E. coli genome manipulation by P1 transduction.
E. coli genome manipulation by P1 transduction.
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DOI：
10.1002/0471142727.mb0116s106
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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
以聚合酶链式反应产物和人工合成的寡核苷酸为底物，通过同源重组，可在体内构建细菌染色体和细菌质粒。这是可能的，因为噬菌体编码的重组蛋白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，而不考虑限制酶切位点的位置。本单元首先描述了表达重组工程功能的电活性细胞的制备及其与双链DNA或单链DNA的转化。然后介绍了描述几种两步选择/反选择方法的支持方案，这些方法进行遗传改变而不在目标DNA中留下任何不想要的改变，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(通过检索进行克隆)到质粒上的方法。其他协议描述了筛选未选择突变的方法，从重组工程菌株中移除有缺陷的原噬菌体，以及其他有用的技术。细菌染色体和质粒可以使用PCR产物和合成的寡核苷酸作为底物，通过同源重组在体内进行工程。这是可能的，因为噬菌体编码的重组有效地重组了同源性短至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，而不考虑限制位点的位置。本单元首先描述了表达重组工程功能的电活性细胞的制备及其与双链DNA或单链DNA的转化。支持协议描述了一种在不留下任何不想要的改变的情况下进行基因改变的两步法，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)并将其移动到质粒上的方法。还给出了一种筛选未选择突变的方法。其他协议描述了有缺陷的原噬菌体的移除，重组的方法。本单元描述了将部分大肠杆菌基因组从一个基因变体转移到另一个基因变体的过程。约100kb的片段可通过P1噬菌体转移。首先在含有要移动的元件的菌株上培养噬菌体，然后用产生的噬菌体裂解物感染第二个受体菌株。裂解物将包含细菌DNA和噬菌体DNA，在受体菌株酶的催化下进行基因重组，将细菌片段整合到受体染色体中。
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.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.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.