Materials and Methods strain IL1403. plasmid and pG+host Bacterial strains. Lactococcus lactis plasmid, containing the ISS1 transposon were used in this study. strains were grown in L. lactis M17 media (BD 218561) supplemented with 0.5% glucose containing appropriate antibiotics. The plasmid pJP005 containing recT was used for recombineering experiments. NZ9000 was used for recombineering and contains genes needed for expression in pJP005. E.coli nisR nisK culture DH5 was grown in LB media (BD 244620). pGEM plasmid was used for subcloning the fragment. Oligos used in this study are shown in Table I. nisRK To prepare our strain for mutagenesis, was transformed Plasmid preparations. L.lactis with plasmid pLNRK-RIG. For plasmid extraction, a Qiagen Miniprep Kit (27106) was used. An overnight culture was pelleted and resuspended in 250 of buffer P1 (50mM Tris-HCl, 10mM μl EDTA, 100µg/ml RNaseA) then transferred to microcentrifuge tube. Then 250 of buffer P2 μl (200 mM NaOH, 1% SDS) was mixed by inverting until solution was clear. Next, 350 of μl buffer N3 (4.2 M Gu-HCl and 0.9 M potassium acetate) was added and the solution was mixed again by inverting. The solution was centrifuged for 10 minutes at 13,000 rpm. The supernatant was poured onto Qiagen columns with collection tubes and centrifuged for 60 seconds. Then 500 of buffer PB (5M Gu-HCL,30% isoproponal) was added and centrifuged for 30 to 60 seconds; μl the flow-through was discarded. Next, 750 of buffer PE (10mM Tris-HCl, 80% ethanol) was μl added, and the flow- through was discarded. The column was put back in the collection tube and
Materials and Methods Bacterial strains. Lactococcus lactis strain IL1403. plasmid and pG+host plasmid, containing the ISS1 transposon were used in this study. L. lactis strains were grown in M17 media (BD 218561) supplemented with 0.5% glucose containing appropriate antibiotics. The plasmid pJP005 containing recT was used for recombineering experiments. NZ9000 was used for recombineering and contains nisR nisK genes needed for expression in pJP005. E.coli culture DH5 was grown in LB media (BD 244620). pGEM plasmid was used for subcloning the nisRK fragment. Oligos used in this study are shown in Table I. Plasmid preparations. To prepare our strain for mutagenesis, L.lactis was transformed with plasmid pLNRK-RIG. For plasmid extraction, a Qiagen Miniprep Kit (27106) was used. An overnight culture was pelleted and resuspended in 250 μl of buffer P1 (50mM Tris-HCl, 10mM EDTA, 100µg/ml RNaseA) then transferred to microcentrifuge tube. Then 250 μl of buffer P2 (200 mM NaOH, 1% SDS) was mixed by inverting until solution was clear. Next, 350 μl of buffer N3 (4.2 M Gu-HCl and 0.9 M potassium acetate) was added and the solution was mixed again by inverting. The solution was centrifuged for 10 minutes at 13,000 rpm. The supernatant was poured onto Qiagen columns with collection tubes and centrifuged for 60 seconds. Then 500 μl of buffer PB (5M Gu-HCL,30% isoproponal) was added and centrifuged for 30 to 60 seconds; the flow-through was discarded. Next, 750 μl of buffer PE (10mM Tris-HCl, 80% ethanol) was added, and the flow- through was discarded. The column was put back in the collection tube and
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