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Golden Gate Cloning Tutorial
Note: To complete the tutorial with the referenced data please download and install the tutorial above into Geneious Prime.
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 Prime: 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)
- 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.
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: Assembly of 6 Parts into a vector
In this exercise we will use the Geneious Golden Gate tool to assemble six sequences and recombine them with a vector “backbone” to create a circular construct.
This exercise has been devised to demonstrate how the rules outlined in the introductory section of this tutorial are implemented, and also, to demonstrate the general procedure for use of the Golden Gate tool.
The six sequences for assembly comprise portions of the green fluorescent protein (GFP) coding sequence (CDS). The various sequences have fusion boundaries defined by existing type IIS sites, primer_bind annotations (with and without extensions) and by blunt ends.
If the assembly goes to plan, Golden Gate-mediated recombination of the six sequences will regenerate a complete CDS encoding the GFP and insert it into a vector “backbone”.
In this exercise we will be using the vector pGoldenGate-SE7. This vector contains two BsaI sites with unique overhangs, that will be used to “receive” our six-Part insert. This vector sequence is provided with this tutorial, click on the above link to select and view the vector sequence.
The six insert fragments, labelled GFP1 to GFP6 are also provided with this tutorial.
Stage 1 – Checking the sequences
With the six insert sequences selected and visible in the sequence viewer, select the Annotations and Tracks tab and ensure ORF, primer_bind and Restriction Site annotations are displayed.
You will see each fragment is annotated with an ORF annotation which defines a region corresponding to a portion of the GFP CDS. The ORF annotations correspond precisely to the regions we wish to assemble. Preexisting BsaI sites are also visible as well as a number of primer_bind annotations.
You will notice that the first sequence, GFP1, is a blunt fragment with a central primer_bind annotation named A Primer. We wish to assemble this entire sequence. However, Geneious will interpret the primer_bind annotation as a fusion boundary (as per Rules 3-6 described in the Tutorial Introduction). We will take steps, described in the next section to ensure Geneious does not use this primer_bind annotation as a boundary.
At this point we should also confirm that none of the sequences contain unwanted internal BsaI sites. Click on the Restriction Analysis tab, select the Type IIS subset of enzymes, then click on Advanced and select BsaI and double check there are no unexpected internal BsaI sites in the six sequences.
Stage 2 – Performing the Assembly
With your backbone vector and six sequences selected, on the Tool bar click Cloning → Golden Gate… to start the Golden Gate tool. This will open the Golden Gate Window.
If not already selected, set the Enzyme: to BsaI.
We wish to use the pGoldengate-SE7 vector as our backbone. By default, Geneious Prime should set the circular sequence as the left most sequence, and set Backbone: to Use Leftmost. If the pGoldenGate-SE7 sequence is not leftmost, then select and drag it to the leftmost position. Alternately, you can use the Backbone: dropdown menu to specify that pGoldengate-SE7 should be the backbone You can also use the Choose… button to navigate to this tutorial folder and select the pGoldengate-SE7 vector sequence as the backbone.
Each sequence is shown as a boxed “Tag” that represents a Golden Gate Part with BsaI-generated overhangs.
If your Parts are in a different order to that shown in the figure above, drag and drop the Tags to rearrange them to the correct order.
If the BsaI sites in the Parts are preexisting, such as the two in the backbone, the Tag will be labelled with Cut by BsaI, or Enzyme. Parts that require design of a primer pair will be labelled PCR product or Primer. Each Tag also provides information on the range of the original sequence that will be incorporated into the final Golden Gate construct.
If preexisting type IIS sites on your fragments are incompatible with sites on adjacent fragments then Geneious will highlight the Tag red and the offending overhangs will also be red. For example, if you switch the order of fragments GFP1 and GFP2, you will see that the Backbone and GFP2 fragments turn red and are labelled Mismatched. This is because the GFP2 sequence has preexisting BsaI sites that are not compatible with the backbone BsaI sites.
Drag and drop to place the GFP1 and GFP2 Tags back in the correct order.
Checking the correct primer_bind boundaries are used
Each part has a dropdown menu accessible via an inverted triangle. This menu can be used to check, and if required, change whether existing primer_bind annotations are used as boundaries. The GFP1 sequence provided for this tutorial contains a primer_bind annotation called “A primer“. Unless told otherwise, Geneious will assume this annotation defines a Part boundary. In this case we want Geneious to ignore this annotation. To do this, click on the triangle on the GFP1 Part and change the “forward” boundary from A primer to Design at 5′ end.
