Difference between revisions of "Part:BBa K2272001"

Latest revision as of 03:34, 2 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(i.e. large amount of oxygen available or significant increase in mitrochondrial respiration) modulation, MnSOD will be upregulated by increasing the corresponding 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 (shown to be ~4 hours 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 to simulate differential oxygen levels. Bacteria in tubes that are covered for longer periods of time will consume the available oxygen over time, resulting in lower dissolved oxygen levels. After the experimental window closes, all relevant parameters were measured (DO, OD600, and RFU output from the optical plate reader) simultaneously. The first trial was conducted on 9/25/2017 and cultures were grown for a total of four hours before being transferred into 14 mL falcon tubes. At four hours, tubes were opened and tested for DO with a Vernier Instruments DO probe and for OD600 with a Genesys 2 spectrophotometer in the order from most hours spent open to least. The reasoning behind this is that the measurement process was time-consuming and exposing cultures that had spent more time open to atmospheric oxygen would minimize the bias caused by re-exposure, as they would be less sensitive than cultures that had more time in an anaerobic environment to changes caused by unsealing tubes. Immediately after all tubes were opened and samples were taken from each strain and time point, the well plate was sealed with an an optical adhesive cover 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. For the relationship between DO and RFU, the coefficient of determination is 0.445, corresponding to a correlation coefficient of -0.667. This indicates that the observed RFU of bacteria containing the Mn-SOD reporter biobrick is negatively correlated with DO with a linear relationship of moderate strength. The relationship between the RFU and time spent exposed to atmospheric oxygen, while positive, was much weaker. The coefficient of determination (R2) was only 0.023, corresponding to a correlation coefficient (R) of 0.152. This is indicative of a weak linear relationship.

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 10/22/2017. 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 9/25/2017, the strain transformed with the Mn-SOD reporter plasmid showed increasing fluorescence over time but a decreasing RFU value as the observed DO content increased. The coefficient of determination and correlation coefficient for the relationship between RFU value and time spend open were 0.503 and 0.709 each. This indicates that the relationship is positive and moderately strong. The relationship between DO and RFU values was negative and the coefficient of determination and correlation coefficient were 0.374 and -0.612. This is indicative of a moderately strong negative linear relationship.

Figure 2: Results from the trial performed over 10/22/2017.

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.These results both contradict and support our original hypothesis and indicate that an unmeasured effect is causing the DO content to decrease in cultures that spend more time exposed to atmospheric oxygen.

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 spent exposed to atmospheric oxygen. In addition, the plot of RFU versus DO shows weaker linear relationship with coefficient of determination and correlation coefficient of only 0.085 and 0.292 when both trials are aggregated into a single dataset. This is weaker than the observed relationship between RFU value and time spent open, which had corresponding R2 and R values of 0.341 and 0.584. This indicates a moderate linear relationship between RFU value and time spent open. In addition, Mn-SOD was tested in P. denitrificans and the results corroborated claims in the literature and results found by experimentation. The R and R2 values for the relationship between RFU value and time spent open were 0.198 and 0.445. The relationship between DO content and RFU value had R and R2 values of 0 and 0. These results were mildly positive and indicate to us that Mn-SOD generally behaves the same way in P. denitrificans as it is reported to in E. coli.

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

DH5a Untransformed

In order to compare expression against a control baseline, we also measured the fluorescence of an untransformed culture of DH5a E. coli cells. When a line of best fit was calculated for the data, there was almost no significant relationship between observed relative fluorescence and either the dissolved oxygen content and the time spent open and exposed to the atmosphere. This was most evident when the data from both replications were aggregated. The coefficient of determination for relative fluorescence versus dissolved oxygen content was 0.008. The coefficient of determination for relative fluorescence versus time spent open is 0.041. These values correspond to correlation coefficients of 0.0894 and 0.202 respectively. These are both very weak linear relationships.

Figure 7: Aggregated data from both trials for fluorescence measurements of untransformed DH5a E. coli.

When the data was split by trial run, however, there were also contradictory findings. Data from the first trial conducted on 9/25/2017 suggest a positive relationship between relative fluorescence and hours spent open and exposed to atmospheric oxygen, while the relationship between dissolved oxygen and relative fluorescence was close to nonexistent. The coefficients of determination for each were 0.198 and 0.094. These correspond to correlation coefficients of 0.444 and 0.306 respectively. This means the relationship between relative fluorescence and hours spent open to the atmosphere is moderately strong while the relationship between relative fluorescence and dissolved oxygen content is weak.

Figure 8: Scatterplots with trends fit to the data for untransformed DH5a bacteria.

Results from the second trial conducted on 10/22/2017 indicate that time spent exposed to atmospheric oxygen has an opposite effect on relative fluorescence. There is a moderate to weak relationship between time and relative fluorescence. The relationship between relative fluorescence and dissolved oxygen content, however, is non-existent just as it is in the first trial. The coefficients of determination were found to be 0.207 and 0.017. These correspond to correlation coefficients of 0.455 and 0.130. This means a weak to moderate negative relationship between dissolved oxygen and relative fluorescence and a weak relationship between relative fluorescence and dissolved oxygen.

Figure 9: Scatterplots with trends fit to the data for untransformed DH5a bacteria.

Sequence and Features