Calcium is a mineral necessary for the body to maintain healthy bones. Bone loss can lead to metabolic bone diseases, such as osteoporosis. This situation often occurs in the elderly population, especially in China. The calcium intake of the Chinese elderly commonly cannot meet the Nutrition Society's recommended standards, because vegetables account for a large proportion of the traditional diet. More severely, vegetables are rich in oxalate that cause calcium precipitation and impede calcium from absorbing.

So, we hope to develop a stable and sustainable endogenous drug delivery method that can assist calcium absorption, therefore truly improve the quality of life and health of human beings, especially for the elderly, promoting the progress of SDG Goal3: Good health and well-being.

Recently, people have attracted great interest in enhancing calcium solubility and subsequent calcium absorption by food substances (especially peptides). Combining calcium with peptides to prevent calcium precipitation can effectively increase calcium absorption in the body. So far, Calcium-binding peptides have been found and selected in various food sources, including egg yolks, cow cheese protein, soybeans, wheat germ, and so on. One of the most widely known is the casein phosphopeptides (CPPs) in milk. CPPs can promote calcium absorption by chelating calcium with phosphoserine residues, so protein kinases are required. However, given that protein kinases play an essential role in signal transduction, and they may not be able to fold and function correctly in the chassis, it is challenging to phosphorylate peptides in bacteria. Therefore, we choose short peptides without phosphoserine residues to promote calcium absorption.

CaAP consists of several peptides that can enhance intestinal calcium absorption by binding calcium to their Asp and Glu residues. Through literature review, we selected five short peptides GPAGPHGPVG, FDHIVY, YQEPVIAPKL^[1], NDEELNK^[2] and DHTKE^[3], to assemble our CaAP. We are doing experiments to show that our CaAP performs better than these peptides individually.

Asn-Asp-Glu-Glu-Leu-Asn-Lys (NDEELNK) is derived from the trypsin hydrolysate of sea cucumber eggs^[2]. This peptide can spontaneously bind calcium at a stoichiometric ratio of 1:1, and the calcium-binding site may involve the carboxyl oxygen and amino nitrogen atoms of two glutamic acids and one aspartic acid residues in the NDEELNK peptide. Besides, it is reported in the literature that the DEELNK-calcium complex undergoes decomposition and self-aggregation in a smaller-sized grid during the digestive process of the gastrointestinal tract, which may help to absorb calcium across the Caco-2 cell monolayer.

Asp-His-Thr-Lys-Glu (DHTKE) pentapeptide comes from the hydrolysis of egg white^[3]. This peptide can spontaneously bind calcium at a stoichiometric ratio of 1:1, and the calcium-binding site corresponds to the carboxyl oxygen, amino nitrogen, and imidazole nitrogen atoms of the DHTKE peptide. Besides, Caco-2 cells were used in vitro to study the DHTKE-calcium complex's effect on improving calcium absorption. Compared with the control without calcium, the results show that the DHTKE-calcium complex can promote the penetration of calcium into the cytoplasm and further increase the Caco-2 cell monolayer's calcium absorption by more than seven times.

To enable CaAP to be secreted into the intestinal environment, we plan to add a signal peptide to the N segment of CAAP to guide the extracellular environment's secretion. Our candidates are NSP4, OmpA, DsbA, PelB, PhoA. We hope to obtain a suitable signal peptide through comparison so that CaAP can be efficiently secreted into the periplasm.

The entire designed gene fusion fragment length is about 500 bp, and the gene loop is organized as follows.

Based on previous work, We decided to use luxI/luxR and TetR to construct a quorum sensor system with the antimicrobial peptide microcin B17 (MccB17) expression system to achieve the "on" and "off" effects of controlling the growth of engineered bacteria (schematic diagram).

MccB17 is a short peptide microcin produced by an endogenous plasmid found in Escherichia coli . It acts on DNA topoisomerase and gyrase, preventing bacterial from DNA replication and transcription and promoting double-strand breaks. McbABCDEFG jointly completes the expression, processing, and secretion of MccB17.

Among them, McbA is precursor of MccB17, and McbBCD is responsible for the post-translational modification of McbA to produce MccB17. McbEF expresses the channel protein to secrete MccB17. McbG is accountable for the immunity against MccB17.

TldD/TldE is a metalloprotease that is ubiquitous in bacteria. When the surrounding bacteria absorb MccB17, TldD/TldE will activate MccB17 and result in the death of the bacteria.

In our antimicrobial peptide expressing system, mcbABCD is under the control of a constitutive promoter, and PtetR regulates the expression of mcbEFG. When the number of engineered bacteria in the intestine is low, few heterdimers are formed by the expression products of luxI and luxR genes, which cannot effectively activate the luxPR promoter. Thus, luxPR driven TetR and CaAP expression are both silent. But, McbEFG is expressed and MccB17 is produced, which inhibit the growth of surrounding bacteria.

When the number of bacteria reaches a threshold, the heterdimers formed by AHL (product of luxI) and LuxR will accumulate and effectively activate luxPR. At this time, tetR is produced, which bind to PtetR promoter and turn off its downstream genes. As a result, mcbEFG stops expressing, and CaAP begins to be produced. Since the population number has reached a certain level, the engineered bacteria can express CaAP more efficiently. Meanwhile, MccB17 is still continuously expressed but cannot be effectively effluxed, which will cause the toxins to gradually accumulate in the engineered bacteria and prevent them from continuously proliferation.

