code r. § 3  (LexisNexis Canada)

Journal: Infection and Immunity

Article Title: Characterization of RNA Helicase CshA and Its Role in Protecting mRNAs and Small RNAs of Staphylococcus aureus Strain Newman

doi: 10.1128/IAI.01042-15

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: The following antibiotics were used when appropriate: erythromycin (2.5 μg/ml), chloramphenicol (10 μg/ml), tetracycline (2.5 μg/ml), rifampin (200 μg/ml), and kanamycin (50 μg/ml) for S. aureus strains and ampicillin (100 μg/ml) and kanamycin (100 μg/ml) for E. coli strains. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Genotype or characteristic(s) Reference or source Strains S. aureus RN4220 Mutagenized strain 8325-4 that accepts foreign DNA 25 Newman Isolated from human infection in 1952 26 ALC6094 Newman with an IPTG-inducible T7 polymerase gene at geh locus 21 ALC6096 ALC6094 containing pG164:: mazF sa 21 ALC7201 ALC6094 Δ cshA :: kan 23 ALC7214 ALC7201 with pG164:: mazF sa This study ALC7252 ALC7201 complement 25 ALC7273 ALC7252 with pG164:: mazF sa This study E. coli DH5α F − endA1 ϕ80 lacZ ΔM15 Δ( lacZ YA-arg F ) U169 rec A1 hsd R17 (r K − m K + ) pho A sup E44 λ − thi - 1 gyr A96 rel A1 BL21(DE3)(pLysS) F − ompT hsdSB (r B − m B − ) gal dcm (DE3)(pLysS ) (Cam r ) Rosetta(DE3)(pLysS) F − ompT hsdSB (r B − m B − ) gal dcm (DE3)(pLysSRARE) (Cam r ) Plasmids pET15b Ap r , p T7lac , His 6 coding sequence (5′), thrombin cleavage site Novagen pET22b(+) Ap r , p T7lac , His 6 coding sequence (3′) Novagen pET28a(+) Kan r , His 6 coding sequence (5′ and 3′), thrombin cleavage site Novagen pMAD E. coli-S. aureus shuttle vector containing temp-sensitive origin of replication, bgaB Erm r Ap r 51 Open in a separate window Strains and plasmids used in this study

Techniques: Plasmid Preparation, Isolation, Infection, Sequencing

Journal: Infection and Immunity

Article Title: Characterization of RNA Helicase CshA and Its Role in Protecting mRNAs and Small RNAs of Staphylococcus aureus Strain Newman

doi: 10.1128/IAI.01042-15

Figure Lengend Snippet: Interaction of sarA mRNA with truncated versions of CshA. (A) Depiction of the different truncated versions of CshA. Each truncated fragment was created by PCR, cloned to pET22b with NdeI and BamHI, and expressed using E. coli BL21(pLysS). (B) SDS-PAGE (12%) analyzing cell lysates before (at an OD600 of 0.7) and after addition of IPTG to a final concentration of 0.2 mM and grown for 3 h [indicated by (−) and (+), respectively]. (C) Northwestern blot showing labeled sarA mRNA hybridized to protein bands.

Article Snippet: The following antibiotics were used when appropriate: erythromycin (2.5 μg/ml), chloramphenicol (10 μg/ml), tetracycline (2.5 μg/ml), rifampin (200 μg/ml), and kanamycin (50 μg/ml) for S. aureus strains and ampicillin (100 μg/ml) and kanamycin (100 μg/ml) for E. coli strains. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Genotype or characteristic(s) Reference or source Strains S. aureus RN4220 Mutagenized strain 8325-4 that accepts foreign DNA 25 Newman Isolated from human infection in 1952 26 ALC6094 Newman with an IPTG-inducible T7 polymerase gene at geh locus 21 ALC6096 ALC6094 containing pG164:: mazF sa 21 ALC7201 ALC6094 Δ cshA :: kan 23 ALC7214 ALC7201 with pG164:: mazF sa This study ALC7252 ALC7201 complement 25 ALC7273 ALC7252 with pG164:: mazF sa This study E. coli DH5α F − endA1 ϕ80 lacZ ΔM15 Δ( lacZ YA-arg F ) U169 rec A1 hsd R17 (r K − m K + ) pho A sup E44 λ − thi - 1 gyr A96 rel A1 BL21(DE3)(pLysS) F − ompT hsdSB (r B − m B − ) gal dcm (DE3)(pLysS ) (Cam r ) Rosetta(DE3)(pLysS) F − ompT hsdSB (r B − m B − ) gal dcm (DE3)(pLysSRARE) (Cam r ) Plasmids pET15b Ap r , p T7lac , His 6 coding sequence (5′), thrombin cleavage site Novagen pET22b(+) Ap r , p T7lac , His 6 coding sequence (3′) Novagen pET28a(+) Kan r , His 6 coding sequence (5′ and 3′), thrombin cleavage site Novagen pMAD E. coli-S. aureus shuttle vector containing temp-sensitive origin of replication, bgaB Erm r Ap r 51 Open in a separate window Strains and plasmids used in this study

