TP25_GoldenGateCloning\TP25_GoldenGateCloning
Golden Gate cloning
Golden Gate is a popular method for joining two or more DNA fragments together. This method is probably one of the less expensive ones, since only short DNA oligonucleotides are needed comparing to methods like Gibson assembly or homologous recombination. It is also a practical way to make combinatorial assemblies of genes.
Typically, only one type IIs restriction enzyme is used (Figure 1), cutting on one side of the recognition sequence. The entire reaction is usually made in one test tube (one pot assembly), saving time and work.
The restriction enzyme BsaI (Figure 2) is often used in Golden Gate cloning. The cut site differs with the orientation as for all IIs enzymes.
A simple Golden Gate strategy is depicted in Figure 3. A plasmid (Destination vector) contains two cut sites for BsaI (Figure 2). There is an insert molecule (blue) that also has two cut sites. Take careful note of the direction of each site. The cut sites are shown in brown and green. These are the sequences producing the stycky-ends. In the vector, the actual recognition sites are present in the short gray part. In the insert, the recognition sites are present in the gray flanking sequences.
The DNA sequences of the two molecules shown in Figure 3 are available in the two files “Destination_vector.gb“ and “insert.gb” in the folder of this file. Figure 4 and Figure 5 show both sequences open in ApE. The BsaI sites are shown in UPPERCASE without color while the rest of the sequnce is in lowercase. The brown and the light green sequences are the cut sites (Figure 3).
By copying and pasting of the appropriate text from Figure 5 and pasting it into the Destination vector (Figure 4), we can simulate the outcome of the Golden Gate experiment (Figure 6).
The size of the resulting vector is 1558 bp and the cSEGUID checksum should be vEV8QVHMfaId332lBo_l1YmGbMw. This sequence is available in the file “result.gb“.
Note that the BsaI recognition site is not present in the final DNA molecule. Look at Figure 7 for a detailed view of the process.
There are two files called “Destination_vector2.gb“ and “insert2.gb” in the folder of this file. They contain two sequences similar, but not identical to the examples shown previously.
Use these to replicate the example using the same principles. The size of the resulting molecule should be 1561 bp and the first five characters of the cSEGUID should be 7eGKs.
What are the last five characters of the cSEGUID checksum?
Since restriction cut sites are almost never present where we need them, we usually have to introduce them by PCR.
This is done by adding the desired nucleotides to the 5’ end of primers for the target insert, much in the same way as for normal type II restriction enzymes (see TP5). The only difference is that you alos have to design the sequence of the cut sites (taat and gaac in Figure 7).
These two YouTube videos has a nice introduction to Golden Gate cloning. Click on the images to watch:
ApE has a Golden Gate assembler that can both design and assemble automatically. If you open at least two ApE windows simultaneously, you can select Tools>Golden Gate Assembler (Figure 8). Using this tool is optional.
Select the BsaI restriction enzyme and click “ok” (Figure 9).
The resulting sequence can be seen in Figure 10.
This is an individual question for each student. Follow this link that points to a Google Spreadsheet. Find your name in the leftmost column.
The column called geneZ contains a DNA sequence that represents a double stranded linear DNA molecule that is also an open reading frame.
Your task is to design two primers (fp and rp) for geneZ that will amplify the entire sequence. The primers should anneal with the template with a length of 18-22 bp.
Each primer should contain one recognition site for the BsaI restriction enzyme. The restriction sites should produce overhangs compatible with the pGG golden gate vector. The pGG vector can also be found in the spreadsheet.
Put your results in the indicated cells for forward primer (fp), reverse primer (rp). Put the sequence of the resulting PCR product in the PCR product cell. Put the resulting vector in the pGG_geneZ column.
Please answer with raw DNA sequences as indicated for the first example student "Max Maximus".