Difference between revisions of "Part:BBa K4225006"

Revision as of 15:58, 12 October 2022

katGp-RFP-Pc-OxyR

This is a composite part that consists of katGp with RFP as a reporter protein and its transcription factor, OxyR, that is induced by a constitutive promoter Pc (BBa_J23109). The OxyR protein reacts with H2O2, forming a disulfide bond in the protein, and hence OxyR is activated. The activated OxyR can then bind to katGp and activate transcription. In this case, RFP is produced. Future teams can use this construct for their project or experiments that are related to hydrogen peroxide, such as identifying the presence of hydrogen peroxide from a reaction.

Contribution: HKUST 2022

Summary

We analyzed the fluorescence intensity of RFP in an increasing concentration of H2O2. Since RFP is regulated under katGp that responds to change of [H2O2] with the assistance of OxyR, we would expect a gradual increase of RFP fluorescence with increasing concentration of H2O2. We analyzed the influence of [H2O2] on the fluorescence intensity of RFP regulated under katGp. Coupled with OxyR, a gradual increase of RFP fluorescence is expected when H2O2 concentration is increased.

Experiments

katGp-RFP-Pc-OxyR (BBa K4225006) constructs are tested with 5000, 1000, 200, 40 and 0 μM of H2O2 alongside katGp-RFP (BBa K4225005) 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 561 nm and emission was read at 610 nm for RFP.

Results and Discussion

From Figure 1, we can observe that the katGp-RFP-Pc-OxyR construct has a gradual increase in fluorescence.. It is important to notice that the fluorescence graph of katGp-RFP-Pc-OxyR constructs is above the plot for the positive and negative controls. This proves the presence of OxyR is required for the katGp to produce high fluorescence.

In addition, based on Figure 2, we can observe that the katGp is activated at a higher H2O2 concentration range of 40 - 200 μM, while oxySp is activated at 0 - 40μM. This shows that katGp has a higher activation threshold of hydrogen peroxide compared to oxySp and is less sensitive than the latter.

This is a part of our engineering cycle. For more information on our engineering cycle, refer to this link