Part:BBa_K2158004
Uricase (from B.subtilis)
It can express Uricase[1.7.3.3].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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.
The growth tests in minimal media
The growth tests in minimal media containing purines of B. subtilis were performed. The result was shown in Fig3. As expected, B. subtilis grew well with uric acid and ammonium sulfate. We confirmed that Bacillus subtilis utilized these intermediate as nitrogen sources.
Fig3. The growth of B. subtilis in minimal media
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 1.0 mmol/L EDTA and detergent (pH8.5). 0.5 mL 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 nm and 25℃ were continuously measured. The result was shown in Fig4. We couldn’t confirm this enzyme catalyzes uric acid into allantoin.
Fig4. The change of absorbance at 290 nm
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 was shown in Fig5. No significant differences were observed in minimal media with 2mM uric acid and w/o ammonium. However, wild type showed more positive growth in minimal media containing ammonia sulfate. We could’t understand why this incident happened.
Fig5. The growth of E. coli and recombinant E. coli in minimal media
SDS-PAGE
The result of SDS-PAGE was shown in Fig6. The marker show the point, 200kDa, 116.2kDa, 66.2kDa, 45.0kDa, 31kDa, 21kDa, respectively. Because Uricase from B. subtilis is 56.5kDa, BBa_K2158004 was synthesized.
Table1. About each lane in SDS-PAGE
| Lane | Explanation |
|---|---|
| 3 | The device includedBBa_K2158004 (supernatant) |
| 4 | The device includedBBa_K2158004 (precipitation) |
| M | Marker |
Fig6. SDS-PAGE
Determination of recombinant uricase in the supernatant and the deposition
In Fig6, expressed uricase was 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 was shown in Fig7. There was a possibility that device BBa_K2158004 has enzyme activity. Uric acid in the reaction mixture may be catalyzed into allantoin.
The result of SDS-PAGE shows that Uricase was mainly in the precipitation.
We centrifugated the sonicated cell extraction and separated into the supernatant and the precipitation. We verified the activity of the Uricase in each solution. We also prepared the reaction mixture that borate buffer was added instead of the enzyme solution (Negative).
Fig7.
Fig8.
The decreased absorbance by the precipitation was significant. The expressed B . subtilis Uricase in E. coli must be agglomerated but still has slight activity.
Combine this two figures, even though the activity is lower than reagent Uricase, the activity of Uricase which expressed by our recombinant E. coli can be detected.
| None |