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. 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).

Fig. 1: Luminescence measurement of Anderson promoters in the reporter vector pSB_Bs3C-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.

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.

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.

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-<font color="#000000"; size="2"><p align="justify"> '''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. </p></font>
+<font color="#000000"; size="2"><p align="justify"> '''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. </p></font>
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-In this result at least some of the Anderson promoters do not seem so weak as in the results of the luminescence measurements (Fig. 1). This experiment should be repeated where we measure a constitutive promoter e.g. P<sub>''liaG''</sub> in the same experiment. The luminescence measurements are in this case more reliable because they were repeated three times with two independent clones.
+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<sub>''liaG''</sub>, in the same experiment. But we think the luminescence measurements are more reliable because they were repeated three times with two independent clones.
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