Team:Edinburgh OG/Collaborations

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As part of our collaboration, we characterized two biobricks from the Newcastle team (whom we met in person at the iGEM Scotland and Northern England meet-up). BBa_K1895000, also known as “Electrically induced promoter system v1” consists of the heat shock promoter BBa_J45504 driving expression of a two-gene operon made up of the rpoH Sigma-32 transcription factor and superfolded GFP. BBa_K1895001, also known as rpoH, is the wild-type RBS for the rpoH Sigma-32 transcription factor.

Colonies were picked and grown overnight at 37 °C, 250 RPM in 50 mL conical tubes in 10 mL of LB medium containing 34 ug/mL chloramphenicol. Cell density was measured in a Jenway 7305 spectrophotometer and the obtained data was processed using Excel. Cells were then subcultured into opaque-walled 96 well plates and adjusted to OD600 0.05 in 290 µl of LB medium supplemented with relevant antibiotics.

Plates were then placed in the Fluostar omega microplate reader from BMG labtech, and grown at 37 °C for 20 hours at 300 RPM. Aside from monitoring OD600, fluorescence was determined by measuring emission wavelengths using a fixed excitation filter (485nm) and a fixed emission filter (538nm) every 30 minutes. Six replicates were performed. The data is presented in relative fluorescence units.