Part:BBa_K774007
Mammalian-Bacterial promoter with RFP reporter: CArG promoter sequence + BBaK216005 + BBa_K08101
Our hybrid promoter hopes to add to the systems already in the registry by creating a hybrid promoter that combines the bacterial promoter PyeaR and the mammalian CArG element , in the orientation mammalian to bacterial, both of which respond to exogenous nitrogenous species. Combining the two would allow a more modular NO sensor that can be used in mammalian and bacterial cells interchangeably. The hybrid promoter has been attached to the reporter: Red Fluorescent Protein (RFP). The hybrid promoter has been characterised by observing expression of flourescent protein, and found to have increased transcription in response to increasing concentrations of potassium nitrate.
E. coli containing this part were then grown in media at 0 mM, 5 mM, 10 mM, 15 mM and 20 mM potassium nitrate concentrations over night before being lysed. The lysed product was then run through a fluorometer in order to gain data of the intensity of expression at different concentrations of substrate.
NOTE: All data for the fluorometer has had the equivalent 0 mM reading subtracted from it in order to nulify the affects of light scattering due to cell debris
The graph above shows the flourescence measured from the expression of RFP due to the response of the mammalian-bacterial promoter to different concentrations of potassium nitrate. The wavelength reading which corresponds to RFP is between 600-650nm. The graph clearly demonstrates that between 0mN and 15mM there is a proportional relationship between fluorescence intensity and potassium nitrate concentration. It has been found that for all BioBricks apart from the mammalian-bacterial promoter ligated to eCFP at a 20mM concentration the intensity of fluorescence sharply decreases. This may be due to the cell overexpressing eCFP up to the point at which the excess protein begins to form inclusion bodies which can no longer fluoresce; alternatively, this could be due the potassium nitrate concentration reaching the critical concentration at which it becomes toxic to the cell. This data differs to the readings taken from the bacterial-mammalian ligated to eCFP, as well as the hybrid promoters to RFP, which may suggest there is a difference in the molecular mechanisms that these promoters function by; however at this point the change in intensity at 20mM is inconclusive and is an area which we would like to look into further.
We were initially unsure of the effect that the orientation of the bacterial (PyeaR) and the mammalian (CaRG) genes would have in gene expression, therefore we synthesised two hybrid promoters in the orientation bacterial-mammalian and mammalian-bacterial. The graph above compares the intensity of fluorescence of the two hybrid promoters (BBa_K774007 and BBa_K774005) ligated to RFP. There appears to be no pattern if the difference between the intensities of these two promoters; however both promoters do show a decrease in intensity at 20mM potassium nitrate and decrease from a maximum intensity of 82a.u. (bacterial-mammalian) and 66a.u. to approximately 36a.u.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 91
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 792
Illegal AgeI site found at 904 - 1000COMPATIBLE WITH RFC[1000]
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