Part:BBa_K774005
Bacterial-Mammalian promoter with RFP reporter: BBaK216005 + BHN + CArG promoter sequence E9-ns2 + B
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: 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 bacterial-mammalian 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. A similar pattern can be seen here as for the mammalian- bacterial promoter with eCFP as at a 20mM concentration the intensity of fluorescence sharply decreases, however the intensity here decreases down to a level between 10mM and 15mM potassium nitate concentration. There is also only a small difference between 5mM and 10mM potassium nitrate, which differs to the pattern seen with the bacterial-mammalian promoter ligated to eCFP. 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.
Flow Cytometry
Flow cytometry was used with part BBa_K774005 to quantify the number of cells which fluoresced (RFP) in response to induction by potassium nitrate.
Three tubes of media were inoculated with E. coli transformed by the B-M + RFP biobrick (BBa_K774005). Each tube then had potassium nitrate added to it at different concentrations; 0 mM, 1 mM and 10 mM respectively. The E. coli were grown over night and then spun down, fixed in 4% PFA and re-suspened in 500ul PBS. The samples were then analysed in an Acuri C6 flow cytometer.
[http://2012.igem.org/Team:NRP-UEA-Norwich/Protocol Full Protocol]
Flow cytometry data for B-M RFP transformed E. coli that were grown in either 0 mM, 1 mM or 10 mM potassium nitrate. Top row: Scatter plot of raw data and gating strategy utilised. Middle row: RFP Fluorescence profiles of samples. Lower left: Fluroescence profiles of the three samples overlai on the same plot. As the graph indicates, the number of cells which fluoresce is proportional to the concentration of potassium nitrate that the cells are exposed to. Suggesting that our hybrid promoter is functional as expected.
Sequencing
BLAST Analysis
To review the success of the sequencing of the Biobrick sample, the predicted DNA sequence was compared to the sequence data using a Pairwise BLAST analysis ([http://blast.ncbi.nlm.nih.gov/Blast.cgi bl2seq]). The predicted DNA sequence was entered as the Query and the data from the sequencing analysis was entered as the Subject. The summary information at the top of the image identifies how many bases are identical between the two sequences. Lines joining the two sets of sequences indicate identical bases, and the absence of a line indicates a difference between the two sequences.
Usage and Biology
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- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 101
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Illegal AgeI site found at 904 - 1000COMPATIBLE WITH RFC[1000]
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