Abstract
Construction of vector plasmids is an essential technique for molecular biology. Here, we present an improved protocol for efficient introduction of multiple DNA fragments to produce a <20kbp insertion with several genes in a binary plasmid vector. Previously we developed a protocol for DNA fragment ligation on streptavidin-coated magnetic-beads. In this study, we combined the protocol with the MultiRound Gateway system to produce a series of binary vector plasmids which carried several sets of Arabidopsis thaliana transcription factor genes under an inducible promoter with estrogen. The transcription factor genes used for the multiple ligation on the vectors were selected by co-expression analysis of Arabidopsis genes which are expressed coordinately with some physiological phenomena. We are currently introducing these vector plasmids into Arabidopsis suspension-cultured cells with Argobacterium-mediated transformation, to evaluate the expression of each transcript and examine metabolic changes in the cells using metabolome analysis.
