Faster Bacterial Gene Cloning Using the Brick into the Gateway (BiG) Protocol

Abstract

Cloning systems like Gateway and Golden Gate/Braid are known because of their efficiency and accuracy. While the main drawback of Gateway is the expensive cost of the enzymes used in its two-step (LR and BP) reaction, Golden Gate requires non-reusable components due to their specific restriction sites. We present the Brick into the Gateway (BiG) protocol as a new cloning strategy, faster and more economic method that combines (i) reusable modules or bricks assembled by the GoldenBraid approach, and (ii) Gateway LR reactions [recombination of attachment sites: attL (L from left) and attR (R from right)] avoiding the BP reaction [recombination of attachment sites: attP (P from phage) and attB (B from bacteria)] usually necessary in the Gateway cloning. The starting point is to perform a PCR reaction to add type IIS restriction sites into DNA fragments generating specific fusion sites. Then, this PCR product is used to design GoldenBraid bricks, including the attL Gateway recombination sites. Using the Golden Gate method, these bricks are assembled to produce an attL1 -gene of interest- attL2 fragment, which is integrated into a compatible vector producing a Gateway entry vector. Finally, the fragment containing the target gene is recombined by LR reaction into the Gateway destination vector. This protocol was validated in: Plasmid (2022), DOI: 10.1016/j.plasmid.2022.102630 Graphical abstract.

Keywords: DNA cloning; Gateway; Golden Gate; GoldenBraid; LR reaction; brick assembly.