Team:LMU-Munich/Data/Anderson

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Revision as of 15:53, 25 September 2012

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Anderson Promoters

Fig. 1: Luminescence measurement of Anderson promoters in the reporter vector pSBBs3C-luxABCDE. OD600 (right), Lumi (middle) and Lumi per OD600 (left) depending on the time (h) are shown for two different clones (green/blue). Data derive from three independent experiments, so graph shows the mean with the standard deviation. Curves were fitted over each other (t=0, OD600=0,3) and smoothed by taking average of three neighboring values.

Eleven of the nineteen promoters of the [http://partsregistry.org/Part:BBa_J23100 Anderson collection] (J23100, J23101, J23102, J23103, J23106, J23107, J23113, J23114, J23115, J23117, J23118) were evaluated. Therefore, we used the reporter vector pSB_Bs3C-luxABCDE from the BioBrickBox containing the lux operon as a reporter for promoter activity. The promoter activity leads to the expression of the lux operon and to the production of the enzyme luciferase. The luminescence, which is produced by the luciferase, can be measured with the plate reader Synergy2 (BioTek) (Fig.1). All clones show a usual growth behaviour. The activity of the promoters increases during transition from log to stationary phase. The maximum (t=1h) reaches from 200Lumi/OD₆₀₀ (promoter J23115) to a maximum of 1500 Lumi/OD₆₀₀ for the strongest promoter (J23101). Afterwards the activity goes down to the beginning level (t=2h). The oscillation of luminescence (Lumi/OD₆₀₀) in the beginning of the curves are due to the small OD₆₀₀ values and do not mean high promoter activity. One clone of J23107 and J23114 shows significantly lower promoter activity. Therefore, additional clones should be measured. In comparison to all the other evaluated Bacillus promoters, these Anderson promoters showed a very low acitivity in B. subtilis.

Fig. 2: β-galactosidase assay (up) and growth curve (down) of the Anderson promoters J23100, J23102, J23103, J23106 in B. subtilis. Results derive from one experiment and are only shown for one Bacillus clone.

To evaluate the activity not only with the lux reporter operon, four promoters of the Anderson collection were cloned into the reporter vector pSB_Bs1C-lacZ to do β-galactosidase assays and then to compare the results of the strength of these promoters in B. subtilis (Fig. 2). The results were compared to the results from the luminescence measurements.