Contribution

To find out more about optimal concentrations of arabinose when using the pBAD promoter we measured BBa_K750000 induced by different concentrations. This was done by first transforming it into BL21(DE3) cells and then growing them in 50 ml conical tubes a couple of hours in 37°C. This culture were then distributed on a 96 well plate and analyzed for 19 hours with measurements at 15 minute intervals. Fluorescence was measured by excitation at 488 nm and emission at 510 at an automatically optimized z-position with top-down measurements in the plate reader. Optical density (OD) was measured at 595 nm and at the same time as fluorescence. Upon analysis we concluded that measurements after ca 6 hours were unreliable as the OD values of exact replicates started to differ greatly, therefore the graph below is cropped at ca 6 hours. The graph is presented in micromolar eGFP molecules per OD which was calculated in the following way: First concentration of pure eGFP (from another source than BBa_K750000) was determined by absorption at 488 nm and using Lambert-Beer’s law with an extinction value of 56000 M-1cm-1[1]. This solution were then diluted to 1.0, 0.8, 0.6, 0.4, 0.2 and 0 µM followed by distribution on a 96 well plate in triplicate and measured in the same plate reader with the same settings. The resulting graph (shown below as figure 2) was used to generate a trendline (done with GraphPad Prism [2]): \(k_1, k_2, ...\)