Microbial cell-surface display system is a system that acts on the surface of the cell membrane or cell wall of the receptor microorganism by combining specific foreign functional proteins with anchorin. Fusion proteins can be directly expressed on the surface of host cells for practical application purposes and are often used to demonstrate the adsorption and removal of excessive and excessive heavy metals by metal binding proteins. Cell-surface display technology can also be used to enhance the stability of catalytic hydrolases to resist temperature changes, and the fusion proteins of surface display as catalysts can be recycled, which is cost-effective compared to the high recovery cost of immobilized enzymes. Studies have shown that after intestinal colonization of the host bacteria, the surface display of enzyme catalysts can play a specific role in the human and animal intestinal tract, which can further improve the absorption and metabolism to a certain degree.
Hence, our experiment uses anchored proteins with specific hosts to localize foreign proteins on the surface of cell membranes. While enhancing the stability and reaction efficiency of reagent enzymes, we also prevents the degradation of foreign proteins by intracellular enzymes produced by the host, eliminating the disadvantages such as the inability of certain proteins or peptides to undergo specific folding within the cell, as well as eliminating the additional cost of enzyme purification and immobilization.
In recent years, the discovery of selenium supplement with high meat material ratio and meat to egg ratio has been widely used in livestock and poultry breeding. However, the reasons of poor selenium supplementation effect, toxic side effects and the high cost of preparing low toxic nanoparticles have become difficulties in promotion. After Dr. Zhang Jinsong found that the inert red nano selenium also has a very good selenium supplement effect, more and more nano is used in practical applications.However, considering the recovery cost of SeNPs preparation in vitro selenite, we proposed an emerging method that combines synthetic biology and intestinal repair to supplement engineered probiotics that reduce selenite to convert dietary supplemented selenium into direct absorption in the gut of livestock.
Therefore, after the applying of cell surface display system carrying anchored membrane selenite reductase in EcN to enhance absorption advantages, we proposed the food additives scheme to the feeding of broiler, make the engineering strain direct reduction function of selenite to generate nano selenium directly in chicken intestine. Our theoretical basis of this novel approach includes higher chicken tissue selenium retention and less selenium loss in chicken dung, higher immunity, antioxidant and intestinal repair, further selenium supplement restoration. After this feeding process, high Se content chicken and eggs can serve as daily dietary food, providing enough Selenium supplement for a wide range of people.
In order to reach this expectation, we came up with specific raising and feeding procedure to the customers and production factories, with the specific setting of the amount of strain to test the efficacy. The Na2SeO3 we plan to use is bought from Solarbio; the purity is above 99%. The fodder is produced and approved by Lanzhou Zhengda Corporate. We plan to set the concentration of Na2SeO3 as 0.5mg/kg. We designed three steps of experiments with 4 parallel groups(EcN, EcN-IS, EcN-IS + Se, Se) to examine the colonization of strain in the broiler’s gut, which can be directly applied by users:
(1) The whole feeding process will be divided into two intervals: 0-14d is the experimental adaption period feeding normal fodders and 14-50d is the testing period using Selenite and strain additive fodder. After the whole process, we suggest the Histological dissection by examining various tissues and fecal samples including liver, chicken breast and cecal contents to test the restoration of selenium. And exventually the animal carcasses and dungs will be strictly transported to the unified collection site of Lanzhou University Medical Campus for processing to elinimate the release of engineered strain. In the future, the sites implementing our Se synthesis approach must be strictly contracted in written form to the nearest excretadisosal site as the requirement in each country.
(2) Extraction of the fecal DNA contents. For different samples, the total DNA of bacteria in each group was taken by the fecal DNA kit (Tiangen Corporate, Beijing)(Sample fecal samples at day 14,28,35,49 from the 4 parallel groups: Control, EcN-IS, EcN-IS+Se, and Se at day 49).
(3) Construction of recombinant plasmids. Primers were designed using gene sequences of INP-SerV01 and 16S rDNA as template and the target fragments were amplified using PCR and ligated with the PSB-18T vector (TaKara, Japan) to convert them to E.Coil DH5a, respectively. Single clones were verified by the PCR method. Plasmid concentration was determined by Nanodrop and copy number of recombinant plasmid was calculated using formula, and finally plasmid diluted separately.
The ultimate goal of the project is to achieve the means of using the average human selenium content in selenium-rich chicken as a selenium supplement. Tests comparing selenium-rich chicken and controls can be carried out to explore whether the products obtained in this experiment can have selenium intake in a balanced diet and be a good way to solve selenium deficiency. In addition, after the successful animal experiment, we can try to redesign the structure of bacteria and make this engineered bacteria adaptive to human intestine environment. This new form of bacteria in the future can be implanted in human intestine and assist human body to assimilate selenium. The improved products may be used to assist in the treatment of selenium deficiency diseases, such as, Kershan disease, thyroid hormone disorders, cartilage disease, to explore whether the medical costs in the treatment process can be reduced and to improve the health of intestinal flora. However, this assumed future work should be carefully examined during exploring in aspects of various effects to human intestinal flora, ethic problems, environmental problems, etc. The road ahead will be long, and our climb will be steep. There will be a long journey and considerable challenges we should take over while trying to finding a better version for our product. Meanwhile, we always place ethics, safety in environment and physiology, and governmental laws in priority, which are our bottom line of research.