Lambda Red recombinase-mediated integration of the high molecular weight DNA into the Escherichia coli chromosome.
Lambda Red recombinase-mediated integration of the high molecular weight DNA into the Escherichia coli chromosome.
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LAMBDA红色重组酶介导的高分子量DNA整合到大肠杆菌染色体中。
DOI：
10.1186/s12934-016-0571-y
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发表时间：
2016-10-05
影响因子：
6.4
通讯作者：
Ajioka JW
中科院分区：
文献类型：
--
作者：
Juhas M;Ajioka JW
大肠杆菌K-12是许多合成生物学和生物技术应用的常用宿主，也是开发最小细胞工厂的底盘。将高分子量DNA整合到大肠杆菌中的新方法。因此，大肠杆菌染色体将极大地促进在这种细菌中的工程化努力。我们开发了一个可靠和灵活的基于λ Red重组酶的系统，该系统利用重叠的DNA片段将高分子量DNA整合到E. coli染色体。我们的染色体整合策略可用于将可变长度的高分子量DNA整合到大肠杆菌中的任何非必需位点。coli染色体。利用这种方法，我们将编码蔗糖催化剂和乳糖代谢和转运操纵子的15 kb DNA整合到大肠杆菌鞭毛区3b的fliK位点。coliK 12 MG 1655染色体。此外，利用该系统，我们将50 kb的枯草芽孢杆菌168 DNA整合到E. coliK 12 MG 1655染色体。染色体整合到fliK位点的效率很高，抑制运动，对E.杆菌除了合成生物学装置的合理设计外，我们的高分子量DNA染色体整合系统将促进E.杆菌 本文的在线版本（doi：10.1186/s12934-016-0571-y）包含补充材料，可供授权用户使用。
Escherichia coli K-12 is a frequently used host for a number of synthetic biology and biotechnology applications and chassis for the development of the minimal cell factories. Novel approaches for integrating high molecular weight DNA into the E. coli chromosome would therefore greatly facilitate engineering efforts in this bacterium. We developed a reliable and flexible lambda Red recombinase-based system, which utilizes overlapping DNA fragments for integration of the high molecular weight DNA into the E. coli chromosome. Our chromosomal integration strategy can be used to integrate high molecular weight DNA of variable length into any non-essential locus in the E. coli chromosome. Using this approach we integrated 15 kb DNA encoding sucrose catabolism and lactose metabolism and transport operons into the fliK locus of the flagellar region 3b in the E. coli K12 MG1655 chromosome. Furthermore, with this system we integrated 50 kb of Bacillus subtilis 168 DNA into two target sites in the E. coli K12 MG1655 chromosome. The chromosomal integrations into the fliK locus occurred with high efficiency, inhibited motility, and did not have a negative effect on the growth of E. coli. In addition to the rational design of synthetic biology devices, our high molecular weight DNA chromosomal integration system will facilitate metabolic and genome-scale engineering of E. coli. The online version of this article (doi:10.1186/s12934-016-0571-y) contains supplementary material, which is available to authorized users.
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影响因子：
4.7
作者：
St-Pierre, Francois;Cui, Lun;Shearwin, Keith E.
通讯作者：
Shearwin, Keith E.
影响因子：
8.4
作者：
Marcellin, Esteban;Chen, Wendy Y.;Nielsen, Lars K.
通讯作者：
Nielsen, Lars K.
DOI：
10.1126/science.1191652
发表时间：
2010-10-01
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Ajikumar PK;Xiao WH;Tyo KE;Wang Y;Simeon F;Leonard E;Mucha O;Phon TH;Pfeifer B;Stephanopoulos G
通讯作者：
Stephanopoulos G
影响因子：
--
作者：
Murphy KC;Campellone KG
通讯作者：
Campellone KG
影响因子：
48
作者：
Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
通讯作者：
Smith, Hamilton O.