Molecular Control and Genetics Section, RNA Biology Laboratory, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland.
formerly with Molecular Control and Genetics Section, RNA Biology Laboratory, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland.
Curr Protoc. 2023 Feb;3(2):e656. doi: 10.1002/cpz1.656.
The bacterial chromosome and bacterial plasmids can be engineered in vivo by homologous recombination using either PCR products or synthetic double-stranded DNA (dsDNA) or single-stranded DNA as substrates. Multiple linear dsDNA molecules can be assembled into an intact plasmid. The technology of recombineering 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 the location of restriction sites and can also be used in combination with CRISPR/Cas targeting systems. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol: Making electrocompetent cells and transforming with linear DNA Support Protocol 1: Selection/counter-selections for genome engineering Support Protocol 2: Creating and screening for oligo recombinants by PCR Support Protocol 3: Other methods of screening for unselected recombinants Support Protocol 4: Curing recombineering plasmids containing a temperature-sensitive replication function Support Protocol 5: Removal of the prophage by recombineering Alternate Protocol 1: Using CRISPR/Cas9 as a counter-selection following recombineering Alternate Protocol 2: Assembly of linear dsDNA fragments into functional plasmids Alternate Protocol 3: Retrieval of alleles onto a plasmid by gap repair Alternate Protocol 4: Modifying multicopy plasmids with recombineering Support Protocol 6: Screening for unselected plasmid recombinants Alternate Protocol 5: Recombineering with an intact λ prophage Alternate Protocol 6: Targeting an infecting λ phage with the defective prophage strains.
细菌染色体和细菌质粒可以通过同源重组在体内进行工程改造，使用 PCR 产物或合成的双链 DNA（dsDNA）或单链 DNA 作为底物。多个线性 dsDNA 分子可以组装成一个完整的质粒。重组技术之所以成为可能，是因为噬菌体编码的重组蛋白能够有效地重组具有 35 到 50 个碱基短同源序列。重组技术可以在不考虑限制酶切位点位置的情况下插入或删除 DNA 序列，并且还可以与 CRISPR/Cas 靶向系统结合使用。© 2023 Wiley Periodicals LLC。本文由美国政府雇员做出贡献，其工作在美国属于公有领域。基本方案：制作电转化感受态细胞和转化线性 DNA 支持方案 1：用于基因组工程的选择/反选择 支持方案 2：通过 PCR 构建和筛选寡核苷酸重组体 支持方案 3：筛选未选择重组体的其他方法 支持方案 4：含有温度敏感复制功能的重组质粒的消除 支持方案 5：通过重组技术去除带有噬菌体 备选方案 1：在重组技术后使用 CRISPR/Cas9 作为反选择 备选方案 2：将线性 dsDNA 片段组装成功能性质粒 备选方案 3：通过缺口修复将等位基因转移到质粒上 备选方案 4：使用重组技术修饰多拷贝质粒 支持方案 6：筛选未选择的质粒重组体 备选方案 5：使用完整 λ噬菌体进行重组技术 备选方案 6：用缺陷噬菌体株靶向感染性 λ噬菌体。