The quorum sensing system described above was originally proposed last year by 19Fudan. Since MccB17 plays a vital role in the entire quorum sensing system, the better the MccB17 is expressed (optimal ammount), the faster the engineered bacteria will switch from quorum sensing to CaAP production. However, we found that the expression of MccB17 tested by Team Fudan 2019 last year was so low that the engineered bacteria cannot be competitive enough even in a test tube.

To solve the problem, we have proposed some improvements. We thought about using the high-copy plasmids to express MccB17, but they are more difficult to sensitively regulate and respond, and will greatly increase the expression burden of engineered bacteria.

By researching the literature and talking with Professor Lin, who is an expert in prokaryotic expression at the Fudan University, we decided to use low-copy plasmids to reduce the expression burden to the engineered bacteria. We tried to keep the expression of MccB17 (McbA processed by McbBCD) relatively low and highly express the immune part (McbEFG).

In our design, the highly expressed channel proteins (McbE,F) will export more MccB17 and reduce the risk of accumulating antimicrobial peptides in the engineered bacteria cells, thereby inducing them to produce more antimicrobial peptides.

Lin recommended a series of promoters (P1 to P14 for short) from an addgene collection which contains combinations of specific constitutive bacterial promoters that vary in strength to us, for driving the expression of mcbABCD to achieve better performance of the system.

For more information, please check /Team:Fudan/Results.

In order to ensure our engineered probiotic will not jeopardize the environment, we integrated a cold triggered toxin/antitoxin Kill Switch to deprive of the survivability of engineered Nissle in the environment when excreted from the human intestine. After carefully considering, we selected the toxin/antitoxin system from killer systems and RNA thermometer from the response systems.

The toxin/antitoxin system is a mature and robust system that is extensively applied both inside and outside of the iGEM competition. We first chose RelE/RelB system of type II TA systems, but replace it with MazF/MazE system later. Please read our note added to 19BNU part BBa_K3036004 page.

RNA thermometers are outstandingly because of their programmable reaction temperature range, high sensitivity and high efficiency. Moreover, they do not rely on extra proteins or are affected by food intake. Here we selected NoChill-06 from 19Rice whose documented data is really impressive.

In summary, our Kill Switch consists of a toxin/antitoxin system MazF/MazE and an RNA thermometer NoChill-06 to regulate it to deprive of the survivability of engineered Nissle in the environment when excreted from the human intestine. One thing we want to point out is that once the Kill Switch was implanted, the engineered bacteria could no longer be frozen as a glycerol stock, and it must be maintained in the culture media above 30 deree.

The antitoxin MazE is liable and expressed at a relatively high level. The MazF toxin is constitutively co-expressed with the antitoxin under the control of an RNA thermometer NoChill-06. Under the body temperature (37℃)，NoChill-06 unfolds and exposes its ribosome binding site (RBS) to express. MazE and MazF neutralize each other by protein-protein interaction and form a stable complexity in a one-to-two ratio. When the bacteria encounter a cold shock(30℃), MazE is degraded rapidly by an ATP-dependent serine protease ClpAP and releases MazF. The toxin MazF acts as a site-specific endoribonuclease to almost all cellular mRNAs, therefore resulting in cell growth arrest and finally cell death ^[4]. The antitoxin MazE is liable and expressed at a relatively high level. The MazF toxin is constitutively co-expressed with the antitoxin under the control of an RNA thermometer NoChill-06. Under the body temperature (37℃)，NoChill-06 unfolds and exposes its ribosome binding site (RBS) to express. MazE and MazF neutralize each other by protein-protein interaction and form a stable complexity in a one-to-two ratio. When the bacteria encounter a cold shock (30℃), MazE is degraded rapidly by an ATP-dependent serine protease ClpAP. Released toxin MazF acts as a site-specific endoribonuclease to almost all cellular mRNAs, therefore resulting in cell growth arrest and finally cell death^[4]. Click here to see more.

[1] Three Newly Isolated Calcium-Chelating Peptides from Tilapia Bone Collagen Hydrolysate Enhance Calcium Absorption Activity in Intestinal Caco-2 Cells. Wanwen Liao, Hui Chen, Wengang Jin, Zhennai Yang, Yong Cao, and Jianyin Miao. Journal of Agricultural and Food Chemistry 2020 68 (7), 2091-2098. DOI: 10.1021/acs.jafc.9b07602
[2] In vitro digestion profile and calcium absorption studies of a sea cucumber ovum derived heptapeptide–calcium complex. Pengbo Cui, Songyi Lin, Ziqi Jin, Beiwei Zhu, Liang Song and Na Sun. Food Funct., 2018,9, 4582-4592. DOI: 10.1039/C8FO00910D.
[3] An Exploration of the Calcium-Binding Mode of Egg White Peptide, Asp-His-Thr-Lys-Glu, and In Vitro Calcium Absorption Studies of Peptide–Calcium Complex. Na Sun, Ziqi Jin, Dongmei Li, Hongjie Yin, and Songyi Lin. Journal of Agricultural and Food Chemistry 2017 65 (44), 9782-9789. DOI: 10.1021/acs.jafc.7b03705
[4] Yamaguchi, Y., Inouye, M. Regulation of growth and death in Escherichia coli by toxin–antitoxin systems. Nat Rev Microbiol 9, 779–790 (2011). https://doi.org/10.1038/nrmicro2651