E. coli genome manipulation by P1 transduction.
E. coli genome manipulation by P1 transduction.
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DOI：
10.1002/0471142727.mb0116s78
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发表时间：
2007-07-01
影响因子：
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通讯作者：
Court, Donald L
中科院分区：
文献类型：
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作者：
Thomason, Lynn C;Costantino, Nina;Court, Donald L
细菌染色体和质粒可以通过使用 PCR 产物和合成寡核苷酸作为底物的同源重组在体内进行工程改造。这是可能的，因为噬菌体编码的重组功能可以有效地重组同源性短至 35 至 40 个碱基的序列。这种重组工程允许插入或删除 DNA 序列，而无需考虑限制性位点的位置。本单元首先描述了表达重组工程功能的电感受态细胞的制备及其用 dsDNA 或 ssDNA 的转化。支持方案描述了一种在不留下任何不需要的变化的情况下进行遗传改变的两步方法，以及一种从大肠杆菌染色体或共电穿孔 DNA 片段中检索遗传标记（克隆）并将其转移到质粒上的方法。还提供了一种筛选未选择突变的方法。其他协议描述了缺陷原噬菌体的去除、重组工程的方法。细菌染色体和细菌质粒可以通过使用 PCR 产物和合成寡核苷酸作为底物的同源重组在体内进行工程改造。这是可能的，因为噬菌体编码的重组蛋白可以有效地重组同源性短至 35 至 50 个碱基的序列。重组工程允许插入或删除 DNA 序列，而无需考虑限制性位点的位置。本单元首先描述了表达重组工程功能的电感受态细胞的制备及其用 dsDNA 或 ssDNA 的转化。然后，它提出了支持方案，描述了几种进行遗传改变而不在目标DNA中留下任何不需要的变化的两步选择/反选择方法，以及一种从大肠杆菌染色体或共电穿孔DNA片段中检索到质粒上的遗传标记（通过检索克隆）的方法。其他协议描述了筛选未选择的突变、从重组工程菌株中去除有缺陷的原噬菌体的方法以及其他有用的技术。本单元描述了用于将大肠杆菌基因组的部分从一种遗传变体转移到另一种遗传变体的程序。 P1 噬菌体可以转移大约 100 kb 的片段。噬菌体首先在含有待移动元件的菌株上生长，所得噬菌体裂解物用于感染第二个受体菌株。裂解物将含有细菌 DNA 以及噬菌体 DNA，在受体菌株的酶的催化下，基因重组会将细菌片段整合到受体染色体中。
The bacterial chromosome and plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination functions efficiently to recombine sequences with homologies as short as 35 to 40 bases. This recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. Support protocols describe a two-step method of making genetic alterations without leaving any unwanted changes, and a method for retrieving a genetic marker (cloning) from the E. coli chromosome or a co-electroporated DNA fragment and moving it onto a plasmid. A method is also given to screen for unselected mutations. Additional protocols describe removal of defective prophage, methods for recombineering.The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using PCR products and synthetic oligonucleotides as substrates. This is possible because bacteriophage-encoded recombination proteins efficiently recombine sequences with homologies as short as 35 to 50 bases. Recombineering allows DNA sequences to be inserted or deleted without regard to location of restriction sites. This unit first describes preparation of electrocompetent cells expressing the recombineering functions and their transformation with dsDNA or ssDNA. It then presents support protocols that describe several two-step selection/counter-selection methods of making genetic alterations without leaving any unwanted changes in the targeted DNA, and a method for retrieving onto a plasmid a genetic marker (cloning by retrieval) from the Escherichia coli chromosome or a co-electroporated DNA fragment. Additional protocols describe methods to screen for unselected mutations, removal of the defective prophage from recombineering strains, and other useful techniques.This unit describes the procedure used to move portions of the E. coli genome from one genetic variant to another. Fragments of approximately 100 kb can be transferred by the P1 bacteriophage. The phage is first grown on a strain containing the elements to be moved, and the resulting phage lysate is used to infect a second recipient strain. The lysate will contain bacterial DNA as well as phage DNA, and genetic recombination, catalyzed by enzymes of the recipient strain, will incorporate the bacterial fragments into the recipient chromosome.