Difference between revisions of "Part:BBa K2272000"

Latest revision as of 03:33, 2 November 2017

promoter VHb

A promoter that is maximally induced under microaerobic condition and regulates the expression of VHb, a bacterial hemoglobin that acts as a terminal oxidase.

Usage and Biology

VhB has been described in literature and previous iGEM projects as being maximally active in hypoxic conditions, ideally at 2% dissolved oxygen saturation. When tested with the above describe protocol, results are inconclusive. The first trial was conducted on 9/25/2017 and yielded positive data that corroborated previous evidence of maximal induction under hypoxic conditions. As the length of time the culture tube was open to aeration increased, measurements of RFU decreased, indicating inhibition of GFP expression. This followed a linear relationship with R and R2 values of 0.044 and -0.210. There was almost no observed relationship between relative fluorescence and dissolved oxygen content, which had R and R2 values of 0.022 and 0.148. Both relationships were very weak and indicate little relationship between RFU value and either DO or time.

Figure 5: Scatterplots of relative fluorescence versus time and dissolved oxygen content DH5a E. coli transformed with the VhB reporter biobrick.

When tested on 10/22/2017, there is a clear positive relationship between time spent exposed to atmospheric oxygen and the intensity of the relative fluorescence. This data is not only contradictory to the other data collected on 9/25/2017, but it is also contradictory to the results found in literature and by other iGEM teams. Because of this, it is reasonable to conclude that there may be a bias in the measurement of fluorescence emitted by cells expressing the VhB reporter biobrick. The relationship between time and RFU value had R and R2 values of 0.369 and 0.607. This is a moderately strong relationship. The relationship between DO and RFU was almost negligible and its R and R2 values were only 0.007 and -0.0837.

Figure 6: Scatterplots of relative fluorescence versus time and dissolved oxygen content DH5a E. coli transformed with the VhB reporter biobrick.

When all data is aggregated between trials, the R and R2 values are much stronger. For the relationship between RFU and time, they are 0.393 and 0.627. For RFU and DO they are 0.236 and 0.486. It must be stressed, however, that these results are contradictory to previous findings and there is strong likelihood of bias in the results. One potential source may be due to the actual versus observed DO level. Volumes used for testing were smaller than the volume recommended for use by the Vernier DO probe. There was also the issue of the time taken to complete all required measurements. The total time between removal of samples from the shaking incubator and the start of the qPCR protocol to measure relative fluorescence was between one and one and a half hours. In that time, gene transcription and translation could have changed significantly.

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