Part:BBa_K2334001

J23100 + RiboJ + B0034 + pucL, Urate Oxidase Generator

Urate oxidase (uricase, EC 1.7.3.4) is an enzyme with copper bonds that catalyze the oxidative opening of the purine ring of uric acid to form allantoin which is 5–10 times more soluble than uric acid. In Bacilus Subtilis, the gene is coded by pucL gene. This enzyme can be used therapeutically to reduce toxic urate accumulation. In our project, uricase is used as the most important enzyme in urate degradation, whose expression is driven by J23100. RiboJ is used for the quantitive pathway construction.

The CDS of uricase in the part is the same as the part K2158000 submitted by Gifu iGEM Team 2016.

The metabolic pathway design of the project. The dotted lines represent the enzymes which can accelerate the spontaneous reaction processes. Urate oxidase (uricase, EC 1.7.3.4) is an enzyme with copper bonds that catalyzes the oxidative opening of the purine ring of uric acid to form 5-hydroxyisourate. HIU hydrolase (3.5.2.17) catalyzes the second reaction of the pathway, hydrolyzing 5-hydroxyisourate (HIU), a product of uricase in the first step to form 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU). OHCU is catalyzed by OHCU decarboxylase (4.1.1.97).

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.

We improved the project of 2016 Gifu iGEM team at the same time. We used the same pathway in our project but had different goals. They intended to utilize the urate metabolic pathway to clean the birds’ dropping, which is rich in urate.

In Gifu’s project, they intended to reduce the urate concentration outside the cell as well. However, they didn't succeed in the end. In our 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.

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

Added by Sesame_Shenzhen

Validating the functionality of Urate Oxidase We constructed E.coli 1917 with only urate oxidase (oxidative system only, no promoter) as the experimental group, and transferred an empty plasmid into another strand as the control group.

Figure 1 validating the catabolic effect of Urate Oxidase

1. Cultivate the engineered strain in LB liquid medium and adjust the uric acid concentration to the corresponding concentration. N=3

2. incubation condition at 37 degrees Celsius, PH=7, 220 rpm, culturing for 48 hours.

3. Centrifuge at 3500 rpm for 5 minutes, and extract the supernatant.

4. use of uric acid detection kit to collect results.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]