Gateway Cloning Tutorial: Simulating Gateway multisite cloning

The files for this exercise are provided in a subfolder called Multisite Gateway cloning. Select this folder to see the example files required for this exercise in the Document Table.

As part of the work described by Peterson and Stowers (2011), a Gateway multisite reaction was used to place the yeast GAL4 CDS between two regulatory elements derived from the D. melanogaster trpA1 gene. The final Gateway construct was then integrated into into the D. melanogaster genome to demonstrate that the trpA1 regulatory elements upregulated GAL4 expression, and consequently UAS-GFP (green fluorescent protein) expression, in larval sensory neurons.

Confocal image showing trpA1-GAL4 driven expression of GFP in D. melanogaster sensory neurons. Image taken from Figure 4 of Peterson and Stowers (2011).


In this exercise we will simulate the work of Peterson and Stower and use the Geneious Gateway tool to perform a Gateway multisite reaction to combine separate upstream and downstream D. melanogaster trpA1 regulatory elements (trpA1 UP and trpA1 Down) with the yeast GAL4 transcriptional activator CDS and with the D. melanogaster-specific destination vector pDESTHaw.

The required part order (with att sites used) is depicted below:

In this exercise we will:

  1. Design primers for PCR-mediated addition of unique attB sites to each of the trpA1 UPstream, GAL4, and trpA1 DOWNstream parts. Primer binding regions will be designed to have a predicted Tm of around 55°C.
  2. Create individual entry clones for each part using specific multisite pDONR vectors.
  3. Recombine the 3 entry clones with the destination vector pDESTHaw, using the Gateway cloning tool, to create a sequence suitable for PhiC31 integrase (attB)-mediated integration into the genomes of D. melanogaster embryos.

Part 1: Primer design and PCR

Step 1: Primer design for trpA1 5' regulatory element (trpA1 UP)

The trpA1 5' regulatory element will be the first part of a 3 part reaction. As per the table in the Multipart Overview we need to use PCR to add flanking attB1 and attB4 sites to the first trpA1 UP sequence.

The 5'-terminus of the trpA1 UP sequence contains a short palindromic motif that results in primers designed across this region having a high predicted Hairpin Tm. To successfully design PCR primers to amplify this sequence using the Design new Primers tool we will need to adjust the default Primer Design settings.

Select the trpA1 UP sequence, click the Primers button in the Toolbar and choose Design new Primers.

Use the settings shown below. Make sure the Max dimer Tm setting is high (60°C) to ensure the forward primer design does not fail due to the presence of the palindromic motif.

To add Gateway extensions to the primers, expand the Advanced section of the Setup window, then click on the 5' Extensions: Fwd: button, click the Reset defaults to remove any previous settings, then click the Gateway site... button and from the dropdown menu choose Site: attB1.

Click OK, and you will see that the extension will comprise 4 G nucleotides and an attB1 site.

Click the 5' Extensions Rev: button, click the Reset to Defaults button to clear existing settings, then click the Gateway site button and add an antisense AttB4 site.

Click OK and OK again to run the Primer Design tool and new forward and reverse primers should be added to the sequence. Hit Save.

Now select the trpA1 UP fragment and go Primers -> Extract PCR Product to generate the PCR product amplified by the new primers. This PCR product will be used in the next section for recombination with the entry vector pDONR221 P1-P4.


Step 2: Primer design for GAL4 CDS sequence

The GAL4 CDS will be the second part of the 3 part multisite reaction. As per the table in the Multipart Overview we need to add flanking attb4r and attb3r sites to the GAL4 sequence.

Select the GAL4 sequence file, and click the Primers button in the Toolbar and choose Design new Primers.

Perform the same steps as in Step 1, this time adding a sense attB4r site to the forward primer and an antisense attB3r to the reverse primer.

The GAl4 sequence contains a 7 nucleotide polyA-region at it's 3' end in the region where a primer will be designed. To prevent the Primer Design tool failing due to the poly-A region set the Max Poly-X value to 7.

Once primers are added to the GAL4 sequence, select and go Primers -> Extract PCR Product to generate the PCR product amplified by the new primers. This PCR product will be used in the next section for recombination with the entry vector pDONR221 P4r-P3r.


Step 3: Primer design for trpA1 3' regulatory element (trpA1 DOWN)

The trpA1 DOWN 3' regulatory region will be the third part of a 3 part multisite reaction. As per the table in the Multipart Overview we need to add flanking attB3 and attB2 sites to the trpA1 DOWN sequence.

Select the trpA1 DOWN sequence file, and click the Primers button in the Toolbar and choose Design new Primers.

Perform the same steps as those in step 1, this time adding a Sense attB3 site to the forward primer and an antisense attB2 to the reverse primer.

Once primers are added to the trpA1 DOWN sequence, select and go Primers -> Extract PCR Product to generate the PCR product amplified by the new primers. This PCR product will be used in the next section for recombination with the entry vector pDONR221 P3-P2.

You should now have three PCR products ready for integration into donor vectors. use the link below to step to Exercise 5.


General Overview: Overview of tutorial
Exercise 1: Designing primers for Gateway cloning
Exercise 2: Simulating an "BP" Entry clone reaction
Exercise 3: Simulating a "LR" Destination clone reaction<
Multisite Overview: Overview of Gateway multisite cloning
Exercise 4: Multisite Part 1: Primer design and PCR
Exercise 5: Multisite Part 2: Creation of Entry and Destination vectors
Gateway Vectors: Preparing Gateway vector sequences

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