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首页> 外文期刊>Applied Microbiology >Properties and Construction of Plasmid pFW213, a Shuttle Vector with the Oral Streptococcus Origin of Replication
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Properties and Construction of Plasmid pFW213, a Shuttle Vector with the Oral Streptococcus Origin of Replication

机译：具有口服链球菌复制起点的穿梭载体pFW213的特性和构建

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Streptococcus parasanguinis is among the most successful colonizers of the human body. Strain FW213 harbors a 7.0-kb cryptic plasmid, pFW213, with a copy number at 5 to 10 per chromosome. Sequence and functional analyses of pFW213 revealed that the open reading frame (ORF) encoding the replication protein (Rep) is essential for the replication of pFW213, and the putative plasmid addiction system (RelB and RelE) and an ORF (ORF6) with no known function are required for its stability. The minimal replicon of pFW213 contains the rep gene and its 5′-flanking 390-bp region. Within the minimal replicon, an A/T-rich region followed by 5 contiguous 22-bp repeats was located 5′ of the ATG of rep. No single-stranded replication intermediates were detected in the derivatives of pFW213, suggesting that pFW213 replicates via the theta replication mechanism. The minimal replicon was unstable in streptococcal hosts without selection, but the stability was greatly enhanced in derivatives containing the intact relBE genes. A Streptococcus - Escherichia coli shuttle vector, pCG1, was constructed with the pFW213 replicon. Plasmid pCG1 features a multiple cloning region and a spectinomycin resistance determinant that is expressed in both Streptococcus spp. and E. coli . Various streptococcal DNA fragments were cloned in pCG1, and the recombinant constructs were stably maintained in the streptococcal hosts. Since pCG1 is compatible with the most widely used streptococcal replicon, pVA380-1, pCG1 will provide a much needed tool allowing the cloning of two genes that work in concert in the same host.
机译：副血链球菌是人体最成功的定居者之一。 FW213菌株带有一个7.0-kb的密码质粒pFW213，每条染色体的拷贝数为5至10。对pFW213的序列和功能分析表明，编码复制蛋白（Rep）的开放阅读框（ORF）对于pFW213的复制至关重要，假定的质粒成瘾系统（RelB和RelE）和一个ORF（ORF6）尚不清楚功能是其稳定性所必需的。 pFW213的最小复制子包含rep基因及其5'侧翼390 bp区域。在最小复制子内，一个富A / T区，后接5个连续的22bp重复序列位于rep的ATG的5'。在pFW213的衍生物中未检测到单链复制中间体，表明pFW213通过theta复制机制复制。在没有选择的情况下，最小复制子在链球菌宿主中不稳定，但在含有完整relBE基因的衍生物中，其稳定性大大增强。用pFW213复制子构建链球菌-大肠杆菌穿梭载体pCG1。质粒pCG1具有一个多克隆区和一个在两个链球菌中均表达的壮观霉素抗性决定簇。和大肠杆菌。将各种链球菌DNA片段克隆到pCG1中，并将重组构建体稳定地保持在链球菌宿主中。由于pCG1与最广泛使用的链球菌复制子pVA380-1兼容，因此pCG1将提供一个急需的工具，允许克隆在同一宿主中协同工作的两个基因。

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