The Parts drop-down menu also provides an option to Reverse Complement Parts that are in the wrong orientation. You also have the option to Reset reaction if you have previously specified non-default boundary options for the Part.
All of the Parts in this exercise, with the exception of the Backbone and GFP2, will require primer design and PCR in order to generate DNA suitable for the Golden Gate reaction. Therefore, make sure the option to Save used Primers is checked. This will provide you with a file for each primer sequence designed by the tool. These sequences can then be submitted to an oligonucleotide synthesis company.
Uncheck the Save intermediate products option as we do not need to see intermediate products.
Finally, we will save our results to the same folder so make sure the option to Save in sub-folder is unchecked.
For more information on the various options available in the Golden Gate window, you can click on the Help button in the bottom left corner of the window.
Completing the Golden Gate Assembly
Press OK to run Golden Gate.
The Geneious Golden Gate tool will then complete the analysis and save a circular construct that represents your assembled sequences, plus the primer sequences required to create the Parts.
Stage 3 – Checking the Assembly
Select the newly created sequence named pGoldenGate-SE7 – GFP1 – GFP2 and 4 other sequences and view it in the Sequence View panel. In the Annotations and Tracks tab ensure the Concatenated Sequences annotation type is checked. To reduce the screen clutter turn off display of Primer Bind and Ligation annotations.
To confirm that our six fragments have recombined precisely to reform the GFP CDS We will define a CDS annotation for the region and examine the gene product.
To do this, select the Ligation of GFP1 – GFP2 – GFP3 – GFP4 – GFP5 – GFP6 into pGoldenGate-SE7 file.
Select the GFP1 ORF annotation (in orange), then hold down shift and select the GFP6 ORF annotation (in orange). This should select the range 2679 to 3395. Hit the “Zoom to Selection” button to zoom in on this region.
Then, click Add Annotation, and define a new annotation for this range called GFP Reassembled of Type: CDS, and click ok.
A new yellow CDS annotation should appear. Click on the yellow GFP reassembled annotation and hover over it to bring up a yellow tool tip showing the details of the Annotation. You should see the CDS translates to the complete GFP gene product starting MRK…, ending …LYK*, with no internal stop codons.
If you wish, you can extract the CDS to a file and perform a pairwise alignment between the GFP reassembled sequence, and the original GFP sequence , which has also been provided with this tutorial. The alignment will show you that the reassembled sequence is identical to the original GFP sequence.
Q. Can the Geneious Golden gate tool consider multiple type IIS restriction enzymes?
A. No. The Geneious Golden gate tool can only use a single type IIS enzyme.
Q. Why aren’t all of the primers designed by the Golden Gate tool annotated onto my final Golden Gate construct?
A. The Golden Gate tool will only map the primer_bind annotations to the output, if the primer_bind annotation matches the sequence 100%. In some cases, for instance when a preexisting type IIS site is utilised, the primer bind region will lie external to the fragment that is assembled, and so will be removed by digestion, and not be present in the final assembly.
Q. Does the Geneious Golden Gate tool exclude adding palindromic overhangs?
A. The Golden Gate tool currently does not exclude using palindromic overhangs.
Q. Does the Geneious Golden Gate tool exclude using overhang combinations where the same three or more consecutive nucleotides are present in another overhang used in the assembly?
A. The Golden Gate tool currently does not check for similarity between overhangs.
Q. Will the Autoarrange button sort based on sequence names?
A. No. The Autoarrange option will try to identify a unique sorting of the sequences based on the available overhangs generated via preexisting sites in your Parts. It does not sort based on sequence name.
Q. I want to have my part synthesized for Golden Gate rather than do PCR – what should I do?
A. Check the option to Save Intermediate products. This will create a PCR product sequence for each Part. Submit this PCR product sequence to you custom synthesis provider of choice. The example below shows the GFP5 PCR product Part that would be generated during the exercise provided in this tutorial.
Q. What criteria are used to design the primer_bind regions of the primers output by the Golden Gate tool? Will they all have similar melting temperatures in the initial rounds of PCR?
A. The Geneious Golden Gate tool uses fairly relaxed settings when designing Golden Gate primers to ensure a primer is designed. The settings used are shown below.
If you wish to design your Golden Gate primers with your own specific primer bind parameters, then you should design and add your own flanking primer_bind annotations to your sequences prior to performing assembly. This will force Geneious to use the annotations as Part boundaries.