Techniques: Clone Assay, SDS Page, Concentration Assay, Labeling

Journal: Infection and Immunity

Article Title: Characterization of RNA Helicase CshA and Its Role in Protecting mRNAs and Small RNAs of Staphylococcus aureus Strain Newman

doi: 10.1128/IAI.01042-15

Figure Lengend Snippet: Binding of labeled sarA mRNA to S. aureus proteins. (A) Fractionated protein samples of S. aureus ALC6094 containing pG164-MazFsa with 1 mM IPTG from the heparin-Sepharose column were hybridized with [γ-32P]ATP end-labeled sarA mRNA on Northwestern blots. A protein band of interest (∼60 kDa) in fraction 49 is indicated by an arrow. (B) E. coli BL21 cell lysates expressing different ORFs from vectors pET22b(+), pET22b::sa0778, pET22b::sa1118, and pET22b::sa1885 were analyzed on 16% SDS gel with (+) and without (−) induction with 0.2 mM IPTG. Two controls from MazFsa-expressing S. aureus were used: crude extract collected after cell disruption and fraction 49, which represents one of the fractions from heparin-Sepharose column chromatography reactive with the sarA probe (A). (C) An identical gel transferred to a blot hybridized with the 375-nt sarA mRNA probe. E. coli BL21(pLysS) crude cell lysates harboring pET22b alone without IPTG did not react with the sarA mRNA probe (not shown).

Article Snippet: The following antibiotics were used when appropriate: erythromycin (2.5 μg/ml), chloramphenicol (10 μg/ml), tetracycline (2.5 μg/ml), rifampin (200 μg/ml), and kanamycin (50 μg/ml) for S. aureus strains and ampicillin (100 μg/ml) and kanamycin (100 μg/ml) for E. coli strains. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Genotype or characteristic(s) Reference or source Strains S. aureus RN4220 Mutagenized strain 8325-4 that accepts foreign DNA 25 Newman Isolated from human infection in 1952 26 ALC6094 Newman with an IPTG-inducible T7 polymerase gene at geh locus 21 ALC6096 ALC6094 containing pG164:: mazF sa 21 ALC7201 ALC6094 Δ cshA :: kan 23 ALC7214 ALC7201 with pG164:: mazF sa This study ALC7252 ALC7201 complement 25 ALC7273 ALC7252 with pG164:: mazF sa This study E. coli DH5α F − endA1 ϕ80 lacZ ΔM15 Δ( lacZ YA-arg F ) U169 rec A1 hsd R17 (r K − m K + ) pho A sup E44 λ − thi - 1 gyr A96 rel A1 BL21(DE3)(pLysS) F − ompT hsdSB (r B − m B − ) gal dcm (DE3)(pLysS ) (Cam r ) Rosetta(DE3)(pLysS) F − ompT hsdSB (r B − m B − ) gal dcm (DE3)(pLysSRARE) (Cam r ) Plasmids pET15b Ap r , p T7lac , His 6 coding sequence (5′), thrombin cleavage site Novagen pET22b(+) Ap r , p T7lac , His 6 coding sequence (3′) Novagen pET28a(+) Kan r , His 6 coding sequence (5′ and 3′), thrombin cleavage site Novagen pMAD E. coli-S. aureus shuttle vector containing temp-sensitive origin of replication, bgaB Erm r Ap r 51 Open in a separate window Strains and plasmids used in this study

Techniques: Binding Assay, Labeling, Expressing, SDS-Gel, Column Chromatography

Journal: Infection and Immunity

Article Title: Characterization of RNA Helicase CshA and Its Role in Protecting mRNAs and Small RNAs of Staphylococcus aureus Strain Newman

doi: 10.1128/IAI.01042-15

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: The following antibiotics were used when appropriate: erythromycin (2.5 μg/ml), chloramphenicol (10 μg/ml), tetracycline (2.5 μg/ml), rifampin (200 μg/ml), and kanamycin (50 μg/ml) for S. aureus strains and ampicillin (100 μg/ml) and kanamycin (100 μg/ml) for E. coli strains. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Genotype or characteristic(s) Reference or source Strains S. aureus RN4220 Mutagenized strain 8325-4 that accepts foreign DNA 25 Newman Isolated from human infection in 1952 26 ALC6094 Newman with an IPTG-inducible T7 polymerase gene at geh locus 21 ALC6096 ALC6094 containing pG164:: mazF sa 21 ALC7201 ALC6094 Δ cshA :: kan 23 ALC7214 ALC7201 with pG164:: mazF sa This study ALC7252 ALC7201 complement 25 ALC7273 ALC7252 with pG164:: mazF sa This study E. coli DH5α F − endA1 ϕ80 lacZ ΔM15 Δ( lacZ YA-arg F ) U169 rec A1 hsd R17 (r K − m K + ) pho A sup E44 λ − thi - 1 gyr A96 rel A1 BL21(DE3)(pLysS) F − ompT hsdSB (r B − m B − ) gal dcm (DE3)(pLysS ) (Cam r ) Rosetta(DE3)(pLysS) F − ompT hsdSB (r B − m B − ) gal dcm (DE3)(pLysSRARE) (Cam r ) Plasmids pET15b Ap r , p T7lac , His 6 coding sequence (5′), thrombin cleavage site Novagen pET22b(+) Ap r , p T7lac , His 6 coding sequence (3′) Novagen pET28a(+) Kan r , His 6 coding sequence (5′ and 3′), thrombin cleavage site Novagen pMAD E. coli-S. aureus shuttle vector containing temp-sensitive origin of replication, bgaB Erm r Ap r 51 Open in a separate window Strains and plasmids used in this study

Techniques: Plasmid Preparation, Isolation, Infection, Sequencing