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Related Experiment Video

Updated: May 26, 2026

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Published on: February 5, 2021

Recombination-based DNA assembly and mutagenesis methods for metabolic engineering.

Xiquan Liang¹, Lansha Peng, Billyana Tsvetanova

¹Life Technologies Corporation, Carlsbad, CA, USA.

Methods in Molecular Biology (Clifton, N.J.)

|December 7, 2011

Summary

This study introduces novel protocols for seamless DNA assembly, efficiently joining DNA fragments of various sizes up to 100,000 base pairs using homologous recombination in vitro or in vivo.

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Last Updated: May 26, 2026

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Published on: February 5, 2021

Area of Science:

Molecular Biology
Synthetic Biology
Genetics

Background:

Growing interest in precise assembly of diverse DNA fragments, including chromosomes.
Limitations of current commercial DNA assembly solutions for extreme fragment sizes.
Need for flexible and efficient methods for gene expression and protein activity fine-tuning.

Purpose of the Study:

To present protocols for seamless, simultaneous, flexible, and highly efficient DNA assembly.
To enable assembly of DNA elements across a wide range of sizes (up to 100,000 base pairs).
To describe an enhanced site-directed mutagenesis protocol.

Main Methods:

Utilizing homologous recombination for DNA fragment assembly.
Protocols applicable both in vitro and within living cells.
Adaptable to DNA fragments with or without shared homology at their ends.

Main Results:

Demonstrated seamless, simultaneous, flexible, and highly efficient assembly of DNA elements.
Successful assembly of DNA fragments ranging widely in size.
An efficient site-directed mutagenesis protocol enhanced by homologous recombination.

Conclusions:

The developed protocols offer a versatile solution for complex DNA assembly tasks.
These methods overcome limitations of existing commercial solutions for large and small DNA elements.
The protocols facilitate precise control over gene expression and protein activity.