Part:BBa_K3007000

HucR binding site

It is a special DNA sequence which can interact with HucR protein and repress the expression of downstream gene when uric acid is absent. If uric acid presents, the formation of HucR-hucO complex can be reduced and restore downstream gene expression. In our design, we used the HucR-hucO system as a detector for uric acid.

Contribution

Group: QHFZ-China iGEM 2019
Author: Cheng Li
Design:

Figure 1. Schematic cartoon of the DNA construct of BBa_K3007000

Documentation:
This year, QHFZ-China designed a UA monitor system in E. coli (Fig. 1). Pc is a constitutive promoter, Pcp6 promoter, and it promotes the expression of HucR and YgfU. If the concentration of uric acid (UA) in environment is low, HucR will bind to hucO sequence (located in PhucR), which inhibits the expression of downstream reporter, dsRed or sfGFP. When extracellular UA is present, YgfU can transport UA into the cytoplasm, which leads HucR dissociates from hucO, and induces the fluorescent protein expression.

Figure 1. Working mechanism of the uric acid detection system in E. coli. (A) Schematic diagram of the gene circuit design. (B) Structure of PhucR, showing the location of BBa_K3007000.

We used the process shown in Fig. 2 to test if the UA detection system works well.

Figure 2. Work flow chart for subsequent experiments

Two clones with UA detection system with dsRed as a reporter were tested. The original gene circuit was able to response to UA in a range of 0 to 200 μM (Fig. 3A), and the clone 1 showed much better dynamics than the other (Fig. 3B). Time course experiments showed that the fluorescence intensity became quite strong at 4 to 6 hours after UA induction, and became stable at 10 to 12 hours (Fig. 3C). Even if we removed UA by replacing fresh LB medium, after 48 hours shaking, the fluorescence would be still notable (Fig. 3D) and there was not significant difference of dsRed fluorescence / OD600 between before and after UA removing (Fig. 3E). All the data meant our design could detect high UA concentration quickly and stably.

Figure 3. Response of UA detection system after different concentration of UA induction. (A) A photo to visualize the fluorescence induced by UA under a blue light. (B) Responding curve about the dsRed fluorescence / OD600 to different UA concentration of two E. coli clones. Data were shown as mean ± SD. N = 3 technical repetitions. (C) Time course experiments about the dsRed fluorescence / OD600 of E. coli after 0, 20 or 100 μM UA addition. Data were shown as mean ± SD. N = 3 technical repetitions. (D) A photo to visualize the fluorescence after UA removal under a blue light. (E) Quantitative measurement of dsRed fluorescence / OD600 before and after UA removal.

We also tried more conditions to test if this system could work well in different environment. In the range of pH 6.0 to 8.0, response of the gene circuit was relatively stable (Fig. 4A). However, the volume of the reaction system would influence the response to UA (Fig. 4B). A possible explain was the relative surface area of the liquid level changed and consequently the dissolved oxygen changed. This result meant the experiments for UA detection should be done at the same reaction system volume. In other experiments, 1 mL reaction volume was used.

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]