pAF3

Protocol tips

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	B. subtilis was transformed according to the method of Anagnostopoulos and Spizizen (1961) with some modifications. To obtain naturally competent cells, B. subtilis 168 was grown in the Spizizen minimal medium (SMM): 80 mM K2HPO4, 45 mM KH2PO4, 15 mM (NH4)2SO4 and 3.8 mM Na3-citrate, supplemented with 5 mM MgSO4, 5 g l-1 glucose, 0.5 g l-1 tryptophan and 0.1 g l-1 casaminohydrolysate. For efficient DNA uptake of pAF3 and pMSP3535 (negative control) by B. subtilis, the plasmid DNA (1 μg) was linearized by NsiI and self-ligated in vitro to generate multimeric plasmidic forms. After dilution of competent cells (10-1) in SMM containing 20 mM MgCl2 and 5 g l-1 glucose, pAF3 or pMSP3535 plasmid DNA multimers were added, and the samples were incubated for 20 min at 37°C. Transformation mixtures were subsequently spread on LB agar containing erythromycin (5 μg ml-1).	B. subtilis transformants were screened for the ability to produce phytase activity on LB agar supplemented with phytic acid (3 mM) by using the well-known two step counterstaining treatment (Bae et al. 1999). Colonies surrounded by clear zones were tested by PCR to confirm the presence of the phy US417 gene.

Protocol tips
B. subtilis was transformed according to the method of Anagnostopoulos and Spizizen (1961) with some modifications. To obtain naturally competent cells, B. subtilis 168 was grown in the Spizizen minimal medium (SMM): 80 mM K2HPO4, 45 mM KH2PO4, 15 mM (NH4)2SO4 and 3.8 mM Na3-citrate, supplemented with 5 mM MgSO4, 5 g l-1 glucose, 0.5 g l-1 tryptophan and 0.1 g l-1 casaminohydrolysate. For efficient DNA uptake of pAF3 and pMSP3535 (negative control) by B. subtilis, the plasmid DNA (1 μg) was linearized by NsiI and self-ligated in vitro to generate multimeric plasmidic forms. After dilution of competent cells (10-1) in SMM containing 20 mM MgCl2 and 5 g l-1 glucose, pAF3 or pMSP3535 plasmid DNA multimers were added, and the samples were incubated for 20 min at 37°C. Transformation mixtures were subsequently spread on LB agar containing erythromycin (5 μg ml-1).
Downstream tips
B. subtilis transformants were screened for the ability to produce phytase activity on LB agar supplemented with phytic acid (3 mM) by using the well-known two step counterstaining treatment (Bae et al. 1999). Colonies surrounded by clear zones were tested by PCR to confirm the presence of the phy US417 gene.

Publication protocol

B. subtilis was transformed according to the method of Anagnostopoulos and Spizizen (1961) with some modifications. To obtain naturally competent cells, B. subtilis 168 was grown in the Spizizen minimal medium (SMM): 80 mM K2HPO4, 45 mM KH2PO4, 15 mM (NH4)2SO4 and 3.8 mM Na3-citrate, supplemented with 5 mM MgSO4, 5 g l-1 glucose, 0.5 g l-1 tryptophan and 0.1 g l-1 casaminohydrolysate. For efficient DNA uptake of pAF3 and pMSP3535 (negative control) by B. subtilis, the plasmid DNA (1 μg) was linearized by NsiI and self-ligated in vitro to generate multimeric plasmidic forms. After dilution of competent cells (10-1) in SMM containing 20 mM MgCl2 and 5 g l-1 glucose, pAF3 or pMSP3535 plasmid DNA multimers were added, and the samples were incubated for 20 min at 37°C. Transformation mixtures were subsequently spread on LB agar containing erythromycin (5 μg ml-1). B. subtilis transformants were screened for the ability to produce phytase activity on LB agar supplemented with phytic acid (3 mM) by using the well-known two step counterstaining treatment (Bae et al. 1999). Colonies surrounded by clear zones were tested by PCR to confirm the presence of the phy US417 gene.

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