(Updated for Geneious Prime)
This tutorial was developed by: Dr Moreland Gibbs (Biomatters Ltd.)
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.
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)
If Geneious detects a pair of appropriately orientated type IIS sites with unique overhangs, then it will assume you wish to use them. Geneious will also assume that you have DNA available to use, and so will not design primers for PCR.
If only one type IIS cut site is detected, then Geneious will assume you wish to use it. A primer will be designed which incorporates the site. A second "opposite orientation" primer for PCR will be designed based on rules 3-6.
2. Existing Overhang(s)
If Geneious detects a pair of valid overhangs compatible with the specified type IIS site, then it will assume you wish to use them. Geneious will also assume that you have this "sticky ended" DNA available and so will not design primers for PCR.
3. Existing primer_bind annotation(s) with valid type IIS cut site(s) on the extension
If Geneious detects a pair of inward facing primer_bind annotations with valid compatible type IIS sites then it will assume you wish to use them. Geneious will assume that you already have the corresponding primers, and new primers will not be designed for the region.
If Geneious detects a single primer_bind annotation with a suitable type IIS site Geneious will assume you wish to use it and a new primer will not be designed. However, a second "opposite orientation" primer will be designed, based on the appropriate rule, for PCR.
4. Existing primer_bind annotations with extension
If Geneious detects a primer annotation with an extension which does not contain a valid type IIS site then the 5' terminus of the extension will be considered the fusion point and the extension will be extended to introduce a valid type IIS recognition site, resulting in a new primer sequence.
5. Existing primer_bind annotation(s) without extension(s)
If a primer_bind annotation without an extension is found, then an extension will be appended to introduce a valid type IIS recognition site, resulting in a new primer sequence.
6. Blunt ends
If Geneious finds a blunt end, and no suitable type IIS sites or primer_bind annotations are present, then a primer with an appropriate type IIS site extension will be designed. The fusion point will be the terminus of the blunt end fragment.
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