Difference between revisions of "Part:BBa K2272000"

Revision as of 04:50, 1 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 and yielded positive data that corroborated previous evidence of maximal induction under hypoxic conditions. As time spend open 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, 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, 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.

Sequence and Features