After discussion, we have decided to use intracellular digestion. The principle of it is to bring uric acid from human body (i.e. intestinal tract) into our new recombinant cell. Then, our recombinant plasmid will express the gene of uricase. Uricase produced will break down uric acid inside the cell effectively.
Why we didn’t choose to adopt extracellular digestion? The main reason is that uricase is an enzyme, which is a kind of protein. Enzymes may have different working rates under different environment. If uricase is produced and secreted out of the cell, the environment may not have the optimum temperature and pH value for it to function. Instead, there is a high possibility for uricase to denature due to the extreme PH value and temperature. Intracellular digestion will solve the above major problem.
As a result, we designed our E.coli to perform intracellular digestion when it gets to our body. To achieve, we conducted lots of experiments and checkings after successfully transforming our recombinant plasmid into our E.coli.
Use of Uricase
Among the current treatments of hyperuricemia and its associated diseases including gout, there is one treatment that functions relatively well on reducing uric acid in human body, which is intaking pegloticase. In fact, pegloticase is a recombinant porcine-like uricase, which turns uric acid to a more soluble of allantoin.
Sadly, this medicine had some very big side effects and drawbacks. One of them is that this medicine will help inducing anti-pegloticase antibodies to lose the uric acid lowering efficacy. Therefore, we decided to use the gene of uricase, which theoretically can be a better alternative and better medicine for lowering uric acid level in human body.
Use of E.coli for cloning plasmid
DH5α is the E.coli we chose for cloning large amount of plasmid for our further experiments. According to essays we have read, this kind of E.coli is proved to be a good, or probably the best among all E.coli, for cloning large amount of plasmids. Due to its high successful rates, we chose it for cloning our new recombinant plasmid.
Genes used in our experiments
HIU hydrolase is a crucial enzyme to speed up the conversion of uric acid to a more soluble form of allantoin. Uric acid is firstly converted to HIU by uricase. Uricase can be converted into allantoin without other enzymes assisting, but with a very slow rate. Therefore, add gene of expressing HIU hydrolase into our new recombinant plasmid can theoretically speed up the conversion of uric acid to allantoin very greatly.
Sensor genes are inserted into two ends of our recombinant plasmid. The sensor genes are designed to be controlling the gene expression of uricase. Firstly, our E.coli will bring in the uric acid from intestinal tract into the cell. Then, our sensor will sense that uric acid is present in patient’s intestinal tract. Therefore, gene of uricase will be expressed and uricase will stay inside the cell to break down the uric acid. Therefore, if there is no uric acid inside the cell, the gene of uricase won’t be expressed. This can effectively ensure a stable and successful gene expression and enzymatic activity.