Team:LMU-Munich/Data/Anderson

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Anderson Promoters

Luminescence measurements

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. For that purpose, 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 bioluminescence, which is produced by the luciferase, can be measured with the microtiter plate reader Synergy2 (["http://www.biotek.com/"] BioTek) (Fig.1).

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 derived from three independent experiments, graph shows the mean with standard deviation. Curves were fitted over each other (t=0, OD600=0,3) and smoothed by taking average of three neighboring values.

All clones show a typical growth behaviour. The activity of the promoters increases during transition from log to stationary phase. The maximal promoter activity (t=1h) reaches 200 to 1500 Lumi/OD₆₀₀ for promoters J23115 and J23101, respectively. Afterwards, the activities drop to the initial levels (t=2h). The deviations of luminescence values (Lumi/OD₆₀₀) in the beginning of the curves are an experimental artifact due to the small OD₆₀₀ values. One clone of J23107 and J23114 showed significantly lower promoter activities. Therefore, additional clones need to be measured. In comparison to all the other evaluated Bacillus promoters, the Anderson promoters showed a rather low acitivity in B. subtilis.

β-galactosidase assay

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.

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.

Verification of the promoter activity by β-galactosidase assays revealed that the Anderson promoters do not seem to be as weak as measured by luminescence (Fig. 1). For direct comparison we should measure a constitutive promoter, e.g. P_liaG, in the same experiment. But we think the luminescence measurements are more reliable because they were repeated three times with two independent clones.