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Golden Gate Cloning

(Updated for Geneious Prime)

This tutorial was developed by: Dr Moreland Gibbs (Biomatters Ltd.)

Introduction

Golden Gate Background:

Golden Gate cloning is a strategy that allows 'single-tube' ordered assembly of a vector (Backbone) and one or more DNA fragments (Parts) into a single, usually circular, construct which is suitable for direct transformation of a bacterial host.

The Golden Gate method uses Type IIs restriction enzymes in combination with DNA ligase. Type IIS restriction enzymes cut DNA at a location adjacent to their non-palindromic recognition site. For example BsaI creates a 4 nucleotide 3'-recessed overhang adjacent to the recognition site (See Figure below). The overhang can comprise any nucleotides, which allows BsaI to create 256 unique overhangs.

The use of type IIS restriction sites allows for the design of PCR primers for production of Parts, that when digested with a Type IIS enzyme, will allow directional, ordered and scarless assembly of the Parts using DNA ligase.

Example of a DNA fragment with 2 BsaI sites. Digestion with BsaI releases a central fragment with unique 4 nucleotide overhangs and no longer contains a BsaI motif.

Golden Gate Parts may be linear or circular. Once you have your Parts "built", the in vitro assembly method involves combining the parts in equimolar concentrations, along with a suitable type IIS enzyme and DNA ligase, then cycling between a temperature that favours restriction enzyme activity, and a temperature that favours ligation. This cycling promotes ordered assembly and ligation of the Parts and backbone into a single, usually circular, fragment.

Golden Gate in Geneious: The Basics

Type IIS site selection

Prior to using the tool you should decide which type IIS restriction enzyme you are going to use. The following rules apply regardless of the type IIS enzyme selected for assembly. Usually the "receiving" type IIS sites already present in your backbone will define the site you will use. Note that the Geneious Golden gate tool currently does not allow the use of multiple type IIS restriction enzymes for assembly.

Assumptions made by Geneious

Geneious will analyse your backbone (if defined), and each sequence passed to it, and will detect existing type IIS restriction sites, overhang annotations, primer_bind annotations and blunt ends. Geneious will then choose one, or a combination of these, in order of precedence (see rules 1 to 6 below) to define the insert boundaries to be used for Golden gate recombination. If required, Geneious will then design a primer pair for PCR amplification of each Part.

Rules, in order of precedence:

1. Existing type IIS cut site(s)

2. Existing Overhang(s)

3. Existing primer_bind annotation(s) with valid type IIS cut site(s) on the extension

4. Existing primer_bind annotations with extension

5. Existing primer_bind annotation(s) without extension(s)

6. Blunt ends

Important

Overriding the default use of primer_bind annotations as Part fusion points (Rules 4 & 5)

You have the option to ignore or choose alternate primer_bind annotations associated with each Part.  See Part 2 of this exercise, section Checking the correct primer_bind boundaries are used, for information on how to do this.

Removal of unwanted internal Type IIS sites

If one or more of your sequences contain the specified type IIS restriction site/s then Geneious will assume you want to use the site/s in the assembly process and design a strategy accordingly.

If one or more of your sequences contain type IIS restriction sites that you do not want be involved in the assembly then you will need to engineer each site out of your fragment, taking care to avoid altering any gene product sequences.


The following link takes you to an exercise using the Golden Gate tool.

Exercise: Golden Gate Exercise

FAQ: Frequently (and not so frequently) Asked Questions