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

Area of Science:

• Molecular Biology
• Synthetic Biology
• Biotechnology

Background:

• Existing cloning systems like Gateway and Golden Gate/Braid offer efficiency but have drawbacks.
• Gateway cloning involves expensive enzymes and a two-step reaction (LR and BP).
• Golden Gate cloning utilizes specific restriction sites, leading to non-reusable components.

Purpose of the Study:

• To introduce the Brick into the Gateway (BiG) protocol, a novel, faster, and more economical cloning strategy.
• To combine the advantages of GoldenBraid assembly with Gateway LR recombination.
• To circumvent the limitations of existing cloning methods, specifically the cost of Gateway enzymes and the non-reusability of Golden Gate components.

Main Methods:

• PCR amplification of DNA fragments incorporating type IIS restriction sites for specific fusion.
• Design and assembly of GoldenBraid bricks containing Gateway attL recombination sites.
• Assembly of bricks using Golden Gate methodology to create an attL1-gene of interest-attL2 fragment for Gateway entry vector construction, followed by LR recombination into a destination vector.

Main Results:

• The BiG protocol successfully integrates GoldenBraid reusable modules with Gateway LR reactions.
• This strategy bypasses the need for the Gateway BP reaction, simplifying the cloning process.
• The method offers a more economical and efficient alternative to traditional Gateway and Golden Gate cloning.

Conclusions:

• The Brick into the Gateway (BiG) protocol presents a validated, cost-effective, and efficient DNA cloning strategy.
• BiG combines reusable modular components with established recombination technology for streamlined molecular cloning.
• This approach enhances accessibility and practicality for various molecular biology applications.