pKUC4

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The vectors were used to transform the B. licheniformis strain MW3 by electroporation [20] resulting in the strains TH3 (pKUC3) and TH4 (pKUC4), respectively. These strains were then cultivated in phosphate-limited BMM as already described.	Culture supernatants for enzyme assays were taken at different time points during the cultivation. The first sample was taken during the exponential growth phase (four hours after cultivation), the second sample during the transient phase and additional samples were taken 2, 4, 6, 8, 10, 12 and 14 h after onset of the stationary growth phase.

Protocol tips
The vectors were used to transform the B. licheniformis strain MW3 by electroporation [20] resulting in the strains TH3 (pKUC3) and TH4 (pKUC4), respectively. These strains were then cultivated in phosphate-limited BMM as already described.
Downstream tips
Culture supernatants for enzyme assays were taken at different time points during the cultivation. The first sample was taken during the exponential growth phase (four hours after cultivation), the second sample during the transient phase and additional samples were taken 2, 4, 6, 8, 10, 12 and 14 h after onset of the stationary growth phase.

Publication protocol

The activity of the PphyL promoter was analyzed by means of translational reporter gene fusions. For this purpose, an approximately 300-bp fragment containing the PphyL promoter from B. licheniformis DSM13 (Additional file 1: Figure S2) was cloned in front of the α-amylase and xylanase reporter genes from B. subtilis 168 with the primer pairs 1/2 and 1/5, respectively (Additional file 1: Table S1). The amyE and xynA genes from B. subtilis 168 were amplified with the primer pairs 3/4 and 6/7, respectively (Additional file 1: Table S1). The PphyL′-′amyE and PphyL′-′xynA fusions were constructed by means of the precise gene fusion polymerase chain reaction strategy described by Yon and Fried [18] by using the primer pairs 1/4 and 1/7, respectively. The PCR-fusion fragments were then inserted into the XbaI and KpnI sites of the multi-copy plasmid pKUC (this shuttle vector is based on the pUC18 and pKTH290 plasmids [19]) resulting in vector pKUC3 and pKUC4, respectively. These vectors were used to transform the B. licheniformis strain MW3 by electroporation [20] resulting in the strains TH3 (pKUC3) and TH4 (pKUC4), respectively. These strains were then cultivated in phosphate-limited BMM as already described. Culture supernatants for enzyme assays were taken at different time points during the cultivation. The first sample was taken during the exponential growth phase (four hours after cultivation), the second sample during the transient phase and additional samples were taken 2, 4, 6, 8, 10, 12 and 14 h after onset of the stationary growth phase.

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