Mini-mu bacteriophage with plasmid replicons for in vivo cloning and lac gene fusing.
Mini-mu bacteriophage with plasmid replicons for in vivo cloning and lac gene fusing.
复制标题
带有质粒复制子的 Mini-mu 噬菌体，用于体内克隆和 lac 基因融合。
DOI：
10.1128/jb.168.1.357-364.1986
复制
发表时间：
1986
影响因子：
3.2
通讯作者：
Casadaban,MJ
中科院分区：
文献类型：
--
作者：
Groisman,EA;Casadaban,MJ
构建了具有质粒复制子的新的mini-Mu转座子，其具有用于体内DNA克隆和大肠杆菌中lac基因融合的额外特征。这些微型Mu复制子可用于克隆DNA，通过使它们与互补Mu噬菌体一起生长，并通过使用所得裂解物来扩增Mu溶原性细胞。这些小型Mu噬菌体具有可选择的基因，用于对卡那霉素、氯霉素和壮观霉素-链霉素的抗性，以及来自高拷贝数质粒pMB 1和P15 A以及低拷贝、宽宿主范围质粒pSa的复制子。这些元件中最有效的元件可用于克隆基因，其频率比先前描述的微型Mu复制子Mu dII 4042高100倍，使得可用少至1微升的含有10（6）个辅助噬菌体的裂解物制备完整的基因库。39-内切酶对Mu头部DNA包装机制限制了形成的克隆的大小。最小的微型Mu元件只有7.9个内切酶对长，允许克隆多达31.1个内切酶对的DNA片段，最大的是21.7个内切酶对，要求克隆携带少于17.3个内切酶对的插入。已构建元件以形成与克隆片段中存在的启动子融合的转录和翻译类型的lac基因。这些元件中的两个还含有来自质粒RK 2的转移起点序列oriT，使得用这些微型Mu噬菌体获得的克隆可以通过接合有效地移动。
New mini-Mu transposons with plasmid replicons were constructed with additional features for in vivo DNA cloning and lac gene fusing in Escherichia coli. These mini-Mu replicons can be used to clone DNA by growing them with a complementing Mu bacteriophage and by using the resulting lysate to transduce Mu-lysogenic cells. These mini-Mu phage have selectable genes for resistance to kanamycin, chloramphenicol, and spectinomycin-streptomycin, and replicons from the high-copy-number plasmids pMB1 and P15A and the low-copy, broad-host-range plasmid pSa. The most efficient of these elements can be used to clone genes 100 times more frequently than with the previously described mini-Mu replicon Mu dII4042, such that complete gene banks can be made with as little as 1 microliter of a lysate containing 10(6) helper phage. The 39-kilobase-pair Mu headful DNA packaging mechanism limits the size of the clones formed. The smallest of the mini-Mu elements is only 7.9 kilobase pairs long, allowing the cloning of DNA fragments of up to 31.1 kilobase pairs, and the largest of them is 21.7 kilobase pairs, requiring that clones carry insertions of less than 17.3 kilobase pairs. Elements have been constructed to form both transcriptional and translational types of lac gene fusions to promoters present in the cloned fragment. Two of these elements also contain the origin-of-transfer sequence oriT from the plasmid RK2, so that clones obtained with these mini-Mu bacteriophage can be efficiently mobilized by conjugation.