Team:Uppsala University/Promoters

This bar diagram shows the differences measured fluorescence between different promoters in each strand, data from one lab strain should not be compared to the other lab strain since these haven't been grown under exactly same conditions.

A promoter couldn't be predicted to have the corresponding strength for the screening systems to give a working artificial small RNA / 5´UTR-YFP mRNA ratio, it was necessary to assemble with promoters of various strengths. For this the promoters J23106, J23110 and PlacIQ was chosen.

To prevent future unnecessary assembling by our or any other team a promoter test was conducted on the promoters used in this years iGEM project, a secondary goal of the promoter-test was to map the strengths of the commonly used promoters Plac and PlacIQ.

The promoter test was conducted by measuring fluorescence expressed by each individual promoter assembled with B0032-SYFP2 (BBa_K864101) though a FACS (Fluorescence Activated Cell Sorter) in lab strains MG1655 and DH5α.

The test was conducted on triplicated cultures of each promoter construct, which were measured in the FACS. For each culture 10⁵ cells fluorescence were measured individually, dead cells and non fluorescent cells were discarded, and an average was calculated. All promoter constructs of each strain were grown under same conditions.

The T5lac promoter is a hybrid promoter between a promoter from pro-phage T5 and the laci-repressor binding sites from the Plac promoter, so the T5lac promoter has the strength of the T5 pro-phage promoter but can be repressed by the laci-repressor.

The variance in expression between MG1655 and DH5α may depend on the J23101-B0032-SYFP2, which is the promoter used as reference. The construct with J23101 in DH5α may have been a weaker than average clone, resulting in higher RPU values compared to other DH5α. Alternatively the maximum protein expression is lower in DH5α due to its lower fitness also resulting in lower expression of SYFP2 in the J23101 construct.