Team:LMU-Munich/Data/Anderson

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Revision as of 15:02, 19 September 2012

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Bacillus BioBrick Box - Promoters

Fig. 1: Luminescence measurement of Anderson promoters in the reporter vector pSB_Bs3C-luxABCDE. OD₆₀₀ (right), LUMI (center) and OD₆₀₀ per LUMI (left) depending on the time (h) are shown for two different clones (green/blue). Data derive from three independent experiments. 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 in the reporter vector pSB_Bs3C-luxABCDE from the BioBrickBox containing the lux operon as a reporter for promoter activity. The gene expression which correlates to the promoter activity leads to the expression of the lux operon with the luciferase. The luminescence which is produced by the luciferase can be measured with the plate reader (BioTek). Data derive from three undependant measurements (Fig. 1). Curves were fitted over each other (t=0, OD₆₀₀=0,3) and smoothed by taking average of three neighboring values. OD₆₀₀ values shown are plate reader units and about one third of the usual OD₆₀₀ values. All clones show a usual growth curves. The activity of the promoters raises during the pass from the transition to the stationary phase. This 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 and do not mean a high promoter activity. The luminescence of one clone of the promoters J23107 and J23114 do not show activity where in future a second clone with promoter activity should be measured. In comparison to all the other evaluated Bacillus promoters these Anderson promoters showed a very low acitivity in B. subtilis.

To measure 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)

Fig. 3: Luminescence measurement of the constitutive Bacillus promoters P_liaG and P_lepA in the reporter vector pSB_Bs3C-luxABCDE. OD₆₀₀ (up), LUMI (center) and LUMI per OD600 (down) depending on the time (h) are shown for two different clones (green/blue). Data come from three independent experiments. Curves were fitted over each other (t=0, OD600=0,3) and smoothed by taking average of three neighboring values.