Regulatory

Part:BBa_K2810002:Experience

Designed by: Geraint Parry   Group: iGEM18_Cardiff_Wales   (2018-08-24)


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Applications of BBa_K2810002

The Cardiff iGEM team of 2018 characterised this promoter using the GUS and mCherry reporter genes. We compared the expression of this promoter to that of the 35S CaMV promoter using the same reporter genes. These can be seen below. We found that using mCherry as a reporter gene, we could quantify the expression of the reporter, and found that the RTBV promoter increased expression levels to 10-100x that of background levels. This is still less than 35S CaMV promoter by a factor of 10-100.



Figure 1) The GUS staining with the upper row of leaves is 35S, and is much stronger than the lower leaves which are using the RTBV promoter instead.




Figure 2) The quantification report of the 35S promoter when linked to mCherry. This shows the raw photon output of leaf tissue in the table below it too, suggesting that the 35S CaMV promoter has expression 1000x that of the background level, and between 10-100x that of RTBV.


We also performed an assay using eGFP to quantify this promoter. We used the RTBV promoter as it is supposed to have higher expression in vascular tissue, which Figure 3 shows. However, the raw quantification report did not show that it had significantly higher expression than control leaves, even though the values were higher.


Figure 2) The quantification report of RTBV linked to eGFP. The raw output shows bright fluorescence in leaf vascular tissue, but these results do not match that of figures 1 and 2. The quantification report in the table show higher raw values, but these are not significant.





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