当前位置： SCI文献检索 > PLASMID期刊下所有文献 > Isolation of a low-molecular-weight, multicopy plasmid, pNHK101, from Thermus sp. TK10 and its use as an expression vector for T. thermophilus HB27.

Isolation of a low-molecular-weight, multicopy plasmid, pNHK101, from Thermus sp. TK10 and its use as an expression vector for T. thermophilus HB27.

Abstract：

:We isolated a small multicopy cryptic plasmid, pNHK101, from Thermus sp. TK10 for use as a replicon of a Thermus expression vector. The nucleotide sequence of pNHK101 revealed that this plasmid was 1564bp long, with a total G+C content of 66.8%, which was in agreement with that of Thermus genomic DNA. The sequence did not show any significant similarities to any other plasmids; also, the amino acid sequences of four putative open reading frames, found in the plasmid, did not show strong similarities to those in the databases, except the ORF1, which had very slight similarities to several replication proteins of plasmids from other bacteria. pNHK101 was able to replicate in Thermus thermophilus HB27 with copy number about 80, and was stably maintained at 60 degrees C, but became unstable at 70 degrees C. Based on pNHK101, we constructed a plasmid vector, pKMH052, containing the highly thermostable kanamycin resistance gene as a selective marker. The copy number of pKMH052 decreased to about one-fourth of that of pNHK101, but stability at 60 degrees C did not alter under non-selective conditions. pKMH052 was compatible with pTT8, and interestingly, the presence of pTT8 in the same cells improved the stability of pKMH052 at 70 degrees C. Cloning of the crtB gene of T. thermophilus HB27 encoding phytoene synthase into pKMH052, and introduction into T. thermophilus cells resulted in a 2.8-fold production of carotenoids, indicating the potential use of this plasmid for overexpression of genes from thermophiles and hyperthermophiles.

journal_name

Plasmid

journal_title

Plasmid

authors

Kobayashi H,Kuwae A,Maseda H,Nakamura A,Hoshino T

doi

10.1016/j.plasmid.2004.12.004

keywords:

subject

Has Abstract

pub_date

2005-07-01 00:00:00

pages

70-9

issue

1

eissn

0147-619X

issn

1095-9890

pii

S0147-619X(04)00145-3

journal_volume

54

pub_type

杂志文章

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