Part:BBa_K4225003

oxySp-GFP-Pc-OxyR, a composite part that is sensitive to H2O2

This composite part is made by digestion and ligation of Pc-OxyR (BBa_K4225000) and oxySp-GFP(BBa_K4225002) composite parts. This composite part is used to characterized oxySp and OxyR, proving that activity of oxySp requires the activation of OxyR by Hydrogen Peroxide. Future teams can use this construct for their project or experiments that are related to hydrogen peroxide, such as characterizing the presence of hydrogen peroxide due to an enzyme activity.

Contribution: HKUST 2022

Summary

We analyzed the influence of [H2O2] on the fluorescence intensity of GFP regulated under oxySp. Coupled with oxyR, a gradual increase of fluorescence is expected with an increase in H2O2 concentration.

Experiments

oxySp-GFP-Pc-OxyR (BBa K4225003) constructs are tested with 5000, 1000, 200, 40 and 0 μM of H2O2 alongside oxySp-GFP (BBa_K4225002) and Pc-OxyR (BBa_K4225000) as positive controls and pSB1C3 construct as negative control. Constructs are inoculated in 5mL LB with chloramphenicol (CHL LB) for 16 hours. The constructs are then subcultured into 5 mL CHL LB and grown until they reach 0.7-1.0 OD600 (log phase). Cultures are subsequently back diluted to 1.0 OD600. These steps are done to synchronize the growth period and the amount of cells for each culture. These cultures are added to different concentrations of H2O2 and incubated for 3 hours. Finally, the samples were excited at 488 nm and emission was read at 530 nm for GFP.

Results and Discussion

Figure 1. Normalized Fluorescence/OD600 of GFP v/s H2O2

From Figure 1, we can observe that the oxySp-GFP-Pc-OxyR construct has a gradual increase in fluorescence. Moreover, the fluorescence for oxySp-GFP-Pc-OxyR constructs is higher than the fluorescence for the positive and negative controls. This proves the presence of OxyR is required for the oxysp to produce high fluorescence.

Figure 2. Fold Change of the Normalized Fluorescence of BBa K4225003 and BBa K4225006 v/s H2O2

Moreover, based on Figure 2, we can observe that the oxySp is activated at a range of 0 - 40 μM, while katGp is activated at a higher concentration of H2O2 compared to oxySp. This shows that oxySp has a lower activation threshold of hydrogen peroxide compared to katGp.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1046
  Illegal NheI site found at 1069
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 811