Osteoporosis and other diseases caused by calcium deficiency are common problems for people, especially the elderly in many areas. CBP (calcium-binding peptide) refers to a class of oligopeptides that are capable of chelating calcium. They promote Calcium uptake in Caco-2 cells, which can help solve the problem of calcium deficiency. Since these oligopeptides are derived from natural plant or animal food, they are biologically safe.

According to some literature, we learned that the entire sequence of the CBP expression peptide contains five different calcium-binding peptides, which are all experimentally proved to significantly increase calcium absorption in vivo or in vitro. The calcium ion spontaneously binds to the Asp or Glu residue, allowing the calcium-peptide complex to promote calcium influx into intestinal epithelial cells. The oligopeptide sequence is expressed as a complete peptide in the engineered bacteria and will be cleaved into functional oligopeptides in the presence of digestive enzymes in the intestinal lumen.

2019 Fudan team tried to make the expression of lactase in intestines controllable to treat lactose intolerance, based on which we establish a safe CBP expression system in Escherichia coli, providing a solution to increase calcium intake.

We have added new parts to the Registry of Parts and conducted experiments to provide new experimental data on the expression of CBP in E. coli, providing references for future teams to use the expression system.

This work is completed by our team members Lu Yiyang and Zhang Zhenru. Enter the team page to learn more about our team members.

The quorum-sensing system makes the bacterial proliferation regulated by its colony number, ensuring safe and controllable growth in human bodies.

In our project, we take advantage of the quorum-sensing system of luxI/luxR and the antimicrobial peptide expressing system to control the number of bacteria and open the CBP expressing system at the proper time. You can find more details on relative pages.

Fudan 2019 team proposed the antimicrobial peptide quorum-sensing system. The expression effect of the antimicrobial peptide system determines whether the flora can colonize as soon as possible and achieve the balance of quorum sensing. The antimicrobial peptide expressing system didn't work favorably last year, so this design still needs feasibility proof. We read the wiki and found that the antibacterial test of the antimicrobial peptide effect failed to achieve a satisfactory effect. Through team communication, we doubt that it might attribute to the low secretion of an antimicrobial peptide.

Therefore, we proposed an improved plan, using low-copy plasmids to reduce the expression burden of engineered bacteria, and adopting a relatively low expression of antimicrobial peptides (mcbA-D) and high expression of the antimicrobial peptide immune portion (mcbE-G) to achieve high secretion of antimicrobial peptides. Through the high expression of channel protein and immune protein to promote the excretion of antimicrobial peptides from the bacteria, we reduce the Microcin accumulation in them, thereby inducing our bacteria to produce more antimicrobial peptides. That's how we achieve our purpose of dynamically and moderately regulating the intensity of antimicrobial peptide expression.

Specifically, we make improvements from the following two aspects:

First, improve the mcbE-G promoter ptetR to achieve a high expression of mcbE-G. We decide to create fusion promoters that few have tried before, fusing the upstream of -35 site, -35~-10 sites, downstream of -10 site, sites near RBS and other proportions of the strong pI promoter in lambda phage with the tetracycline operon tetO. We expect to obtain two new fusion promoters ptetO2 and ptetO3, and try to find the best expression state by changing the number of tetO operons. From the experimental data of the literature, we have learned that these two promoters have low-leakage characteristics, and their peak intensity reaches about 4 times compared with placZ, ensuring a high expression level.

Second, screen a suitable expression promoter for mcbA-D. Since the mcbA-D gene expresses the toxic part of the antimicrobial peptide, an appropriate promoter strength can ensure the expression effect of the antimicrobial peptide without causing excessive expression burden. We used a set of deeply optimized promoters of different strengths for experiments, constructed multiple systems, and screened 14 promoters to obtain 6 promoters suitable for the expression of antimicrobial peptides in E. coli.

We can now stably express antimicrobial peptides and overexpress their channel proteins through the above two improvements. Existing literature proves that increasing the expression of channel protein in high-yield or even wild-type bacteria can significantly increase the production of antibiotics. As the copy number of channel protein increases, it can be detected that the ratio of extracellular to intracellular antibiotics increases, which means discharge efficiency increases. You can find the specific reason analysis on relevant pages.

This improvement not only continues the design of Fudan 2019 team and helps prove the feasibility of this quorum-sensing system, but also facilitates future teams to use this system to control the multiplication of flora. Besides, the methods of obtaining high-expression and low-leakage fusion promoters and constructing a system to screen suitable strength promoters also provide new effective ideas for regulating gene expression efficiently.

We can provide reliable experimental data about related parts, which confirms the feasibility of this quorum-sensing system and also provides reference information for future teams who use the system. For details, read the relevant pages.

This contribution is made by our team members Li Mingwei and Zhang Gaochen. You can enter the team page to learn more about the team members.

Kill switches can be classified by their functions, including controlling the density of flora, protecting users, protecting the environment, etc. Among them, the environment-friendly kill switch is a genetic circuit that promotes the death of the engineered bacteria when they leave the environment where the designer expects it to work. In the intestine, a probiotic kill switch is frequently used to prevent the modified gene from leaking into the environment when probiotics are released.

The kill switch in our project this year applies the toxin-antitoxin system, in which the toxin relE is constantly expressed, while the antitoxin relB is expressed only at body temperature(37℃), which protects the engineered bacteria from toxin. When the environment temperature is lower than 37℃, the antitoxin expression decreases, and the engineered bacteria die. We obtain the data from 19BNU-China for modeling verification. You can read relevant pages for details.

This kill switch has significant advantages compared to 2019 Fudan team. Last year their kill switch reaches the best killing effect at 40°C instead of 37℃, not suitable for our goal. But this year we make it. It allows the bacteria to survive at 37℃ but suicide when the temperature decrease, which is more suitable for working in the internal environment of the human body. Moreover, last year they had to express a variety of proteins to complete the process of temperature control, which caused the bacteria survival burden heavier.

Fortunately, now our kill switch contains fewer essential proteins，helping the bacterial flora stably express genes we insert. Besides, our kill switch has a more sensitive temperature response and higher efficiency than 19BNU-China. We also selected various existing kill switches based on a kill switch database established by 2016 Marlborough team. We analyze and summarize suicide trigger conditions and killing mechanisms of these kill switches, citing the kill switches used by previous teams as examples for reference. This will help future teams to better understand the killing effect and applicable scope of various kill switches. And they could choose the appropriate kill switch according to their own needs to achieve the purpose of experimental safety.

For detailed illustration, please read the Safety page.

This contribution is made by our team member Zhang Jingqi. You can enter the team page to learn more about our team members.