Mar 19, 2013

Modular construction of plasmids by parallel assembly of linear vector components

Analytical Biochemistry
XinZheng GaoYuenan Li

Abstract

Construction of plasmids is the basic and pivotal technology in molecular biology. The common method for constructing plasmids is to cut DNA fragments by restriction enzymes and then join the resulting fragments using ligase. We present here a modified Golden Gate cloning method for modular construction of plasmids. Unlike the original Golden Gate cloning system for cloning from entry vector to expression vector, this method can be used to construct plasmids immediately from linear DNA fragments. After polymerase chain reaction (PCR) amplification for flanking with BsaI sites, multiple linear DNA components (modules) can be parallel assembled into a circle plasmid by a single restriction-ligation reaction using the method. This method is flexible to construct different types of plasmids because the modules can be freely selected and assembled in any combination. This method was applied successfully to construct a prokaryotic expression plasmid from four modules and a plant expression plasmid from five modules (fragments). The results suggest that this method provides a simple and flexible platform for modular construction of plasmids.

  • References25
  • Citations9

References

Mentioned in this Paper

Protein Methylation
Enzymes, antithrombotic
expression vector
Transcription, Genetic
Agrobacterium
Promega
Cytokinesis of the Fertilized Ovum
Promoter
Skeletal System
Gene Amplification Technique

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