Abstract
BackgroundFunctional genomic research always needs to assemble different DNA fragments into a binary vector, so as to express genes with different tags from various promoters with different levels. The cloning systems available bear similar disadvantages, such as promoters/tags are fixed on a binary vector, which is generally with low cloning efficiency and limited for cloning sites if a novel promoter/tag is in need. Therefore, it is difficult both to assemble a gene and a promoter together and to modify the vectors in hand. Another disadvantage is that a long spacer from recombination sites, which may be detrimental to the protein function, exists between a gene and a tag. Multiple GATEWAY system only resolves former problem at the expense of very low efficiency and expensive for multiple LR reaction.ResultsTo improve efficiency and flexibility for constructing expression vectors, we developed a platform, BioVector, by combining classical restriction enzyme/ligase strategy with modern Gateway DNA recombination system. This system included a series of vectors for gene cloning, promoter cloning, and binary vector construction to meet various needs for plant functional genomic study.ConclusionThis BioVector platform makes it easy to construct any vectors to express a target gene from a specific promoter with desired intensity, and it is also waiting to be freely modified by researchers themselves for ongoing demands. This idea can also be transferred to the different fields including animal or yeast study.
Highlights
Functional genomic research always needs to assemble different DNA fragments into a binary vector, so as to express genes with different tags from various promoters with different levels
BioVector is composed of three basic vectors: a gene entry clone (GEC) with one pair of site-specific recombination (SSR) of attL1/ attL2, a promoter entry clone (PEC) with another pair of SSRs of attL3/attL4, and a binary destination vector (BDV) with two pairs of corresponding SSRs of attR1/ attR2 and attR3/attR4
restriction enzyme/ligase (REL) sites were inserted inside site-specific recombination (SSR) sites in GECs, so that it was possible to make a seamless fusion between a gene and a tag in our system, rather than to introduce a SSR spacer between them as widely-used GATEWAY Cloning System does, in which the tags are in destination vectors [1,7,15]
Summary
Functional genomic research always needs to assemble different DNA fragments into a binary vector, so as to express genes with different tags from various promoters with different levels. The primers at the first step need a long extra sequence for recombination reaction, which decreases the efficiency of PCR and BP reaction and increases the cost. Aimed at these problems, higher efficiency vector systems are developed. Multiple Gateway Systems are developed for cloning multiple DNA fragments (such as, promoters, genes and terminators) into a expression vector in one step [5,6,7,8], but it suffers from low efficiency because of many recombination sites. Two excellent papers reported new gene cloning strategies, referred to Golden Gate Cloning (mainly based on the type IIs endonucleases Bsa I and Bbs I) [9] and GoldenBraid (mainly based on the type IIs endonucleases Bsa I and BsmB I) [10], with which it is possible to seamlessly assemble multiple reusable gene modules, including promoters, genes and terminators, together in a binary vector in a single restriction-ligation
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