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Construction of A New ccdB Tolerant Strain ThroughOligonucleotide Mediated Recombineering(PDF)


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Construction of A New ccdB Tolerant Strain ThroughOligonucleotide Mediated Recombineering
Yan ZhenyaChai MeixiaZhang QingLuo XiShang Guangdong
School of Life Sciences,Nanjing Normal University,Jiangsu Engineering and Technology Research Center for Microbiology,Jiangsu Key Laboratory for Microbes and Functional Genomics, Nanjing 210023,China
ccdBR6Ksingle-stranded oligonucleotiderecombineering
Toxic gene ccdB is the most often used counter selection marker,together with R6K replicon,they constitute the highly efficient gene cloning and genome modification genetic elements. To obtain a high transformation efficiency ccdB tolerant strain,in this study,a single-stranded oligonucleotide designed to circumvent the mismatch repair was co-transformed with pUC backbone,ccdB harboring plasmid pMK2010 into Escherichia coli BUN20,a DH10B derivative with pir116 genotype. Seven out of ten strains exhibited the expected GyrA462 genotype. To eliminate pMK2010,a pUC backbone,auto-cleavable vector pLS2750 was constructed. pMK2010 was replaced by pLS2750 under ampicillin selection via incompatibility mechanism,then pLS2750 was self eliminated under the I-SceI self cleavage. The engineered stain LS027 shows an electroporation efficiency of 6.9×108/mg,which is about 100 folder higher than that of the control strain;and R6K plasmid shows high copy number. Serial gene cloning vectors were constructed with LS027 as host,the vector will eliminate self-ligated background. The new ccdB tolerant strain LS027 will find general applications in gene manipulation.


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Last Update: 2016-06-30