Biomedical Engineering Reference
In-Depth Information
Chapter 9
Golden Gate Cloning
Carola Engler and Sylvestre Marillonnet
Abstract
DNA assembly methods are essential tools for biological research and biotechnology. Therefore various
methods have been developed to clone DNA fragments of interest. Conventional methods usually require
several cloning steps to generate a construct of interest. At each step, a single DNA fragment is transferred
from a donor plasmid or PCR product to a recipient vector. In the past few years, a number of methods
have been developed to facilitate and speed up this process. One of these methods, Golden Gate cloning,
allows assembling up to nine fragments at a time in a recipient plasmid. Cloning is performed by pipetting
in a single tube all plasmid donors, the recipient vector, a type IIS restriction enzyme and ligase, and incu-
bating the mix in a thermal cycler. Despite the simplicity of the cloning procedure, the majority of clones
obtained after transformation contain the expected construct. Using Golden Gate cloning however
requires the use of carefully designed donor and recipient plasmids. We provide here a protocol describing how
to design these plasmids and also describe the conditions necessary to perform the assembly reaction.
Key words
DNA assembly, DNA shuffl ing, Combinatorial, Hierarchical, Type IIS restriction
enzymes, Seamless cloning, Modular cloning, Synthetic biology
1
Introduction
In the past few years several methods have been developed to
allow assembly of multiple DNA fragments in a single cloning step
[
1
-
7
]. Most of these methods are based on recombination between
homologous sequences present at the ends of the DNA fragments
to assemble. These methods have the advantage of allowing
seamless assembly of any sequence of choice irrespective of the
presence of restriction enzyme recognition sites. A limitation is
however the need for overlapping sequences of at least 15 nucleo-
tides at the ends of the fragments. Assembly of non-overlapping
DNA fragments therefore requires adding terminal extensions or
using bridging oligonucleotides [
7
]. This is a limiting factor for
combinatorial assembly of multiple DNA fragments of interest
since a correspondingly large number of bridging oligonucleotides
will be required.
