Team:Uppsala University/Promoters

To put synthetic and natural promoters on the same scale, a promoter test was carried out. Every promoter was assembled before B0032-SYFP2 (K864101) in the backbone K592200 (very similar to the pSB3x5 backbones). The test was performed in E coli wild type strain MG1655 and cloning strain DH5α by flow cytometry fluorescence measurements of single cells.

Triplicates of each strain and promoter were inoculated in 2 mL LB media with spectinomycin (50 µg/mL) and grown overnight shaking at 37° C. Samples were equilibrated in PBS solution at 1:160 dilution for one hour, and then measured by a BD Biosciences FACSaria III. 10^5 cells of each sample were individually measured and averaged, with dead and other non-fluorescent cells excluded. Promoter strength is measured relative to the reference promoter's, J23101, strength in corresponding strain.

The T5lac promoter K592008 is a hybrid promoter between a promoter from phage T5 and the lacI-repressor binding sites from the Plac promoter, so the T5lac promoter has the strength of the T5 phage promoter but can be repressed by the lacI-repressor.

Results

Measured fluorescense, relative to J23101.

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