Part:BBa_K3311001

pHucO

The pHucO operator from Deinococcus radiodurans contains a single binding site that combine with HucR.

Reference

1.Liang C , Xiong D , Zhang Y , et al. Development of a novel uric-acid-responsive regulatory system inEscherichia coli[J]. Applied Microbiology and Biotechnology, 2015, 99(5):2267-2275.

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]

Function test

The pHucO operator from Deinococcus radiodurans contains a single binding site that combine with HucR. We added mCherry (BBa_J06504) after pHucO to verify the regulating capacity of pHucO. The plasmid “pHucO-mCherry” was transformed to E. Coli DH5α for function test. As shown in Figure 1, The bacteria turned red after incubating overnight at 37 °C, which means the pHucO works well.

Figure 1 Incubation of E. Coli containing the pSB1C3-pHucO-mCherry

Then EMSA analysis was carried out for testing the affinity between pHucO and HucR-mCherry (BBa_K3311007).The biotin labeled pHucO and HucR-mCherry fusion protein were prepared following the EMSA protocols. The HucR-mCherry and biotin labeled pHucO were incubated together and then separated by EMSA gel. The HucR-mCherry-pHucO complex moves more slowly than non-binding labeled pHucO probes. After adding uric acid, HucR-mCherry-pHucO complex will decrease and could be viewed in EMSA gel. The concentration of HucR-mCherry increases from 0 to 1 g, which means the more HucR-mCherry is, the more the combination is, proving the combination of HucR-mCherry and pHucO (Figure 2A). When uric acid appears in the solution, HucR-mCherry loses its function and falls off from the pHucO. Consequently, the downstream gene can be translated normally. The EMSA shows that, under the same concentration of HucR-mCherry and pHucO, there is a negative relationship between the concentration of uric acid and HucR-mCherry-pHucO complex. As the concentration of uric acid increases from 0 ~ 1000 M, the color of HucR-mCherry-pHucO complex become lighter (Figure 2B ), indicating the effect of uric acid on HucR-mCherry-pHucO complex.

When uric acid appears in the solution, HucR-mCherry loses its function and falls off from the pHucO. Consequently, the downstream gene can be translated normally. The EMSA shows that, under the same concentration of HucR-mCherry and pHucO, there is a negative relationship between the concentration of uric acid and HucR-mCherry-pHucO complex. As the concentration of uric acid increases from 0 ~ 1000 M, the color of HucR-mCherry-pHucO complex become lighter (Figure 2B), indicating the effect of uric acid on HucR-mCherry-pHucO complex.

Figure 2 (A) The combination of pHucO and HucR-mCherry in EMSA gel. (B) The combination of pHucO and HucR-mCherry after uric acid treatment.

Reference

1.Liang C , Xiong D , Zhang Y , et al. Development of a novel uric-acid-responsive regulatory system inEscherichia coli[J]. Applied Microbiology and Biotechnology, 2015, 99(5):2267-2275.