Team:Linkoping Sweden/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 \(56 000 M^{-1} cm^{-1}\) [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]): \(Y = 60185*X + 1371\) This was then inverted by WolframAlpha [3] into this: \(X=(y - 1371)/60185\). All fluorescent data from the characterization were then processed by this equation converting the data from fluorescence to µM eGFP, this was then divided by the OD values for each well at each time point generating the final graph shown in figure 1.
Figure 1. Characterization of BBa_K750000 with 6 different concentrations of arabinose (10, 1, 0.1, 0.01, 0.001, 0 mM), compared to eGFP induced with a strong T7 promoter and empty BL21 (DE3) Codon+ cells. The measurements was done using an infinite M1000 pro plate reader at 37°C. Between measurements the plate was shaking with 216 rpm. Cells with BBa_K750000 were done in 12 replicates, 4 technical and 3 biological (different clones from the same transformation), the empty cells received the same amounts of arabinose as the cells with BBa_K750000 but were measured in technical duplicate (2 of each arabinose conc.) and the eGFP reference all were induced with 0.2 mM IPTG (12 technical replicates).
Figure 2. eGFP standard generated from purified eGFP with known concentration diluted (1.0, 0.8, 0.6, 0.4, 0.2 and 0 µM) and measured in triplicate with an infinite M1000 pro plate reader.
Sources
1. Shaner N, Steinbach P, Tsien R. A guide to choosing fluorescent proteins. Nature Methods [serial on the Internet]. (2005, Dec), 2(12): 905-909.
2. Version 6.01 for Windows, GraphPad Software, La Jolla California USA, www.graphpad.com
3. http://www.wolframalpha.com/
