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Construction of Pseudoalteromonas - Escherichia coli shuttle vector based on a small plasmid from the marine organism Pseudoalteromonas

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Title
Construction of Pseudoalteromonas - Escherichia coli shuttle vector based on a small plasmid from the marine organism Pseudoalteromonas
Authors
Kim, Dockyu
Park, Ha Ju
Park, Hyun
Keywords
Pseudoalteromonas; Cold-active enzyme; Gene transfer; Shuttle vector
Issue Date
2016
Citation
Kim, Dockyu, Park, Ha Ju, Park, Hyun. 2016. "Construction of Pseudoalteromonas - Escherichia coli shuttle vector based on a small plasmid from the marine organism Pseudoalteromonas". Korean Journal of Microbiology, 52(1): 110-115.
Abstract
A small plasmid (pDK4) from the Antarctic marine organism Pseudoalteromonas sp. PAMC 21150, was purified, sequenced and analyzed. pDK4 was determined to be 3,480 bp in length with a G+C content of 41.64% and contains three open reading frames encoding a replication initiation protein (RepA), a conjugative mobilization protein (Mob) and a hypothetical protein. PCR-amplified pDK4 was cloned in high-copy pUC19 to yield the fusion vector pDOC153. The chloramphenicol resistance gene was inserted into pDOC153 to give an ampicillin and chloramphenicol-resistant, Pseudoalteromonas ? Escherichia coli shuttle vector (7,216 bp; pDOC155). The TonB-dependent receptor (chi22718_IV ) and exochitinase (chi22718_III ) genes from Arctic marine P. issachenkonii PAMC 22718 were cloned into pDOC155 to produce pDOC158 and pDOC165, respectively. Both vector derivatives were transferred into plasmid-free Pseudoalteromonas sp. PAMC 22137 by the triparental mating method. PCR experiments showed that the genes were stably maintained both in Pseudoalteromonas sp. PAMC 22137 and E. coli DH5α cells, indicating the potential use of pDOC155 as a new gene transfer system into marine Pseudoalteromonas spp.
DOI
http://dx.doi.org/10.7845/kjm.2016.5054
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