Manipulating and understanding antibiotic production in Streptomyces coelicolor A3(2) with decoy oligonucleotides -- McArthur and Bibb 105 (3): 1020 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

McArthur and Bibb 1010.1073/pnas.0710724105

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T7/DNase I in Vivo Footprinting Protocol
Preparation of DNA Fragments.
All buffers were supplemented with 0.25 mM PMSF and a mixture of protease inhibitors before use.

• To 2 ´ 50-ml flasks, add 20 ml of RNase A plus 20 ml of DNase I (crude DNase I at 80 units/ml) plus 0.5 ml of 3.6% CaCl₂.

• (This step proved necessary because there was heavy contamination with extracellular DNA.)

• Incubate at 30°C for 15 min.

• Wash four times in TES [25 mM Tris[hydroxymethyl]methyl-2-amino-ethanesulphonic acid (pH 7.2), EDTA (add 3 ml of 0.25 M EDTA to each 100 ml; final concentration 7.5 mM EDTA)].

• Resuspend in 10 ml of TES plus detergent (add 5 ml of 10% Nonidet P-40 and 5 ml of 10% Triton X-100 to 100 ml of TES buffer).

• Incubate at 30°C for 15 min.

• Wash in Mc2 [10 mM Tris·HCl, 2.5% sucrose, 5 mM MgCl₂, 1 mM CaCl₂, 0.1 mM DTT, 0.5% Nonidet P-40, 0.5% Triton X-100 (pH 8)].

• Resuspend in 5 ml of Mc2 and digest for 30 min at 30°C in the presence of 500 units of SacI or other enzyme for a conveniently placed upstream restriction site.

• Supplement with 1,250 units of T7 exonuclease (NEB).

• Split into five 1-ml aliquots; add 0, 1, 2, 5, or 10 ml of DNase I (Roche Molecular Biology grade; 10 units/ml).

• Continue incubation for a further 7 min at 30°C.

• To each, add 1 ml of STOP [50 mM Tris·HCl, 5 mM EDTA, 0.2% (wt/vol) SDS, 10 mg/ml proteinase K (pH 8)]. Incubate for 1 h to overnight at 55°C

• The DNA is recovered after phenol-chloroform extraction, digestion with RNase A, and ethanol precipitation

Amplification of DNA Fragments.
A fragment containing the promoter of interest (actII-orf4) was cloned into pGEMT-Easy. Using biotinylated primers either to the M13r or T7 primer binding site in combination with a nonbiotinylated partner, it is possible to produce PCR fragments that only differ in whether they have a biotin at the 5' or 3' end (relative to the genomic orientation of the promoter fragment). Each of these is separately immobilized onto a streptavidin-containing (paramagnetic) matrix, denatured, and used to capture by hybridization fragments from the digested genomic DNA that are derived from the promoter of interest. The fragments captured with the 3' biotinylated PCR fragment are referred to as forward (F), and vice versa.

• Captured fragments are washed three times in 1´ Roche Taq polymerase buffer, and the beads are captured on a magnetic stand.

• The beads are resuspended in 50 ml of 1´ Roche Taq polymerase buffer supplemented with 0.5 mM dNTPs, 10% DMSO, and 0.5 units of Roche Taq polymerase.

• An extension is carried out at 72°C for 15 min.

• The beads are heated to 95°C for 5 min, and the supernatant is isolated after a flash spin.

• Supplement the supernatant with a biotinylated M13r or T7 primer (as appropriate) and allow to cool to 55°C.

• Perform a single extension step at 72°C.

• Capture dsDNA molecule on streptavidin paramagnetic beads (Dynal).

LM-PCR of DNA Fragments.
LM-PCR protocol is based on the methods devised in the Riggs laboratory and their detailed, published protocols.

Mc7F

ctc tgg cgc gcc ttc ctg tcc gta cgt ccg act

c gag acc gcg cgg aag gac agg cat gca ggc tg -Phos.

Mc7R

• Form the adaptor by setting up the following hybridization reaction: 5 ml of Mc7F (stock at 250 pmol/ml), 5 ml of Mc7R (stock at 250 pmol/ml), 12.5 ml of 1 M Tris·HCl (pH 7.6), and 27.5 ml of water.

• Heat to 95° C for 3 min and allow to cool slowly to room temperature.

Resuspend the beads in the following mixture: 1.2 ml of 1 M MgCl₂, 2.7 ml of 1 M DTT, 1.5 ml of 100 mM ATP,1 ml of 100´ BSA, 1 ml of 3 units/ml Promega T4 DNA ligase, 10 ml of Adaptor (250 pmol), and 72.6 ml of water.

• Incubate overnight at 18°C.

• Heat-inactivate at 72°C for 2 h.

• Add to the reaction 17 ml of 3 M NaOAc (pH 5.2), 1 ml of 10 mg/ml glycogen, and 440 ml of EtOH. Precipitate and resuspend in 50 ml of water.

• Perform PCR with the following mixture: 10 ml of 10´ Roche Taq buffer, 1.5 ml of DMSO, 1 ml of 25 mM MgCl₂, 1.2 ml of 10 mM dNTPs, 1 ml of Mc7F (stock at 25 pmol/ml), 1 ml of DIG-labeled M13r or T7 primer (stock at 25 pmol/ml) or alternatively a nested primer, 0.6 ml of 3 units/ml Roche Taq polymerase, 5 ml of DMSO, 27.7 ml of water, and varying amounts of template DNA.

• Cycle with following conditions: 20 times at 95°C for 1 min, 60°C for 2 min, and 72°C for 1 min.

• Add 1 ml of 10´ Roche Taq buffer, 0.5 ml of Roche Taq, and 8.5 ml of water, and incubate at 72°C for a further 10 min.

• Ethanol-precipitate and analyze on a 12% polyacrylamide gel.