当前位置： SCI文献检索 > APPLIED MICROBIOLOGY AND BIOTECHNOLOGY期刊下所有文献 > Transfer of megaplasmid pKB1 from the rubber-degrading bacterium Gordonia westfalica strain Kb1 to related bacteria and its modification.

Transfer of megaplasmid pKB1 from the rubber-degrading bacterium Gordonia westfalica strain Kb1 to related bacteria and its modification.

Abstract：

:Because engineering of the 101.016-bp megaplasmid pKB1 of Gordonia westfalica Kb1 failed due to the absence of an effective transfer system, pKB1 was transferred by conjugation from G. westfalica Kb1 to a kanamycin-resistant mutant of Rhodococcus opacus PD630 at a frequency of about 6.2 x 10(-8) events per recipient cell. Furthermore, pKB1 was transferred to G. polyisoprenivorans strains VH2 and Y2K and to Mycobacterium smegmatis by electroporation at frequencies of 5.5 x 10(3), 1.9 x 10(3), and 8.3 x 10(2) transformants per microgram plasmid DNA. The pKB1-encoded cadmium resistance gene cadA was used for selection in these experiments. Recombinant pKB1-containing G. polyisoprenivorans VH2 and M. smegmatis were then used to engineer pKB1. A kanamycin resistance cassette was inserted into the pKB1-encoded cadA gene, ligated to suicide plasmid pBBR1MCS-5, and the resulting plasmid was electroporated into plasmid-harboring strains. Homologous recombination between cadA on suicide plasmid and the respective sequence in pKB1 led to its integration into pKB1. Thus, two selection markers were accommodated in pKB1 to monitor plasmid transfer into Gordonia and related taxa for analysis of genes essential for rubber degradation and others. In this study, two transfer methods for large plasmids and strategies for engineering of pKB1 were successfully applied, thereby, extending the tool box for Gordonia.

journal_name

Appl Microbiol Biotechnol

journal_title

Applied microbiology and biotechnology

authors

Bröker D,Arenskötter M,Steinbüchel A

doi

10.1007/s00253-007-1262-8

subject

Has Abstract

pub_date

2008-01-01 00:00:00

pages

1317-27

issue

6

eissn

0175-7598

issn

1432-0614

journal_volume

77

pub_type

杂志文章

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