Part:BBa_K376003:Experience

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Applications of BBa_K376003

All measurements were taken using the Tecan Infinite M 1000 spectrophotometer. The following is a graph of the raw fluorescence values recorded by the Tecan for two oxygen promoters, D5 and J6. Each construct was duplicated in the 96-well plate. One was allowed to grow normally under aerobic conditions, while the other sample was covered in 100uL of mineral oil to create an anaerobic environment. Serial dilution of two plates resulted in 3 sets of data for this experiment, as seen below.

The next graph shown is the Raw data for OD for the same constructs shown above. The OD is proportional to the number of cells growing in the media, so dividing the fluorescence by these values is approximately proportional to the fluorescence per cell.

Shown below is the Fluorescence over OD of each sample. As the samples reach steady state, the graph should become a horizontal line. Notice how the graph comes closer and closer to reaching this steady state with each successive plate. Also, it is important to remember that evaporation comes into play here when comparing anaerobic and anaerobic samples with each other. Since the anaerobic samples are covered in mineral oil, it is expected that the evaporation rates of the media underneath is less. This means that, for aerobic cells, the OD becomes artificially inflated as the same number of cells have become concentrated in less and less media. It is not known to what extent the evaporation effect has on these data.

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Next, the fold-change was calculated for each sample. That is, the fluorescence/OD for anaerobic growth of the sample was divided bye fluorescence/OD for the aerobic sample. The same was done for a constitutive promoter expressing AFP. That way, a comparison could be made between constant expression of AFP in an anoxic environment and induced expression.

[http://2010.igem.org/Team:Penn_State/Project#Oxygen_Promoter_J6 Graphical Results]

The results are that both promoters indeed were inducible under anaerobic conditions. The fold-change expression when switching from aerobic to anaerobic is significantly higher than the fold-change expression of the constitutive promoter.

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