Part:BBa_K2158004:Experience

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UNIQ1350c1d6863a6153-partinfo-00000000-QINU UNIQ1350c1d6863a6153-partinfo-00000001-QINU

This part consists of the LacI promoter (BBa_R0010), the RBS (BBa_B0034), the Uricase from Bacillus subtilis (BBa_K2158000) and the double terminator (BBa_B0015). Uricase is the mainly enzyme of the purine metabolism pathway, and catalyzes the oxidation of uric acid to 5-Hydroxyisourate.

Fig1. BBa_K2158004                                          Fig2. Purine metabolism pathway

The growth tests in minimal media
The growth tests in minimal media containing purines of B. subtilis were performed. The result is Fig3. As expected, B. subtilis grew well with uric acid and ammonium sulfate. We confirmed that Bacillus subtilis utilized these intermediate as nitrogen sources.



Determination of expressed uricase activity
We measured the decrease of uric acid when we added uricase to uric acid solution. We prepared 0.001% uric acid solution by dissolving in 50.0 mmol/L borate buffer containing 1.0mmol/L EDTA and detergent (pH8.5). 0.5mL of distilled water was added and the mixture was preheated at 25○C.
E. coli culture were grown in the presence of IPTG. Cell suspensions were sonicated and centrifuged (150rpm, room temperature, 10min). The top layer was removed, and cells were buffered with 50mM borate buffer containing 1mM EDTA and detergent. Uricase from B. subtilis were measured. These crude extractions were diluted 1 to 102 times with the borate buffer. After that, the absorbance at a wavelength of 290.0nm and 25℃ were continuously measured. The result is Figure.4. We couldn’t confirm this enzyme catalyzes uric acid into allantoin.
The growth ability of the recombinant E. coli in minimal media containing uric acid
Growth test in minimal media of wild type E. coli and the recombinant E. coli were conducted to confirm uricase activity which was expressed by the recombinant E. coli. The result of this assay is shown in Fig5. No significant differences were observed in minimal media with 2mM uric acid and w/o ammonium. However, wild type is more positive growth in minimal media containing ammonia sulfate. We could’t understand why this incident happened.


SDS-PAGE
The result of SDS-PAGE is Fig6. The marker show the point, 200kDa, 116.2kDa, 66.2kDa, 45.0kDa, 31kDa, 21kDa.And Uricase from B. subtilis is 56.5kDa. So BBa_K2158004 expressed.
Determination of recombinant uricase in the supernatant and the deposition
In Fig6, expressed uricase is mainly in the deposition. So, we assayed uricase in the supernatant and the deposition of the sonicated cell suspension respectively by the method mentioned above and tried to verify the activity of the uricase. This protein extraction and sample preparation was followed the protocol of SDS-PAGE. (Protocols)The result of absorbance change is shown in Fig7. There is a possibility that device BBa_K2158004 has enzyme activity. Uric acid in the reaction mixture may be catalyzed into allantoin.