulate and activate the luxPR promoter. At this time, expressed TetR transcription factor will turn off PtetR. Thus, when luxPR driven CaAP begins to be produced, mcbEFG stops expressing.
Improved from last year, we: (1) use low-copy plasmid backbone pFAB to reduce the expression burden on engineered bacteria; (2) low expression promoters to drive mcbABCD; (3) PtetR drives mcbEFG from the reverse strand, to avoid translational readthrough; (4) validate individual McbA/B/C/D/E/F/G expression in an IPTG induction setting.
We quantitively showed the antibacterial effect with our favorite compositve part BBa_K3606029, which is RFC[21] assembly compatible.
In the figures above, MEFL is the absolute value of total green fluorescence and particle represents the absolute counts of bacteria, so that Y axis represents green fluorescence per bacteria. In those experiments, we mixed WT E. coli (expressing GFP driven by Plac) with mcbABCD-(mcbEFG-PtetR) at different ratios 5:7, 20:7 and 50:7, then measured MEFL/particle two hours after adding 1 mM IPTG. We used this assay (experimental flow chart at the bottom of this page) to study the antibacterial effect of MccB17 on protein synthesis.
Similarly, we shown that our antimicrobial peptide suppresses WT bacteria growth (data shown on /Team:Fudan/Parts#Composite and the main page of the part in the Registry).
![demo CaAsst](https://static.igem.org/mediawiki/2020/d/d9/T--Fudan--Poster_caasst.png)
Both calcium deficiency or high blood calcium leads to many diseases and severely reduces the quality of life. Therefore, it is important to monitor blood calcium levels when taking calcium supplements.
Traditional blood calcium testing requires at least 5 mL blood and dedicated machines. We developed CaAsst (short from Calcium Assistant) to measure trace calcium ion in the blood, as little as 10 microliters. We use methylthymol blue to convert calcium concentration into 620 nm absorbance (A620). Our experimental results showed a linear relationship between calcium concentration and A620, within 20 mM calcium ion in solution, while the concentration of blood calcium ion is around 2-4 mM.
In addition, we introduced common interference substances to showcase the robustness of our measurement.
Further development may focus on improving the stability of application solutions, making glucometer compatible test strips, and low-cost-and-portable measuring hardware.
![parts](https://static.igem.org/mediawiki/2020/1/15/T--Fudan--Poster_parts.png)
Our part collection offers a complete array of parts for future researchers to develop other probiotic secreted proteins/peptides. This year, we have MccB17 and CaAP secreted and experimentally verified.
We contributed 3 probiotic signal peptides (NSP4, OmpA, DsbA), 14 low-expression promoters (BBa_K3606008 ~ BBa_K3606023) in a low-copy plasmid backbone, PtetR promoter on the reverse strand (BBa_K3606030), mcbEFG on the reverse strand (BBa_K3606821), luxPR promoter driven various proteins, PlacIq_lacI_Ptac promoter driven various proteins, PtetO3 (BBa_K3606007, improved from PtetR), etc.
We not only provide sequences and references for these parts, but also experimental results and procedures. Most of the parts were assembled together using high-fidelity PCR and ClonExpress ligation reactions. All plasmids were sequencing verified and available upon email request igem@fudan.edu.cn.
As outlined below, the majority of parts we contributed this year are either BioBrick RFC[10] or RFC[21] compatible.
For more details, please visit:
![references](https://static.igem.org/mediawiki/2020/0/0e/T--Fudan--Poster_ack.png)
Our project wouldn’t be complete without the help of many dedicated people.
We give special thanks to the institutions, organizations, professors, experts, key stakeholders who have provided tremendous help to us along the way. Our project wouldn’t be complete without their dedication.
We were also fortunate enough to have advisors, experts and senior undergraduate students who offered vital advice and contribution in the making of this project.
We would also like to acknowledge our collaboration with other teams from the iGEM community, including 19Fudan, 19BNU-China, TJUSLS-China, and SCU-China. They have helped us with perfecting education work and guided us through conducting experiments.
References: 只需要核心文献
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