Abstract
Building expression constructs for transgenesis is one of the fundamental day-to-day tasks in modern biology. Traditionally it is based on a multitude of type II restriction endonucleases and T4 DNA ligase. Especially in case of long inserts and applications requiring high-throughput, this approach is limited by the number of available unique restriction sites and the need for designing individual cloning strategies for each project. Several alternative cloning systems have been developed in recent years to overcome these issues, including the type IIS enzyme based Golden Gate technique. Here we introduce our GreenGate system for rapidly assembling plant transformation constructs, which is based on the Golden Gate method. GreenGate cloning is simple and efficient since it uses only one type IIS restriction endonuclease, depends on only six types of insert modules (plant promoter, N-terminal tag, coding sequence, C-terminal tag, plant terminator and plant resistance cassette), but at the same time allows assembling several expression cassettes in one binary destination vector from a collection of pre-cloned building blocks. The system is cheap and reliable and when combined with a library of modules considerably speeds up cloning and transgene stacking for plant transformation.
Highlights
Ever since the first construction of a recombinant plasmid [1,2], genetic engineering and molecular cloning mostly rely on the use of type II restriction endonucleases and DNA ligases
Such a cloning system requires all inserts to be flanked by type IIS recognition sites in an orientation that fragments do not carry the sites after type IIS mediated release
Specific overhang sequences for each class of insert define the orientation of each fragment, and the order in which they will be assembled in the final construct: Dependent on the intended position in the construct, each module is flanked by a different overhang at 59- and 39-end, while the overhangs of adjacent fragments are complementary
Summary
Ever since the first construction of a recombinant plasmid [1,2], genetic engineering and molecular cloning mostly rely on the use of type II restriction endonucleases and DNA ligases. The DNA fragments to be combined are first excised from their precursor molecules via the endonucleases and in a separate reaction re-assembled by the ligase, usually after spontaneous annealing of complementary single-stranded overhangs created during the endonuclease cut While this approach is generally successful, there are certain limitations, especially when it comes to the assembly of complex plasmids from multiple elements, since with increasing numbers of DNA fragments the ligation reaction becomes less and less efficient. Since most recognition sites used in a digestion-ligation cycle remain in the construct, the corresponding enzymes cannot be used for adding further DNA fragments in subsequent steps Many of these recognition sites occur fairly frequently in a given piece of DNA, making the assembly of long constructs even more difficult because of the lack of unique restriction targets. With the advent of high throughput approaches and widespread use of transgenic models to test gene function in vivo, classical restriction based cloning rapidly became a major limitation and alternative technologies began to emerge
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