Studies on transformation of Escherichia coli with plasmids

Hanahan, Douglas

doi:10.1016/s0022-2836(83)80284-8

Cited by 10,355 publications

(4,473 citation statements)

References 64 publications

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“…Moloney murine leukemia virus reverse transcriptase (Mo-MLV-RT) was purchased from Bacterial strains and media M. xanthus FB (DZF1) was grown at 30ЊC in CYE medium and supplemented with streptomycin (500 g ml ¹1 ) or kanamycin (40 g ml ¹1 ) when necessary (Campos et al, 1978). E. coli JM83 (Vieira and Messing, 1982) CL83 (Lerner and Inouye, 1990), SB221 (Nakamura et al, 1982) and DH5␣ (Hanahan, 1983) were used as recipient strains for transformations. E. coli BL21(DE3) was used for the T7 RNA polymerase expression system (Studier et al, 1990).…”

Section: Methodsmentioning

confidence: 99%

Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus

Hanlon

Inouye

1997

Molecular Microbiology

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SummaryThe Myxococcus xanthus gene, pkn9, encodes a protein that contains significant homology with eukaryotic Ser/ Thr protein kinases. The pkn9 gene was singled out of a previously identified family of kinase genes by amplification techniques that displayed differences in kinase gene expression during selected periods of the M. xanthus life cycle. Pkn9 was constitutively expressed during vegetative growth and upregulated during the aggregation stage of early development. It consists of 589 amino acids, and its N-terminal 394 residues show 38% identity with both Pkn1 and Pkn2 of M. xanthus. This region also shows 29, 25 and 29% identity with myosin light-chain kinase, protein kinase C, and cAMP-dependent protein kinase, respectively. A 22-residue hydrophobic transmembrane domain separates the kinase domain from the 173-residue C-terminal domain that resides on the outside of the inner membrane. The C-terminal domain contains two sets of tandem repeats of 13 and 10 residues which have no known function. When expressed in Escherichia coli under the T7 promoter, Pkn9 was found to be phosphorylated on serine and threonine residues. Disruption of the pkn9 kinase catalytic subdomains I-III by the insertion of a kanamycin-resistance gene resulted in slightly delayed, smaller and more-crowded fruiting bodies, while spore formation was normal. Total deletion of the pkn9 gene caused severely reduced progression through development resulting in light loose mounds that become slightly more compact over time. Development progressed further at the centre than at the edge of the spot, and spore formation was significantly reduced. Twodimensional gel analysis revealed that both the disruption and the deletion of pkn9 prevented the expression of five membrane proteins (KREP9-1-4). These results suggest that the loss of Pkn9 kinase activity caused altered fruiting-body formation, the absence of the KREP9 proteins in the membrane, and reduced spore production.

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Section: Methodsmentioning

confidence: 99%

Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus

Hanlon

Inouye

1997

Molecular Microbiology

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“…Plasmids were propagated in E. coli (DH5a cells), following the procedure described by [17], and plasmid DNA was extracted-purified with the Wizard Ò Plus SV Minipreps DNA Purification System (Promega), following the manufacturer's instructions.…”

Section: Bacteria Transformation and Dna Extractionmentioning

confidence: 99%

Cloning, characterization and in vitro and in planta expression of a glucanase inhibitor protein (GIP) of Phytophthora cinnamomi

Martins

Belo

et al. 2014

Mol Biol Rep

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Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. They are able to secrete a glucanase inhibitor protein (GIP) that inhibits the activity of endoglucanases (EGases) involved in defense responses against infection. One of the most widely distributed and aggressive Phytophthora species, with more than 1,000 host plants is P. cinnamomi. In this work we report the sequencing and characterization of a class of GIPs secreted by Phytophthora cinnamomi. The gip gene from P. cinnamomi has a 937 bp ORF encoding a putative peptide of 312 deduced amino acids. The expression of this gene was studied during growth in different carbon sources (glucose, cellulose and sawdust), by RT-qPCR and its level of expression was evaluated at five time points. The highest expression of gip gene occurred in sawdust at 8 h of induction. In vivo infection of C. sativa revealed an increase in gip expression from 12 to 24 h. At 36 h its expression decreased suggesting that a compensatory mechanism must occur in plant.

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“…The pGFPuv vector (100 ng; BD Biosciences, San Jose, CA, USA) was transformed into the competent E. coli O91:H21 strain B2F1. Competent cells were prepared using rubidium chloride as previously described [19]. Reactions were plated on an L-agar plate containing ampicillin (100 mg/mL), and transformed colonies were identified by fluorescence when exposed to UV light.…”

Section: Bacterial Strainsmentioning

confidence: 99%

Proposal for effective treatment of Shiga toxin-producing Escherichia coli infection in mice

Amran

Fujii

Kolling

et al. 2013

Microbial Pathogenesis

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Studies on transformation of Escherichia coli with plasmids

Cited by 10,355 publications

References 64 publications

Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus

Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus

Cloning, characterization and in vitro and in planta expression of a glucanase inhibitor protein (GIP) of Phytophthora cinnamomi

Proposal for effective treatment of Shiga toxin-producing Escherichia coli infection in mice

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