PLASMID CLONING VECTORS FOR THE CONJUGAL TRANSFER OF DNA FROM ESCHERICHIA-COLI TO STREPTOMYCES SPP
PLASMID CLONING VECTORS FOR THE CONJUGAL TRANSFER OF DNA FROM ESCHERICHIA-COLI TO STREPTOMYCES SPP
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DOI：
10.1016/0378-1119(92)90627-2
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发表时间：
1992-07-01
期刊：
影响因子：
3.5
通讯作者：
SCHONER, BE
中科院分区：
文献类型：
--
作者：
BIERMAN, M;LOGAN, R;SCHONER, BE
我们已经构建了从大肠杆菌到链霉菌的DNA接合转移的克隆载体。所有载体均含有来自IncP质粒RK 2的760-bp oriT片段。传递函数需要由E. coli供体菌株。我们已经将在链霉菌属中起作用的选择性抗真菌标记（Am（R）、Th（R）、Sp（R））并入这些载体中。和其他特征，应允许：（i）通过同源重组在克隆的DNA和链霉菌属之间的整合。染色体，（ii）自主复制，或（iii）在噬菌体phi-C31附着位点的位点特异性整合。用于构建基因组文库的穿梭质粒和能够接受约1000个氨基酸的噬菌体P1克隆载体。还描述了100-kb片段。本文介绍了一种简单的交配方法，用于从大肠杆菌中进行接合转移。大肠杆菌对链霉菌属（Streptomyces spp.）包括将供体菌株和受体菌株的萌发孢子或菌丝体片段接种。我们已经表明，这些载体中的几个可以引入到链霉菌fragrance，是出了名的难以通过PEG介导的原生质体转化转化的菌株。
We have constructed cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp. All vectors contain the 760-bp oriT fragment froin the IncP plasmid, RK2. Transfer functions need to be supplied in trans by the E. coli donor strain. We have incorporated into these vectors selectable antibiotic-resistance markers (Am(R), Th(R), Sp(R)) that function in Streptomyces spp. and other features that should allow for: (i) integration via homologous recombination between cloned DNA and the Streptomyces spp. chromosome, (ii) autonomous replication, or (iii) site-specific integration at the bacteriophage phi-C31 attachment site. Shuttle cosmids for constructing genomic libraries and bacteriophage P1 cloning vector capable of accepting approx. 100-kb fragments are also described. A simple mating procedure has been developed for thc conjugal transfer of these vectors from E. coli to Streptomyces spp. that involves plating of the donor strain and either germinated spores or mycelial fragments of the recipient strain. We have shown that several of these vectors can be introduced into Streptomyces fradiae, a strain that is notoriously difficult to transform by PEG-mediated protoplast transformation.