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

극지해양 Pseudoalteromonas 유래의 소형 플라스미드에 기반한 Pseudoalteromonas - Escherichia coli 셔틀벡터 제작

  • Kim, Dockyu (Division of Life Sciences, Korea Polar Research Institute) ;
  • Park, Ha Ju (Division of Life Sciences, Korea Polar Research Institute) ;
  • Park, Hyun (Division of Life Sciences, Korea Polar Research Institute)
  • 김덕규 (극지연구소 극지생명과학연구부) ;
  • 박하주 (극지연구소 극지생명과학연구부) ;
  • 박현 (극지연구소 극지생명과학연구부)
  • Received : 2015.11.19
  • Accepted : 2016.01.12
  • Published : 2016.03.31

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 $DH5{\alpha}$ cells, indicating the potential use of pDOC155 as a new gene transfer system into marine Pseudoalteromonas spp.

Keywords

Pseudoalteromonas;cold-active enzyme;gene transfer;shuttle vector

Acknowledgement

Supported by : Korea Polar Research Institute (KOPRI)

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