Team:Shanghai United/Design
Design
Para-cresol sulfate (PCS) is a protein-binding uremia toxin derived from intestinal food protein. Among patients who have chronic kidney disease, the ability of the kidney to excrete PCS is significantly reduced, and the binding of PCS to albumin in the blood is significantly increased. Also, the serum concentration of PCS was highly correlated with the progression of chronic kidney disease, major adverse cardiovascular events, cardiovascular mortality and all-cause mortality.
Because PCS has a high affinity with albumin, it is not easy for PCS to dissociate from albumin and cannot be discharged by the kidney compensatively, which is more likely to cause the accumulation of PCS in the body than those toxins with low binding rate to albumin.
The intestinal anaerobe converts phenylalanine and tyrosine in food into 4-hydroxyphenylacetic acid, also known as the decarboxylated to p-cresol. Most of the p-cresol is absorbed by the intestinal mucosa and converted to PCS under the action of sulfonyl transferase in the intestinal epithelial cells.
Therefore, we use engineering probiotic bacteria which excessive express uremic toxins (BHT) degradation enzyme, polyphenol oxidase, induced by food source of amino acids, tyrosine and phenylalanine. The enzyme can reduce the cases of chronic kidney disease in the gut PCS precursor of cresol content, and as far as possible to reduce the formation of PCS, treatment, and alleviate the symptoms of chronic kidney disease and processes.
We planned two routes for our experiment. First, polyphenol oxidase (PPO) genes on the p15A plasmid are amplify and express in Escherichia coli Nissle 1917 (ECN). In this route, p-cresol can be degraded by the PPO expressed by ECN. Second, pchC- and pchF- as well as pchA- are isolated from Pseudomonas citronellolis and express them in ECN cells. These genes encode p-cresol methylhydroxylase (PCMH) and p-hydroxybenzaldehyde dehydrogenase (PHBDD), both of which are enzymes that metabolize p-cresol. We don’t directly use P. citronellolis for route 2 due to its potential risk to infect the host.
Advanced technologies were applied to our experimenting procedure, such as PCR (polymerase chain reaction), homologous recombination, plasmid extraction, gel electrophoresis, gel extraction, protein purification. Mathematical modeling is also used for further observations.
One possible defect of our project appears to be the fact that few people have explored this topic before, so that we didn’t know exactly which route would be successful before we started experiment. There might be a possibility that none of the methods succeed, particularly taking the probabilities of the techniques (PCR, homologous recombination, etc.) a factor. For example, it’s hard to decide how we link the gene segments separated from P. citronellolis, a eukaryotic fungus composed of macromolecules.
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