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

Author(s)

Pierdona, Flaviani G. ; Carbajal, Yajahaira ; Vicente, Mateus H. ; Ferigolo, Leticia F. ; Nogueira, Fabio T. S.

• Document type: Journal article
• Source: BIO-PROTOCOL; v. 12, n. 24, p. 10-pg., 2022-12-20.

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. (AU)

• FAPESP's process: 18/17441-3 - Molecular control of shoot architecture: interplay between microRNAs, transcription factors and phytohormones
• Grantee: Fabio Tebaldi Silveira Nogueira
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 20/12940-1 - Study of the interaction between the microRNA156/SPL module and two hormones (brassinosteroid and gibberellin) during early hypocotyl growth
• Grantee: Flaviani Gabriela Pierdoná
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 19/20157-8 - Evaluation of possible interactions between photoperiodic, age and gibereline flowering pathway with SP/SFT module and its impacts on tomato architecture (Solanum lycopersicum L.)
• Grantee: Mateus Henrique Vicente
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 21/14640-8 - Light-based regulation of the miRNA156/SPL9 module in the early hypocotyl elongation
• Grantee: Flaviani Gabriela Pierdoná
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate