Part:BBa_K2752003

HucR

Binding to hucO in the absence of uric acid prevents transcription. In the case of uric acid, it binds to uric acid and does not prevent transcription.

Sequence and Features

Characterization

HucO is regulated by HucR. When uric acid is abscent, HucR can bind to hucO making downstream gene silencing. Lysis1 and Lysis2 are lyases. Lysis1siRNA286 can inhibit Lysis1, and Lysis2siRNA106 can inhibit Lysis2. When both plasmids are simultaneously expressed, the lyases are inhibited. But if any plasmid is lost or HucR combined hucO，part3 will not be expressed. And the bacterium is cleaved by the Lysis2 . A series of things happens when bacteria are excreted: First, the concentration of uric acid get lower. Second, the hucO of part3 is suppressed by HucR and cannot work. Next, Lysis2 siRNA106 cannot inhibit Lysis2. Finally, the bacteria are cleaved by Lysis2. To test the lethal ability of part2 and part3, and the regulation of uric acid on part3 we did four sets of experiments.

Figure 1. Plasmid map of part2 and part3。

[[File:Figure 16-colony result.png|400px|thumb|Figure 2. Colony number regulation experiment result。Blank is a plasmid-free BL21 strain. Part3 is a BL21 strain containing the part3 plasmid. Part2 is a BL21 strain containing the part2 plasmid. Part3-with uric acid is a BL21 strain containing part3 plasmid cultured with a uric acid （The final concentration of uric acid was adjusted to 4×10-4M.）

result

The promoter should be fully activated at the highest uric acid concentration safe to human body (0.465mM). The activity of the promoters were determined by measuring the fluorescence intensity of the enhanced green fluorescent protein (EGFP). The results showed that the Phuc2 had the highest sensitive and activity at the conditions of 0.1mM and 0.01 mM of uric acid. Then, the EGFP coding sequence was replaced by the uricase gene to check whether the module could sense and metabolize the uric acid. 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. 3 Comparison of induction strength of the promoter Phuc1 (circle), Phuc2 (square), and Phuc3 (triangle) by measuring green fluorescence of EGFP at 0.1 mM of the inducer uric acid (with 1:100 dilution of culture inoculation). Error bars represent standard deviations.

Fig. 4 Comparison of induction strength of the promoter Phuc1 (circle), Phuc2 (square), and Phuc3 (triangle) by measuring fluorescence of EGFP at 0.01 mM of the inducer uric acid (with 1:100 dilution of culture inoculation). Error bars represent standard deviations.

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.

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 508
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 244
  Illegal SapI.rc site found at 208