Journal: Toxins

Article Title: Pore-Forming Cardiotoxin VVA2 (Volvatoxin A2) Variant I82E/L86K Is an Atypical Duplex-Specific Nuclease

doi: 10.3390/toxins14060392

Figure Lengend Snippet: Re-VVA2 I82E/L86K cleaves dsDNA non-specifically with cleavage preference in dG-dC-rich regions. ( A ) The schematic diagram of the experimental design for mapping the cleavage sites of nuclease Re-VVA2 I82E/L86K. The cleaved products (pUC19 and pET28a, mainly in RF II) were digested by S1 nuclease and cloned into pre-linearized pBluescript II SK (+) vector by EcoRV. Colonies were selected and sequenced using universal primer M13F. The detailed protocol is stated in the Materials and Methods. ( B ) Cleavage sites of Re-VVA2 I82E/L86K on pUC19 and pET28a were marked on the related plasmid map. Specific cleavage sites are shown in . ( C ) Re-VVA2 I82E/L86K has cleavage preference in dG-dC-rich regions. The sequences around cleavage sites were aligned with WebLogo 3. The proposed cleavage sites were marked on the figure.

Article Snippet: Plasmid: pET28a, pUC19, pBluescript II SK (+) vector (Addgene, Watertown, MA, USA).

Techniques: Clone Assay, Plasmid Preparation

Journal:

Article Title: Modification of Type IV Pilus-Associated Epithelial Cell Adherence and Multicellular Behavior by the PilU Protein of Neisseria gonorrhoeae

doi: 10.1128/IAI.70.7.3891-3903.2002

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: All E. coli strains and their recombinants were maintained in Luria-Bertani medium supplemented with the appropriate antibiotics. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant characteristic(s) Reference or source N. gonorrhoeae strains N400 Derived from VD300, a contains the IPTG-inducible recA6 allele b 11 GF2 (PilF − ) pilF ::m-Tn 3erm 11 GT3 pilT ::m-Tn 3erm 40 GT7 pilT ::m-Tn 3erm 40 GT17 pilT ::m-Tn 3cm at position 73 c This study GT26 pilT ::m-Tn 3erm 40 GT50 pilT ::m-Tn 3erm at position 1105 c This study GT101 pilT dud1 40 GT102 pilT Δ QSL 40 GT103 pilT ::m-Tn 3erm 40 GT104 pilT ind 40 GT105 pilT fs164 40 GTU2 pilT ::m-Tn 3cm pilU ::mTn 3erm This study GTXE1 pilT :: xylE This study GUXE2 pilU :: xylE This study GU2 pilU ::m-Tn 3erm at position 1591 c This study GU4 pilU ::m-Tn 3erm at position 1714 c This study GU5 pilU ::m-Tn 3erm at position 2102 c This study GU21 pilU ::m-Tn 3erm at position 1314 c This study GU33 pilU ::m-Tn 3erm at position 2541 c This study GU127 pilU ::m-Tn 3erm at position 1192 c This study E. coli strains BL21(DE3) F − ompT r B − m B − with prophage λ carring the T7 RNA polymerase gene 32 HB101 F − mcrB mrr hsdS20 (r B − m B − ) recA13 supE44 ara14 galK2 lacY1 proA2 rpsL20 (Sm r ) xyl5 λ − leu mtl1 GIBCO BRL Plasmids pBluescript II SK Cloning vector, Amp r 32 p6/16/11 1.1-kb Sal I fragment in pBluescript II SK This study p11/2/13 3.3-kb EagI/Xba I fragment in pBluescript II SK This study pHSS6 Vector for shuttle mutagenesis, Kan r 27 p1-49 3.3-kb Eag I/ Xba I fragment in pHSS6 This study pT7-5 T7 RNA polymerase promoter λ10, Amp r 32 pT7U pilU in pT7-5 This work pT7U (Δ Sal I) Sal I deletion subclone of pT7U This work Open in a separate window a VD300 is an MS11 derivative ( 15 ). b All remaining Gc strains are derived from N400. c Nucleotide positions correspond to those in GenBank no. {"type":"entrez-nucleotide","attrs":{"text":"S72391","term_id":"632717","term_text":"S72391"}} S72391 .

