A mini-Mu transposon-based method for multiple DNA fragment integration into bacterial genomes |
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Authors: | Xiao-Xing Wei Zhen-Yu Shi Zheng-Jun Li Lei Cai Qiong Wu Guo-Qiang Chen |
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Institution: | (1) Department of Biology, School of Life Sciences, Tsinghua University, Beijing, 100084, China;(2) Department of Chemistry, University of Melbourne, Parkville, Melbourne, VIC, 3052, Australia; |
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Abstract: | A method for construction of bacterial strains with multiple DNA inserted into their chromosomes has been developed based
on the mini-Mu transposon and FLP/FRT recombination. Exogenous DNA can be integrated by Mu transposition with an FRT cassette
containing selection marker and conditional replicative origin (R6Kγori). Subsequently, with the introduction of a helper plasmid bearing gene of FLP recombinase, drug-resistant selection marker
is excised from the chromosome. Cells cured of the helper plasmid can undergo the next cycle of transposition and excision
of selection marker. Each cycle can add further foreign gene(s) to the chromosome. As an example, resistance genes of chloramphenicol,
tetracycline, and gentamicin were successively integrated into the chromosome of Escherichia coli BW25113 by three cycles of insertion and excision as described above. This method proved to be simple and time-saving, which
could be applicable to a variety of microorganisms. |
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