Bierman M, Logan R, O'Brien K, Seno E T, Rao R N, Schoner B E
Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, IN 46285-0424.
Gene. 1992 Jul 1;116(1):43-9. doi: 10.1016/0378-1119(92)90627-2.
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 from 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 (AmR, ThR, SpR) 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 the 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.
我们构建了用于将DNA从大肠杆菌接合转移至链霉菌属细菌的克隆载体。所有载体均含有来自IncP质粒RK2的760 bp oriT片段。转移功能需由大肠杆菌供体菌株反式提供。我们已将在链霉菌属细菌中起作用的可选择抗生素抗性标记（AmR、ThR、SpR）以及其他一些特性整合到这些载体中，这些特性应允许：（i）通过克隆DNA与链霉菌属细菌染色体之间的同源重组进行整合，（ii）自主复制，或（iii）在噬菌体phi C31附着位点进行位点特异性整合。还描述了用于构建基因组文库的穿梭粘粒和能够接受约100 kb片段的噬菌体P1克隆载体。已开发出一种简单的交配程序，用于将这些载体从大肠杆菌接合转移至链霉菌属细菌，该程序包括接种供体菌株以及受体菌株的萌发孢子或菌丝片段。我们已证明，其中几种载体可导入弗氏链霉菌，该菌株通过聚乙二醇介导的原生质体转化法进行转化非常困难。
