design

As the global economy develops, and people's living standards continue to improve, the incidence and mortality rates of chronic metabolic diseases are on the rise. BUCT has also recognized this issue and, after brainstorming, decided to focus on chronic kidney disease. The plan is to use genetic engineering techniques to create probiotic strains of engineered bacteria to help reduce the levels of metabolic waste products, such as creatinine and urea, in the bodies of kidney disease patients, ultimately improving their quality of life.

Chronic kidney failure is the final outcome of various kidney diseases. In addition to the primary symptoms, its main manifestations are the accumulation of metabolic byproducts and disturbances in water and electrolyte balance. Once chronic kidney failure occurs, the deterioration of kidney function is related to the activity of the underlying disease. However, even if the primary disease stops its activity, kidney function can still continue to decline. The main reason is that uremia not only exacerbates kidney damage but also causes damage to various organs throughout the body. Toxins at high concentrations in the bloodstream enter the gastrointestinal tract, causing changes in the gut microbiota, including alterations in the quantity and types of bacteria.

Due to the excess toxins entering the intestines, an imbalance in the gut microbiota occurs. Proliferating gut bacteria metabolize substances like creatinine and urea as an energy source for their growth. These bacteria produce metabolites such as methylguanidine, which worsen the condition and cause more severe damage. The goal is to convert creatinine into creatine with the catalysis of creatinine hydrolase and then further convert creatine into creatine amine with creatine hydrolase. The genes encoding these two enzymes will be fused and transferred into probiotic bacteria in the gut. These genetically modified bacterial strains containing these genes will be turned into bio-preparations and introduced into the digestive tract of kidney disease patients. This will result in the breakdown of creatinine into creatine amine, which is not only non-toxic but also a nutritious substance. Research has shown that it can protect the nervous system and improve memory. Through this approach, the goal is to reduce blood creatinine levels in chronic kidney disease patients and improve their nutrition.

In E. coli carriers, the expression of exogenous proteins has been hindered by the formation of inclusion bodies due to the lack of modification systems. Previous attempts to express urease in E. coli carriers from sources like Helicobacter pylori and Proteus mirabilis led to the formation of inclusion bodies (although the structural gene ureABC of urease was soluble, the accessory genes ureEFGD were essential for efficient nickel ion transport, which is critical for urease). The BUCT team attempted to increase the solubility of urease using DNA molecular chaperone methods to enhance its activity.

Polyglutamic acid (γ-PGA) is a safe and non-toxic high molecular weight polymer that has been widely used in the food and pharmaceutical industries. It has been reported to maintain gut microbiota and promote intestinal peristalsis. Currently, there is no effective research indicating a mechanism for the human body to metabolize γ-PGA. Therefore, excreting creatinine and urea nitrogen in this form can effectively reduce the burden on the kidneys of CKD patients, ultimately improving their quality of life and disease prognosis. To reduce the load on the EcN carrier, in the glutamate dehydrogenase overexpression module, the BUCT team used CRISPR-cas9 technology to integrate ygay-50trc-gdhA into Nissle 1917 and replace the constitutive promoter to enhance its expression. BUCT used the polyglutamic acid synthetase (pgs promoter) from Bacillus subtilis to express γ-PGA for the first time in the EcN carrier. Since γ-PGA synthesis requires equal amounts of L-Glu and D-Glu, glutamate racemase from Bacillus cereus was used to produce racemic glutamic acid as a substrate for glutamic acid polymerization to increase its yield.

In E. coli K12, nitrogen regulatory elements, including glnAP2, ntrB, and ntrC, were identified through transcriptome analysis. Changes in glutamine concentrations were observed to affect this nitrogen control system. NtrB phosphorylates NtrC, forming a hexameric complex. It is known that NtrC is one of the σ factors for glnA, which, under conditions of external nitrogen limitation, induces overexpression of glutamine synthetase. BUCT aims to characterize this original element for the first time by using mCherry fluorescent reporter protein as a means to detect its activity in a low-nitrogen induction gradient. BUCT will conduct random mutagenesis and directed selection of this element to achieve the required detection threshold.

Through the use of this ammonia-responsive promoter, a system for self-destruction of the carrier bacteria can be created. It is reported that the concentration of leaking creatinine and urea nitrogen in the intestines of CKD patients is usually 400mg/L or higher. Based on investigations, the ammonia nitrogen concentration in urban residents' sewage septic tanks is approximately 150mg/L (data from Beijing Municipal Sewage Treatment Center, sampling from septic tanks in various residential areas within the district, with an average ammonia nitrogen level of 126.6mg/L). Therefore, BUCT will use random PCR mutagenesis to obtain a corresponding mutation gene library for the glnAp2 promoter and will further screen for bacterial strains with forward mutations in the detection domain based on fluorescence intensity characterization.

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