Golden Gate cloning is a form of restriction cloning that takes advantage of type IIS restriction enzymes. Type IIS restriction enzymes cleave outside of their recognition sites, leaving fragments with flanking overhangs whose sequence is unrelated to the recognition site. This type of restriction digest provides two key advantages through careful design of the backbone and inserts containing the gene of interest.
First, it allows for custom overhangs’ design, which can be done using PCR amplification. The complementary base pairing between the fragment overhangs is used to direct the orientation and assembly of the backbone and each fragment. By designing fragments with overhangs that complement only one other overhang, many fragments can be included in Golden Gate cloning simultaneously.
And second, after digestion, the desired fragment will not contain the enzyme recognition site, allowing for an irreversible ligation between the fragments. The cut sites for the enzymes exist outside the recognition site, so the resulting desired backbone fragment will not contain the recognition sites. The backbone, also called the destination vector, is designed so the type IIS restriction sites surround the area where the gene(s) of interest will be incorporated.
When designing the inserts, type IIS restriction sites surround the gene of interest. In this case, the cut sites exist between the gene of interest and the recognition sites so that after digestion, the fragment containing the gene of interest will not include the recognition sites. Through digestion and ligation of the fragments, these advantages allow us to use Golden Gate cloning to produce a directional and scarless final vector between a backbone and many genes of interest at once.
Procedure
With Mendelgen, performing a simulation of Golden Gate cloning is simple. Simply, follow the steps outlined below. Here are the dna files in case you want to follow along.
1. Open Mendelgen, https://mendelgen.com and sign up or in.
2. Select a vector from your library as the backbone, also called the destination vector.
The destination vector must already be designed with type IIS restriction sites.When the page opens, the destination vector will be shown in the viewer.
3. Click the cloning tool button, displayed as a circular DNA sequence, in the toolbar at the top of the page.
4. Select ‘Golden Gate Cloning’ in the dropdown.
- A tab will open to the right of the viewer titled ‘Create a Golden Gate Cloning Vector’.
- The cloning tool clicked with Golden Gate Cloning highlighted, and the cloning tab open.
5. The first step is already complete and shows that the destination vector is the sequence displayed in the viewer.
6. Upload/Enter the insert sequence(s) containing the gene(s) of interest. (‘From My Library’ is preselected, but you can also choose ‘From Clipboard’.)
'From My Library' lets you upload a sequence from your library.- Choose a sequence from the dropdown, and it will automatically be added under 'Insert Sequence(s)'.- You can view this sequence in a new tab by clicking the sequence name once it has been added to 'Insert Sequence(s)'.
'From Clipboard' lets you enter a new sequence.1. Enter the Sequence Name.2. Enter the full sequence.3. Click 'Add Sequence' and it will be under 'Insert Sequence(s)'.4. 'From Clipboard' chosen to enter a new sequence as the insert.
- Up to 52 sequences can be added at a time.
- Each sequence must already be designed with the type IIS restriction sites.
- When uploading many sequences, don’t worry about the order in which you enter them. Using the custom overhangs in your designed sequences, we will ensure they are added to the destination vector in the correct order
7. Check the correct insert(s) have been added under ‘Insert Sequence(s)’
- If you want to remove one of the inserts simply click the ‘X’ to the right of the sequence name.
- Many inserts listed under ‘Insert Sequence(s)’.
8. Select the type IIS restriction enzyme(s).
- Choose all enzymes that will be used to digest the destination vector and the inserts.
- Select Enzyme dropdown open to choose the type IIS restriction enzyme.
9. Click the ‘Clone’ button.
- You will not be able to click ‘Clone’ if any errors exist.
Errors and their descriptions.
i. At least one insert must be added.
ii. At least one enzyme is needed.
iii. The backbone must be cut twice. (Every sequence, backbone or insert, must be cut twice.)
iv. The enzyme (enzyme name will be provided) cutting the backbone does not result in a flanking overhang. (Every sequence, backbone or insert, must have a flanking overhang after being cut.)
v. The insert (insert name will be provided) must be cut twice (Every sequence, backbone, or insert must be cut twice)
vi. The enzyme (enzyme name will be provided) cutting the insert, insert name will be provided, does not result in a flanking overhang. (Every sequence, backbone or insert, must have a flanking overhang after being cut.)
vii. The backbone is constructed incorrectly.
The backbone must be constructed in the following way:
1. Surround the area where inserts will be incorporated with type IIS restriction sites, with the cut site occurring outside the recognition sites. 2. CUT_SITE - RECOGNITION_SITE - (INSERTS WILL BE ADDED) - RECOGNITION_SITE - CUT_SITE3. An error showing the backbone isn't constructed correctly is shown.)4. An error showing the backbone isn't constructed correctly is shown.
viii. The insert (insert name will be provided) has not been constructed correctly.
1. The inserts must be constructed in the following way.2. Surround the gene of interest with type IIS restriction sites, with the recognition site occurring outside the cleavage site.3. RECOGNITION_SITE — -CUT_SITE — — GENE_OF_INTEREST — — CUT_SITE — -RECOGNITION_SITE4. An error showing the insert isn’t constructed correctly is shown.
ix. The overhang (overhang sequence provided) occurs in more than 2 sequences.
1. The overhangs, through complementary base pairing, are used to define the order of insert fragments in the resulting cloning vector. If the overhang occurs in more than two sequences then the order of fragments in the resulting cloning vector cannot be determined.2. An error showing the overhang occurs more than twice is shown.
x. The overhang (overhang sequence provided) does not have a match.
1. The overhangs, through complementary base parings, are used to define the order of insert fragments in the resulting cloning vector. If an overhang does not have a match, the sequence cannot be incorporated into the resulting cloning vector2. An error showing the overhang has no match is shown.
10. Bazinga! The final resulting vector will be displayed in the viewer.
- We have simulated the digestion and ligation behind the scenes and produced your final vector.
- Finished cloning simulation with many inserts
Are you looking to simulate another cloning method? Mendelgen supports cloning methods like Restriction, TA, Gibson Assembly, Gateway, and Golden Gate.
Have feedback? Let us know in the comments below.
