Golden GATEway Cloning – A Combinatorial Approach to Generate Fusion and Recombination Constructs

Stephan Kirchmaier,Katharina Lust,Joachim Wittbrodt,Vladimir Korzh

doi:10.1371/journal.pone.0076117

Abstract

The design and generation of DNA constructs is among the necessary but generally tedious tasks for molecular biologists and, typically, the cloning strategy is restricted by available restriction sites. However, increasingly sophisticated experiments require increasingly complex DNA constructs, with an intricacy that exceeds what is achievable using standard cloning procedures. Many transgenes such as inducible gene cassettes or recombination elements consist of multiple components that often require precise in-frame fusions. Here, we present an efficient protocol that facilitates the generation of these complex constructs. The golden GATEway cloning approach presented here combines two established cloning methods, namely golden Gate cloning and Multisite GatewayTM cloning. This allows efficient and seamless assembly as well as reuse of predefined DNA elements. The golden Gate cloning procedure follows clear and simple design rules and allows the assembly of multiple fragments with different sizes into one open reading frame. The final product can be directly integrated into the widely used Multisite GatewayTM cloning system, granting more flexibility when using a transgene in the context of multiple species. This adaptable and streamlined cloning procedure overcomes restrictions of “classical construct generation” and allows focusing on construct design.

Highlights

Plasmid construction is a necessary task and often a serious challenge for molecular biologists
Our Golden GATEway cloning kit is characterized by the assembly of predefined DNA building blocks in two distinct steps (Figure 1)
With our standardized cloning approach, the Golden GATEway cloning kit, we provide a framework that allows fast and efficient assembly of complex DNA constructs by combining Golden Gate cloning with Multisite GatewayTM cloning

Summary

Introduction

Plasmid construction is a necessary task and often a serious challenge for molecular biologists. Fusion proteins with linkers of varying length can have different properties [1], arguing for an evaluation of a number of candidate linkers to find the optimal one for a specific application Often, this requires the careful design of individual cloning strategies for all planned candidates, which is timeconsuming and can be technically demanding. This requires the careful design of individual cloning strategies for all planned candidates, which is timeconsuming and can be technically demanding This process is inherently inflexible to rapid prototyping and the continuous consideration of new results obtained with earlier candidates. This problem becomes evident in experimental approaches that rely on small repetitive elements for targeted genetic recombination. Our goal was to provide a framework for the rapid and flexible generation of these complex transgenesis constructs with a simple, modular design strategy

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Conclusion