Part:BBa_K2334004

J23100 + RiboJ + B0034 + YgfU, Urate Transporter Generator

YgfU is a high-capacity transporter for uric acid in E.coli, which is homologous to nucleobase transporters of the ubiquitous family NCS2. In our project, it's used as the urate transporter in E.coli, whose expression is driven by J23100. RiboJ is used for the quantitive pathway construction.

Figure 1. YgfU wasn’t detected driven by J23100+RiboJ+B0034 gene strucutre. The protein expressed is noted in the figure.

We couldn’t detect the YgfU protein expression via SDS-PAGE. We constructed the part BBa_K2334006, which consists of K2334004 (J23100 + RiboJ + B0034 + YgfU, Urate Transporter Generator) and K2334001 (J23100 + RiboJ + B0034 + pucL, Urate Oxidase Generator).

We tested the function of YgfU by detecting the urate concentration in the overnight cultured LB medium. In this experiment, we used pucL(K2334001), pucL + YgfU(K2334004), eGFP(J23100 + RiboJ + B0034 + eGFP, not submitted) in pSB1C3 vector (transformed to E.coli BL21). The result shows that, those constructions can’t reduce the urate concentration in the LB medium (Figure 2).

Figure 2. pucL, YgfU + pucL & eGFP were cultivated in urate LB medium overnight (Urate concentration was about 500uM). The initial OD600 of the LB medium was controlled at 0.02. Urate concentration in the LB medium was detected by HPLC. The values we showed are the raw peak area data. The figure shows that pucL, YgfU + pucL were not able to reduce the urate concentration in the LB medium.

To our surprise, the result shows that urate did enter the cytoplasm, but at one point, it’s excluded from the cell and won’t be taken in again anymore (Figure 3). If uricase exists in the cytoplasm, it can still work to reduce the urate concentration in the cytoplasm, but even so, urate still can’t be taken in continuously. There’s a possible unknown mechanism to prevent the urate from entering the cell when the mechanism is activated. In previous research, YgfU was expressed driven by T7 promoter, which was a furious expression process that the mechanism couldn’t influence in a short time. When YgfU is constitutively over-expressed, the mechanism can actually act successfully.

Figure 3. pucL and eGFP were cultivated in urate LB medium overnight (Urate concentration is about 1mM). The initial OD600 of the LB medium was controlled at 0.02. Urate concentration in the LB medium was detected by HPLC. Cytoplasm urate concentration was detected by HPLC as well. Diluted cell lysate was used for detection.

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 397
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 579
  
• 1000
  COMPATIBLE WITH RFC[1000]