Golden Gate Cloning - DNA Cloning and Assembly Methods - page 118

original site will be redigested, allowing their components to be
made available for further ligation. This leads to increasing forma-
tion of the desired product with increasing time of incubation, and
results in a high cloning effi ciency. Since the sequence of the over-
hangs at the ends of the digested fragments can be chosen to be
any four nucleotide sequence of choice, multiple compatible DNA
fragments can be assembled in a defi ned linear order in a single
restriction-ligation step.
Golden Gate cloning can be used for gene shuffl ing and for
assembly of any construct of interest. To facilitate construct
engineering, a modular cloning system (MoClo) that uses Golden
Gate cloning for all assembly steps was developed [ 10 ]. The base
of the MoClo system consists of libraries of standard level 0
modules (for details, please refer to the Fig. 1 legend) that each
contains a defi ned genetic element such as a promoter, a 5 untrans-

lated region, and a coding sequence or a terminator (Fig. 1b).
Transcription units are assembled from level 0 modules using a fi rst
assembly reaction and multigene constructs assembled from tran-
scription units using a second cloning step. The process can be
repeated to generate constructs containing larger numbers of tran-
scription units. To be suitable for Golden Gate cloning for all
assembly steps, level 0 modules internal sequences should not
contain restriction sites for any of the type IIS enzymes used for
MoClo. Level 0 modules lacking type IIS restriction sites can be
cloned from native sequences using Golden Gate cloning.
We provide here a protocol for Golden Gate cloning, using as
example the construction of MoClo level 0 modules. The protocol
requires amplifying several fragments by PCR using primers
designed to mutate internal restriction sites, cloning the amplifi ed
fragments and sequencing them, and fi nally assembling the cloned
fragments in a compatible destination vector. The protocol pro-
vided here can also be used for gene shuffl ing or for combinatorial
assembly of various sequences of interest [ 11 ].

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2 Materials

1. Novagen KOD Hot Start DNA polymerase (Merck KGaA,
Darmstadt), supplied with 10× buffer, 25 mM MgSO 4 and
2 mM dNTPs, or any other high fi delity DNA polymerase.
2. Custom-made primers can be ordered from many commercial
vendors.
3. NucleoSpin ® Extract II kit (Macherey Nagel, Düren), for puri-
fi cation of PCR products.

2.1 PCR

1. Restriction endonuclease SmaI (10 U/ L) (NEB, New England

Biolabs, Inc., Ipswich, MA, USA), supplied with 10× NEBuffer
4 (200 mM Tris-Acetate pH 7.5, 100 mM magnesium acetate,
500 mM potassium acetate, 10 mM dithiothreitol).

μ 2.2 Cloning