Techniques: Plasmid Preparation, Derivative Assay, Clone Assay, Mutagenesis

Journal:

Article Title: Homologues of Neisserial Heme Oxygenase in Gram-Negative Bacteria: Degradation of Heme by the Product of the pigA Gene of Pseudomonas aeruginosa

doi: 10.1128/JB.183.21.6394-6403.2001

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: Isopropyl-1-thiol-β- d -galactopyranoside (IPTG; Fisher) was used at a final concentration of 1 mM for E. coli and N. meningitidis and 20 mM for P. aeruginosa . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant genotype and characteristic(s) a Source or reference E. coli DH5α Routine cloning vector Stratagene BL21(DE3) gal ompT , lysogen of DE3 carrying T7 polymerase gene 29 S17-1 (IR747) Carries RP4 tra genes and λ pir gene 25 IR1638 IR747 harboring pWMZ1637 This study P. aeruginosa IA614 PAO1 derivative, siderophore deficient 1 IR1648 IA614 derivative, chromosomal knockout of pigA , Gen r This study IR4309 IR1648, harbors pMMB66HE This study IR4310 IR1648, harbors pMMB66HE This study IR4311 IR1648, harbors pMCR2050( pigA ) This study N. meningitidis IR1072 WT laboratory strain, serogroup C 40 2855 IR1072 derivative, chromosomal knockout of hemO 40 4093 2855 derivative, chromosomal insertion of pigA This study Plasmids pCR 2.1 Topo II PCR cloning vector, Amp r Kan r Invitrogen pBluescript II SK(+) ColE1 cloning phagemid, Amp r Stratagene pRK2013 Km r , ColE1 with Tra + Mob + 6 pET21a T7 expression vector, Amp r Novagene pGMΩ 0.9-kb gentamicin cassette cloned into pHP45Ω, Amp r Gen r 23 pGP704 pir -dependent suicide vector, Amp r 14 pCVD442 pir -dependent suicide vector, Suc s , Amp r 5 pMMB66HE Broad host range, tac P expression vector, Amp r 8 pWMZ1591a 3-kb erm-lacIOP cassette cloned 5′ of hemO 24 pWMZ1605 PCR product encoding PigA on pCR 2.1 Topo II This study pWMZ1606 Bam HI fragment containing pigA in pBluescript II SK(+) This study pWMZ1613 Hin dIII- Sac I fragment from pWMZ1606 in pUC18 This study pWMZ1621 Gentamicin cassette cloned into Kpn I site of pWMZ1613 This study pWMZ1629 Eco RI fragment of pWMZ1621 cloned into pGP704 This study pWMZ1637 Xba I- Sma I fragment from pWMZ1621 cloned into pCVD442 This study pWMZ1614 0.8-kb Eco RI- Hin dIII PCR product of hmb R in pCR 2.1 Topo II This study pWMZ1616 4-kb Eco RI- Kpn I from pWMZ1591 and hmb R from pWMZ1614 in pBlueScript II SK(+) This study pWMZ1619 pCR 2.1 Topo II minus Eco RI restriction site This study pWMZ1635 5-kb Kpn I- Hin dIII fragment of pWMZ1616 into pWMZ1619 This study pWMZ1639a Eco RI pigA fragment of pWMZ1605 in pWMZ1635 This study pWMZ1655 PCR product encoding PigA-His tagged in pCR 2.1 Topo II This study pWMZ1656 Nde I- Xho I fragment from pWMZ1655 cloned into pET21a This study pWMZ1665 PCR product encoding native PigA in PCR 2.1 Topo II This study pWMZ1666 Nde I- Xho I fragment from pWMZ1666 cloned into pET21a This study pMCR2050 Eco RI fragment carrying pigA cloned into pMMB66HE This study Open in a separate window a WT, wild type; Amp, ampicillin; Kan, kanamycin; Gen, gentamicin; Suc, sucrose.

Techniques: Plasmid Preparation, Clone Assay, Knock-Out, PCR Cloning, Expressing