Difference between revisions of "Part:BBa K774004"

Revision as of 16:00, 26 September 2012

Bacterial-Mammalian promoter with eCFP reporter: BBaK216005 + CArG promoter sequence + BBa_E0420

Pellets of E. coli transformed by this part ligated to BBa_E0420 (an RBS and CFP reporter) and grown in media with added concentrations of potassium nitrate. Going from left to right in concentrations of potassium nitrate: 10 mM, 50 mM, 100 mM and 0 mM

Aim: 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 , 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: enhanced Cyan Fluorescence Protein (eCFP). 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.

This image (right) shows competent cells transformed with part: BBa K774004 and grown in media containing potassium nitrate (as a source of nitrates in order to induce promoter activity) at concentrations of 0 mM, 10 mM, 50 mM and 100 mM (from right to left). The E. coli was grown for 6 hours and then added to eppendorf tubes and spun down in a centrifuge in order to produce a pellet. The four samples were then viewed under a UV box to assess for fluorescence; as the photograph to the right shows that the sample at 0 mM potassium nitrate did not fluoresce, however those at 10, 50 and 100 mM potassium nitrate did fluoresce. They also appeared to fluoresce at the same strength, suggesting that 10 mM was equal to or above the maximum sensitivity level of this part.

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 eCFP due to the response of the bacterial-mammalian promoter to different concentrations of potassium nitrate. The wavelength reading which corresponds to eCFP is between 440-500nm. The graph clearly demonstrates that between 0mM and 15mM there is a proportional relationship between fluorescence intensity and potassium nitrate concentration. There appears to be a sharp increase in fluorescence intensity between 5mM and 10mM, and the rate at which intensity increase gradually decreases so that there is only a small increase between 15mM and 20mM.

We were initially unsure of the effect that the orientation of the bacterial (pYEAR) and the mammalian (CaRG) genes would have on 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_K774004 and BBa_K774006) ligated to eCFP. There is a distinct difference between the intensity of fluorescence produced by the bacterial-mammalian promoter and the mammalian-bacterial promoter; which is something that we would like to look into further. It is particularly interesting that at an intensity of 109a.u. the mammalian-bacterial promoter returns to the same level of intensity as the suggested maxiumum of the bacterial-mammalian promoter at 40a.u.

Sequencing

BLAST Analysis