e coli cole1 origin  (Addgene inc)

Journal: Journal of Bacteriology

Article Title: Nucleoside Diphosphate Kinase Escalates A-to-C Mutations in MutT-Deficient Strains of Escherichia coli

doi: 10.1128/JB.00567-19

Figure Lengend Snippet: Purification of EcoNDK from the E. coli mutT null strain and activity analysis on 8-O-dGTP. (A) Representative 15% SDS-PAGE gel showing the purity and migration of the purified EcoNDK. M stands for the protein size markers. (B) Chromatograms indicate NDK-catalyzed reaction on 8-O-dGTP. Reaction analytes were separated on a DNAPac column through high-performance liquid chromatography (HPLC), using a gradient of 1 M LiCl (0% to 40%). The retention times of different analytes are shown on the x axis and the corresponding peak intensities (given in arbitrary units) are shown on the y axis. (i) Control. Shown is the migration profile of ADP and 8-O-dGTP. (ii) Reaction in the presence of NDK. Shown is the emergence of ATP and 8-O-dGDP peaks due to NDK-mediated transfer of terminal phosphate from 8-O-dGTP to ADP.

Article Snippet: Enzymes used for DNA manipulations were procured from Thermo Fisher and New England Biolabs. table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Strain or plasmid Relevant details Reference or source Strains E. coli MG1655 K strain, F − λ − rph-1 44 E. coli MG1655 Δ mutT :: kan MG1655 strain, where the mutT gene is disrupted with a kan cassette 28 E. coli BW25113 F − Δ( araD araB ) 567 Δ lacZ4787 (:: rrnB-3 ) λ − rph-1 Δ( rhaD rhaB ) 568 hsdR514 45 E. coli JW2502-1 Δ ndk :: kan BW25113 strain with Δ ndk-764 :: kan 45 E. coli CC101 F′ ara-600 Δ( gpt lac ) 5 λ − relA1 spoT1 thiE1 F128 − (used to screen for A-to-C or T-to-G mutation) 42 E. coli CC101 Δ mutT :: kan CC101 strain with Δ mutT :: kan allele 28 E. coli CC101 Δ mutT A derivative of CC101 Δ mutT :: kan strain in which the kanamycin marker cassette has been removed from the Δ mutT :: kan locus to create an unmarked deletion of mutT This study E. coli CC101 Δ mutT Δ ndk CC101 Δ mutT cured strain, in which the ndk allele has been disrupted by insertion of kan cassette This study Plasmids pQE60 An E. coli expression vector harboring ColE1 origin of replication, ampicillin resistance marker, and T5 promoter (containing the lac operator) and provides a C-terminal His tag Invitrogen pQE60_ Eco_ndk pQE60 plasmid harboring Eco_ndk cloned (NcoI, BglII) under T5 promoter This study pBADHisB (pBAD) An E. coli expression vector harboring ColE1 origin of replication, ampicillin resistance marker, and an arabinose-inducible promoter Invitrogen pBAD_ Eco_mutT Derivative of pBAD plasmid harboring Eco_mutT cloned (NcoI, NheI) under arabinose promoter 28 pBAD_ Eco_ndk A derivative of pBAD plasmid harboring Eco_ndk cloned (NcoI, BglII) under arabinose promoter This study Open in a separate window Description of strains and plasmids used in the study

Techniques: Purification, Activity Assay, SDS Page, Migration, High Performance Liquid Chromatography, Control

Journal: Journal of Bacteriology

Article Title: Nucleoside Diphosphate Kinase Escalates A-to-C Mutations in MutT-Deficient Strains of Escherichia coli

doi: 10.1128/JB.00567-19

Figure Lengend Snippet: Description of strains and plasmids used in the study

Article Snippet: Enzymes used for DNA manipulations were procured from Thermo Fisher and New England Biolabs. table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Strain or plasmid Relevant details Reference or source Strains E. coli MG1655 K strain, F − λ − rph-1 44 E. coli MG1655 Δ mutT :: kan MG1655 strain, where the mutT gene is disrupted with a kan cassette 28 E. coli BW25113 F − Δ( araD araB ) 567 Δ lacZ4787 (:: rrnB-3 ) λ − rph-1 Δ( rhaD rhaB ) 568 hsdR514 45 E. coli JW2502-1 Δ ndk :: kan BW25113 strain with Δ ndk-764 :: kan 45 E. coli CC101 F′ ara-600 Δ( gpt lac ) 5 λ − relA1 spoT1 thiE1 F128 − (used to screen for A-to-C or T-to-G mutation) 42 E. coli CC101 Δ mutT :: kan CC101 strain with Δ mutT :: kan allele 28 E. coli CC101 Δ mutT A derivative of CC101 Δ mutT :: kan strain in which the kanamycin marker cassette has been removed from the Δ mutT :: kan locus to create an unmarked deletion of mutT This study E. coli CC101 Δ mutT Δ ndk CC101 Δ mutT cured strain, in which the ndk allele has been disrupted by insertion of kan cassette This study Plasmids pQE60 An E. coli expression vector harboring ColE1 origin of replication, ampicillin resistance marker, and T5 promoter (containing the lac operator) and provides a C-terminal His tag Invitrogen pQE60_ Eco_ndk pQE60 plasmid harboring Eco_ndk cloned (NcoI, BglII) under T5 promoter This study pBADHisB (pBAD) An E. coli expression vector harboring ColE1 origin of replication, ampicillin resistance marker, and an arabinose-inducible promoter Invitrogen pBAD_ Eco_mutT Derivative of pBAD plasmid harboring Eco_mutT cloned (NcoI, NheI) under arabinose promoter 28 pBAD_ Eco_ndk A derivative of pBAD plasmid harboring Eco_ndk cloned (NcoI, BglII) under arabinose promoter This study Open in a separate window Description of strains and plasmids used in the study

Techniques: Plasmid Preparation, Mutagenesis, Marker, Expressing, Clone Assay