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From 2012.igem.org

Golden Gate Standard

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On this page, we introduce the Golden Gate Standard to the Registry of Standard Biological parts. We explain in detail, how Golden Gate Cloning works and how it can be made compatible with RFC 10 standard. Moreover, we provide step-by-step protocols for using this new standard.

Introduction

Although BioBrick assembly is a powerful tool for the synbio community because it allows standardized, simple construction of complex genetic constructs from basic genetic modules, it is not the best option when it comes to assembling larger numbers of modules in a short period of time. Furthermore, BioBrick assembly leaves scars between assembled parts, which is not optimal for protein fusion constructs. One popular method which overcomes these obstacles is Gibson Cloning. This method uses an exonuclease to produce sticky ends on overlapping PCR products, a polymerase to fill up single stranded gaps after annealing and a ligase to connect the different parts . Figure 1: Schematic overview of Gibson Assembly (Gibson et al. 2009).

Gibson cloning allows for assembling whole constructs in one reaction and has been used by many iGEM teams over the past years. However, this technique is not compatible with parts provided by the Registry, unless parts are PCR-amplified in order to linearize them and to add overlapping sequences. Furthermore, Gibson cloning requires three different enzymes and can be very tricky. We therefore propose another method called Golden Gate Cloning (or ist derivatives MoClo and GoldenBraid ). Golden Gate Cloning (GGC) can be used to assemble many fragments with very high efficiency in one reaction . Importantly, insert fragments can be cut out of amplification vectors (such as iGEM standard vectors) and assembled in one single reaction.