Engineering large fragment insertions into the chromosome of Escherichia coli.
Engineering large fragment insertions into the chromosome of Escherichia coli.
复制标题
将大片段插入大肠杆菌的染色体中。
DOI：
10.1016/j.gene.2004.02.054
复制
发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Miller,JeffreyH
中科院分区：
文献类型：
--
作者：
Rong,Rui;Slupska,MalgorzataM;Chiang,Ju-Huei;Miller,JeffreyH
An effective DNA replacement system has been established for engineering large fragment insertions into the chromosome of Escherichia coli. The DNA replacement plasmid, pHybrid I, was first constructed based on the bacterial artificial chromosome (BAC) vector. Two fragments of the E. coli genome, 5.5 and 6.5 kb in length, were introduced into the vector for homologous recombination. In addition to the chloramphenicol gene, a second gene neo was introduced for double marker screening for recombinant clones. By shot-gun cloning and homologous recombination techniques, using our new recombinant vector (pHybrid I), a 20-kb fragment from Lactococcus lactis genomic DNA has been successfully integrated into the chromosome of the E. coli strain J93-140. Plating tests and PCR amplification indicated that the integration remained stable after many generations in cell culture. This system will be especially useful for the chromosome engineering of large heterologous fragment insertions, which is necessary for pathway engineering.
