Part:BBa_K2158000

Uricase from Bacillus subtilis

Uricase from Bacillus subtilis.

The following BioBrick information was updated by 2017 iGEM Team SCU-WestChina

We improved the project of 2016 Gifu iGEM team and function of biobrick. We used the same pathway in our project. Though we have different goals but we both wanted to utilize the urate outside. For Gifu, they intended to utilize the urate metabolic pathway to clean the birds' dropping, which is rich in urate. However, they didn't succeed in the end. In SCU-WestChina's project, we constructed the completed pathway and verified the parameters may influence the result to reduce the urate concentration outside the cell, then reduce the urate concentration outside successfully. For the birds’ dropping cleaning, according to our experiment, E. coli Nissle 1917 transformed wth uricase generator (BBa_K2334001), whose expression is driven by constitutive promoter J23100 in M9 medium, is able to achieve the goal to clean the urate in the birds’ dropping by spread the medium culture on the surface of it.

The constitutive promoter determines that the uricase can be generated continiously to clean the urate, which is an improvement compared to use inducible promoter T7. Our uricase sequence is the same as the part BBa_K2158000, which is submitted by Gifu 2016 team.

Figure 1. pucL was expressed successfully driven by J23100+RiboJ+B0034 gene strucutre. The protein expressed is noted in the figure.

To test the function of pucL we expressed, we used the crude bacteria extraction (expressed in E. coli BL21) to test directly. Before we started to react, the total protein quantity of each sample was made to be the same. 100ul reaction extraction was added into 900ul PBS with urate (PH=8.0). HPLC was performed after reaction for 2h and heated for 10min. The components of reaction extraction are described in the table with brackets. (See SCU-WestChina 2017 protocols for more details).

Figure 2. Results of separated crude extraction mix experiment. The numbers refer to the HPLC original peak area.

The result showed that pucL has a strong ability to consume the urate.

To achieve our goal to utilize the urate outside the bacteria, SCU-WestChina cultivated E. coli Nissle 1917 transformed with eGFP, pucL (BBa_K2334001), YgfU+pucL (K2334004 + K2334001), LM4 Full (BBa_K2334017), LM4 Optimum (BBa_K2334017) in M9/LB medium and aerobic & anaerobic environments to explore the paramters may influence the process. The urate concentration measured by HPLC.

The values in different independent repeated experiments are not always the same. But the tendency is the same in every group: For urate utilization ability, M9(Anaerobic)＞M9(Aerobic) ＞LB(Anaerobic)＞LB(Aerobic). It means that bad nutrition environment and anaerobic environment can reduce the urate concentration better. According to our experiment results, our gene constructions (pucL, YgfU + pucL, LM4 Full, LM4 Optimum) all have the ability to reduce the urate concentration outside the cell.

Figure 3. The experiment results of Group 1-3 original HPLC peak area result shown in bar. Green bar is eGFP as control. It's clear that M9 and anaerobic cultivation performed better for urate consuming.

For more details about the part, please visit SCU-WestChina 2017 wiki.

Usage and Biology

result

Group: NEFU_China 2019

As shown in Fig.5, the control group kept constant uric acid levels, but the experimental group displayed reduced uric acid concentrations in a time-dependent manner in the first 3 h of incubation. After that, the uric acid levels kept constant. The data suggested that the expression of uricase was induced at high concentrations of uric acid and repressed at its relatively low concentrations. The result indicated that the expression of uricase was under the control of Phuc2 responsive to the concentration of uric acid.

Fig.5 The cultured bacteria were transferred into M9 medium containing 8 mM uric acid. Samples were collected every 30 min to measure the concentrations of uric acid. The control group was the Nissle bacteria while the experimental group was the Nissle bacteria with the uric acid regulation module. Error bars represent standard deviations.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]