Casein phosphopeptides are phosphorylated casein of digest milk, 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.

Through literature search and database screening, currently selected peptides are GPAGPHGPVG, FDHIVY, YQEPVIAPKL[1], NDEELNK[2], DHTKE[3]. Experiments have determined these peptides can promote higher calcium absorption.

Asn-Asp-Glu-Glu-Leu-Asn-Lys (NDEELNK) hepeptide is derived from the trypsin hydrolysate of sea cucumber eggs. 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. 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.

The five peptides are connected by FR linkers to form a short peptide that promotes calcium absorption so that it can be cleaved and hydrolyzed by digestive tract proteases.

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 expresses the main structure of the antimicrobial peptide, and mcbBCD is responsible for the post-translational modification of the antimicrobial peptide. McbEF expresses the channel protein to secrete the peptide antibiotic. McbG is accountable for the immunity against MccB17. McbEFG is all related to E. coli's immunity while self-producing microcin. TldD/tldE is a metalloprotease that is ubiquitous in bacteria. When the surrounding bacteria absorb MccB17 produced by Escherichia coli, TldD/tldE will activate it and kill the bacteria.

In our antimicrobial peptide expressing system, McbABCD is under the control of a constitutive promoter, but PtetR regulates the expression of McbEFG. When the number of engineered bacteria in the intestine is small, few dimers are formed by the expression products of luxI and luxR genes, which cannot effectively activate the luxpR promoter. Thus, TetR and CaAP expression systems are both silent. However, McbEFG is expressed and transports MccB17, which can inhibit the growth of surrounding bacteria. When the number of bacteria increases to a certain extent, the dimers formed by the products of luxI and luxR will accumulate and effectively activate luxpR. At this time, tetR is turned on, and PtetR is off. As a result, McbEFG stops expressing, and the CaAP system begins to work. 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 unrestricted proliferation.

In order to ensure our engineered probiotic will not jeopardize the environment or users, this year, we integrated two kinds of Kill Switches into our project. The major kind of Kill Switch is cold triggered toxin/antitoxin Kill Switch to deprive of the survivability of engineered Nissle in the environment when excreted from the human intestine.

Kill Switch is a gene device to eliminate bacteria in a short period of time when engineered bacteria escape from the set conditions. Kill Switches commonly are applied on account of three reasons, hindering gene vertical transfer, hindering gene horizontal transfer and preventing the survival of bacteria outside certain environments [1].

The ultimate goal of our project is to produce a commercial calcium supplementary probiotic colonized in the intestines. An eco-friendly commercial probiotic should avoid the occurrence of modified gene vertical transfer and spread widely in the natural environment when excreted from the human intestine. And gene vertical transfer is directly associated with the survival of bacteria. Therefore, we needed to construct a Kill Switch to deprive of the survivability of engineered E. coli outside human bodies.

Simultaneously, we noticed that consumers may want to cease probiotics functioning because of accidental overdose, incidental gastrointestinal distress or any other reasons. We decided to qualify our probiotic for rapid suicide by BBa_K3036005.

Also, concerning hindering gene horizontal transfer, we provided a potential solution other than introducing one more Kill Switch. (Click here to see our solution on the Future Expectation of Safety page.)

Previous experience is a very important source to learn from. So, before we formally started to design our own Kill Switch, we traversed most suicide circuits in intestinal probiotics by iGEM teams and summarized mostly used methods. The Kill Switch database made by team 2016 Marburg has incredibly reduced our workload in searching Kill Switches used from 2007 to 2015. (Click here to see the database.)

After summarizing, we discover that the most commonly used kill switch circuit can be divided into two core components, the killer system and the sponsor system. According to the concept of biobrick in synthetic biology, we catalog parts of the killer system and the sponsor system separately. For parts of resemble mechanism, only the latest and the most efficient ones are listed. In this way, we can easily figure out the fittest part in the two groups to compose our kill switch, and hopefully offer a reference table for the future iGEM team working on intestinal probiotics to design kill switch and choose parts more conveniently.

Table 1. summary of the mostly used killer system in intestinal probiotics project. More “+” represents higher efficiency.

Note 1: The method of can gene knock out by last year’s grand prize winner is ingenious and effective, unfortunately, it can be applied to our project. It is reported that carbonic anhydrase, expression product of can gene, influence the release of PTH, leading to complex effects on bone remodeling. Carbonic anhydrase deficiency is manifested by osteopetrosis in human.[2] Although no concrete evidence has been found to show the relevance between carbonic anhydrase deficiency in intestinal microbiota and osteopetrosis, we decided to give up this method to avoid potential problems.

Table 2. summary of the mostly used response system in intestinal probiotics project. More “+” represents higher efficiency and more “-” represents stronger killing power.

After a throughout analysis of the above two tables, we finally selected the toxin/ antitoxin system from killer systems and RNA thermometer from the response systems.

The toxin/antitoxin system is a mature and stable system that is extensively applied both inside and outside the iGEM competition. By regulating the strength of RBS, we could achieve a state that toxin is totally neutralized and guarantee the security on the host. Here we first chose RelE/RelB system of type II TA systems but replace it with MazF/MazE system later. (Click here to see our consideration in engineering.)

RNA thermometers are apparent outstandingly because of its appropriate 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 which presents to be the most sensitive and efficient one.

So, our Kill Switch consists of a toxin-antitoxin system 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.

Figure 1. cold triggered MazF/MazE Kill Switch under body temperature(37℃) and outside the body(30℃)

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 No-Chill 06. Under the body temperature (37℃)，No-Chill 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 [5]. 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 No-Chill 06. Under the body temperature (37℃)，No-Chill 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 [5].

In the former version of RelE/RelB system, the toxin RelE is a global translational inhibitor that cleaves mRNAs under translating at the ribosomal A site and the antitoxin RelB is degraded rapidly by an ATP-dependent serine protease Lon [6].

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