Shuttle vectors for hyperthermophilic archaea

被引:56
作者
Aravalli, RN [1 ]
Garrett, RA [1 ]
机构
[1] UNIV COPENHAGEN,INST MOL BIOL,DK-1307 COPENHAGEN K,DENMARK
关键词
archaea; hyperthermophile; Pyrococcus Sulfolobus; shuttle vector; alcohol dehydrogenase;
D O I
10.1007/s007920050032
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Progress in understanding the basic molecular, biochemical, and physiological characteristics of archaeal hyperthermophiles has been limited by the lack of suitable expression vectors. Here, we report the construction of versatile shuttle vectors that can be maintained, and selected for, in both archaea and bacteria. The primary construct, pAG1, was produced by ligating portions of the archaeal cryptic plasmid pGT5 and the bacterial plasmid pUC19, both of which exhibit high copy numbers. A second vector construct, pAG2, was generated, with a reduced copy number in Escherichia coli, by introducing the Rom/Rop gene from pBR322 into pAG1. After transformation, both pAG1 and pAG2 were stably maintained and propagated in the euryarchaeote Pyrococcus furiosus, the crenarchaeote Sulfolobus acidocaldarius, and in Escherichia coli. An archaeal selective marker, the alcohol dehydrogenase gene from Sulfolobus solfataricus, was isolated by polymerase chain reaction (PCR) amplification and cloned into the two constructs. They were stably maintained and expressed in the two archaea and conferred resistance to butanol and benzyl alcohol. However, the vector pAG21, deriving from pAG2, proved the more stable in E. coli probably due to its lower copy number in the bacterium. Conditions are presented for the use of the vectors which, potentially, can be used for other hyperthermophilic archaea.
引用
收藏
页码:183 / 191
页数:9
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