一句短语概括整个页面，不要用overview这类无实际意义的词或词组

Due to the COVID-19 and limited time, our project is still in the laboratory stage. But our pace will never stop. We plan to continue implementing our project in project design, product manufacturing, promotion of business plans, and education while considering related ethical and safety issues. We believe our project will fully take advantage of synthetic biology and play a role in human society to promote SDG realization.

Further synthetic biology engineering

We have not fully verified the effect of CaAP and the effect of the final overall project. We first plan further to ascertain the impact of CaAP on complexing calcium ions and promoting the absorption of calcium ions by Caco-2 cells. Suppose efficiency cannot meet our requirements. In that case, we will conduct further analysis and in-depth database retrieval and replace them with other studied CaAPs until the final efficiency meets our needs and expectations.

Finally, we will import CaAP into the quorum sensing system's downstream module to verify whether the whole system can work as expected. This includes:

Whether it can be colonized in a simulated intestinal environment and maintain a specific population.
Whether it can promote calcium absorption by co-cultured Caco-2 cells.
Whether the Kill switch can function normally.

We will implement the engineering design cycle:

Research → Imagine → Design → Build → Test → Learn → Improve → Research...

Finally, we will perfect our project and apply it to the real world.

Target group

Our project comes from the elderly calcium problem. China already has a population of 1.4 billion, and the economy develops rapidly. People are asking for a better life now. Among them, the bone and calcium problem is a problem we can not ignore.

China is an ancient civilization, and Chinese food culture is of great antiquity and well-known globally. However, China's dietary structure also has brought trouble for people's calcium problems. In the traditional diet of East Asians, including Chinese people, vegetarians are the leading food, supplemented by meat, and rarely consume animal dairy products, leading to a low calcium intake. Some plants also contain many oxalates, phosphate, and other elements, which complex calcium ions and reduced calcium ions' absorption rate.

Statistics show that the average Chinese consumes less calcium than the recommended daily intake and many Chinese face serious bone problems. It is estimated that in 2006, there were nearly 70 million osteoporosis patients in China, and the number of bone loss has exceeded 200 million. Despite the lack of recent epidemiological data, estimates of osteoporosis and bone loss in China far exceed these figures. The problem of osteoporosis is more severe in the elderly, and there are deaths due to falls and fractures every year. Therefore, we limited our target population to the elderly.

Product form

Our project aims to design a genetically engineered probiotic that colonizes and secretes a short peptide in the gut. This short peptide binds to calcium ions to increase soluble calcium and increases the absorption rate of calcium in the intestine, thus achieving calcium supplementation.

Our products are basically positioned as probiotics health products. The relatively mature probiotics products in the market include probiotics lozenges, drops, powders, and capsules. And we chose to make our program available to the target population in the form of probiotic capsules. Compared with other forms of probiotics, the capsule has unique advantages, such as high bioactivity and mature technology. Besides, the commonly used capsule shell is plant fiber shell, which is less than the traditional gelatin capsule additive and more suitable for the elderly.

Commonly used probiotic lozenges can add more additives and adhesives, sweeteners, and molding needs heating, which will damage the probiotics' activity. Due to many additives, although easily accepted by children, older people won't care about whether the drug will taste good. A lot of additives can add the burden on the health of the elderly, instead of other forms such as drops, dry powder each have advantages, but there are not easy to save, the shortcomings of high cost, it is not a good choice for the elderly.

Business plan

We have a long-term plan for the realization of our project, and we have won the investors' approval.

The first milestone is two months later. We plan to obtain all plasmids by Cai Lab and prove that all components can function normally. But we still need to contact medical laboratories for the future's clinical trial. If we want our project to apply, it is a necessary step. We also need to contact investment institutions for financial support.
After six months, we are supposed to successfully import all components into the recipient bacteria and make sure that all imported parts function as expected. We also want to consult experts and doctors and pay attention to relevant laws and regulations that enable our project to process smoothly.
Ten months later, we may start our experiment on the mouse, and confirm that the successful preclinical trial has passed the review of FDA in the same period, obtain the approval of clinical trial, and apply for clinical trial.
One year later, we may start our phase I, II, and III clinical trials. And then, we're continually tweaking our programs based on the results of the clinical trials.
Five years later, some pharmaceutical companies may prepare our test data for new drug applications to the FDA following the application procedures. At that time, our products may be used by the public.

Further education

The old is such a more special community in our society, especially in 2020. They are incredibly vulnerable to many diseases, COVID-19, for example. After Bone Appetit, we realized that the old and their needs are often neglected in daily life. We established a special emotional bond with several old people in the nursing house and understood their difficulties. The fast-developing society confuses them. Misinformation fraud and dangerous diseases are ghosts that never fades away. We genuinely hope that every older adults can enjoy their daily life without the shadow of these difficulties. Even though Bone Appetit is coming to an end, we will always remember how vital the whole-life education for the old is. We will keep paying attention and trying our best to protect the unique and beloved community in society.

Considered safety aspects

To solve the problem of insufficient calcium intake and diseases it causes in China, we want to develop a kind of engineered bacteria that can colonize in the human intestine and secrete peptides promoting calcium absorption based on Nissle. In order to enable the bacteria to colonize in the intestine, we introduced the quorum sensing system which can regulate the downstream gene expression. Introduction of the luxI/luxR system and antimicrobial peptide system enables the E. coli to colonize the intestine, reach a sufficient population faster, gain niche advantages, and form intestinal colonization when the population is small. And when the bacterial population reaches a certain level, the antimicrobial peptide system's efflux can be closed to inhibit over reproduction. Once the engineered bacteria have successfully colonized the intestinal tract and its population is high enough to activate the luxpR promoter, a series of short peptides that can bind to calcium and promote calcium absorption by intestinal epithelial cells will be expressed. We have connected an ompA signal peptide sequence upstream of the short peptide sequence, which can use the secretion pathway of E. coli to carry the effector short peptide outside the cell and promote the absorption of calcium by intestinal epithelial cells.

General calcium supplements may increase the risk of cardiovascular diseases when taken in excess, but there is no need to worry about this due to the quorum sensing module in our product. The number of our engineered probiotics in the gut is strictly limited so they have little chance of causing intestinal flora disturbance. The kill switch module will be employed to effectively prevent leakage of bacteria into the natural environment. As for ethical risks, we imagine it will only occur when the product initiated clinical trials. We want to be very mindful of ethical risks about our product and ensure that we meet all requirements posed by the Chinese government as well as international ethical guidelines for conducting clinical research, including but not limited to informed consent, fair subject selection, confidentiality, and informing participants about new information. At that stage, we will also consult experts in this area and take our ethical practices a step further.

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. In order to ensure our engineered probiotic will not jeopardize the environment, this year, we introduced a cold triggered toxin/antitoxin Kill Switch into our project to deprive of the survivability of engineered Nissle in the environment when excreted from the human intestine.

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.)

Signature: Shizheng