Difference between revisions of "Part:BBa K2272001"

Revision as of 02:44, 1 November 2017

promoter MnSOD

A promoter that regulates the production of MnSOD (mitochondrial antioxidant manganese superoxide dismutase), which detoxify radical oxygen produced by mitochondrial respiration. Under oxidative stress or hypoxic modulation, MnSOD will be upregulated by an increase of its promoter activity as well as by other complex transcriptional/post-transcriptional modifications.

Usage and Biology

While Mn-SOD has been described in literature as an oxygen-inducible promoter element in E. coli, the results of experimentation described under protocols (citation, link) have been inconclusive. Following the previously described protocol, bacterial cell cultures were grown from single colonies for four hours to reach early- to mid-exponential growth phase (as shown in the literature and our own results). After four hours, colonies were transferred into 14 mL tubes. Because equipment to modulate the dissolved oxygen environment was not available, tubes were covered after a specified amount of time and kept closed until the end of experimentation when all relevant parameters were measured (DO, OD600, and RFU output from the optical plate reader). The first trial was conducted on 9/25 and cultures were grown for a total of four hours after being transferred into 14 mL falcon tubes. At four hours, tubes were opened and probed for DO and OD600 in the order from most hours spent open to least. Immediately after all tubes were opened and samples were taken from each strain and time point, the well plate was sealed with optical well tape and measured by a BioRad MyiQ optical plate reader. When plotted against hours spent open and exposed to open atmosphere, a steady increase in the relative fluorescence can be observed. When compared against the DO content (in mg/L), however, the opposite trend is observed. As the DO moves from 0.8 to 1.5 mg/L, RFU decreases from ~600 to ~400. This may be explained by the fact that DO is not necessarily correlated with the length time a culture spends open to the atmosphere. The data showed a sharp increase in the DO level, followed by a slow decline. Because of these inconsistencies, the plot of time exposed versus RFU may be more indicative of the Mn-SOD promoter’s response to cellular oxygen levels.

Figure 1: OD600 normalized RFU values between all measurement replicates over time and DO saturation/content (mg/L).

The same pattern is also observed in results taken from the second trial on 10/22. This time, bacterial cell cultures were grown at 37 C in a shaking incubator (220 rpm) for four hours before being transferred into smaller 3.5 mL culture volumes. All aliquots were grown and a new tube was covered every hour as described above, except cultures were grown for a total of six hours. As in the DH5a strain tested on 9/25, the strain transformed with the Mn-SOD reporter plasmid showed increasing fluorescence over time but decreasing fluorescence as the observed dissolved oxygen content increased.

Figure 2: Results from the trial performed over 10/22.

Again, the observation is made that as the duration spent opened increases, the level of relative fluorescence picked up by the optical reader does as well. It is also evident in Figure 2 that as the dissolved oxygen content increases, the relative fluorescence decreases.

Figure 3: Aggregated data for Mn-SOD in P. denitrificans across both trials.

Combining both trials yields stronger evidence for the trends described above, as a positive linear trend is also observed when plotting relative fluorescence against time spend exposed to atmospheric oxygen. In addition, the plot of relative fluorescence is weaker when both trials are aggregated into a single dataset. In addition, Mn-SOD was tested in P. denitrificans and the results corroborated claims in the literature and results found by experimentation.

Figure 4: Scatterplots of relative fluorescence versus time and dissolved oxygen content.

Sequence and Features