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Combinatorial Golden Gate DNA Assembly – Part 1
Combinatorial Golden Gate DNA Assembly – Part 1

Build a small combinatorial DNA library of 8 constructs using the Type II endonuclease (Golden Gate) assembly method

Michael Fero PhD avatar
Written by Michael Fero PhD
Updated over a week ago

Let's build a small DNA library. We will use the combinatorial design view of the Design Editor whiteboard to make the design simple and compact. 

We want to divide each construct into the following components:

  1. Vector backbone

  2. N-terminal Signal Peptide (2 variants)

  3. Gly-Ser Linker (2 variants)

  4. GFP (1 variant)

  5. ssrA 5-prime degradation tag (2 variants)

  6. ssrA 3-prime degradation tag (1 variant)

We will start by creating a new design called Small DNA Library. From the main page, click Designs → Designs → New Design → Blank Design.

Save the new design as "Small DNA Library". 

After saving the design, the design editor will look like the figure below (minus the colored arrows and outlined boxes). 

To add a new component column, click on the plus sign inside the blue box. To add a new variant row, click on the plus sign inside the red box. Details of each bin (or component) can be viewed or modified by clicking the bin icon on the inspector panel on the right. Since each construct has 6 components, let’s add 4 more bins to the current design. In addition, the 2nd, 3rd, and 5th parts each have 2 variants, therefore, we want to add one more row to the current design. Doing so, our combinatorial design now looks like this. 

TeselaGen design editor has a variety of symbols representing different genetic elements that could accommodate the needs of visualizing complex DNA constructs. In this particular design, the 2nd, 3rd, and 4th components are coding sequences; hence, it makes sense to use a “CDS” (coding sequence) icon for these components. For the last two components, we can use a “Protein Stability Element” icon. To change a bin’s symbol, click on that bin and choose a symbol from the list of SBOL icons above. (If you do not have the SBOL Icons at the top, click on View and then Show SBOL Icon Tool) In addition, we can change the name of each component to a more descriptive name by changing its name in the bin’s detail tab. The sketch of our design now looks like the figure below.

This finishes the sketch of our design. Now, let's assign the actual DNA parts. From our DNA Parts Library, we just need to assign the DNA to cells under the proper column headings. Do this by double-clicking a cell you want to assign a DNA part to, the GFP cell for example. A dialog containing the DNA parts will open up, which allows us to search for "GFP", find the DNA part we are looking for, and assign it with a double-click or by hitting the OK button at the lower right corner of the dialog.

For the components with variants (e.g. 2nd, 3rd, and 5th parts), assign the variants to the cells of the same column but on different rows. For example, for the N-terminal signal peptide, the DNA part could be either a BMC_nterm_sig_pep or a ccmN_nterm_sig_pep. Following these rules to assign DNA parts to all the cells in the design, we obtain something like the figure below. Now that all the cells have been assigned a part, the “Submit for Assembly” button turns green and becomes enabled. 

Now, here is a trick. It turns out that these little degradation tags are pretty short. We need to do some PCR to pull some of our desired DNA out of their host vectors, so why not embed these little guys into the forward and reverse primers? We can tell the assembler to do this by assigning a "Forced Assembly Strategy". You can select the forced assembly strategy options for any part by selecting the part of interest, then select the part icon on the inspector panel on the right to view the part’s details. For our example, let’s click on the "ssrA_tag_3prime" cell, then pick "Embed in primer forward" from the "Forced Assembly Strategy" dropdown menu. For the two DNA part options in the ssrA_5primeTag column, pick "Embed in primer reverse". The design editor uses colored markers to indicate different assembly strategies. In our example, the “Embed in primer forward” has the green color bar, while “Embed in primer reverse” has the purple color bar. These colors add a layer of information to the visualization of the design without overwhelming the users. 

Alternatively, you can right-click the part, and choose a strategy from the “Forced Assembly Strategy” menu as shown in the figure below.

We can add one more feature to this design. It turns out that some of these parts are contiguous in their hosts. The TeselaGen assembler is smart enough not to break everything apart, just to put it all back together again. However, the assembler will do a cost tradeoff between direct synthesis and PCR based on the cost of synthesis. To tell the assembler not to consider direct synthesis, you can impose a "Direct Synthesis Firewall" to a column. To do this, select a bin and click on the bin icon on the right panel to view its detail. Check the option for “Direct Synthesis Firewall”. You should see a red line appear on the right of the selected bin indicating the direct synthesis firewall (DSF) is in effect. In our example, we do this for columns 2 and 5. Our final design looks like the figure below.

Alternatively, you can achieve the same thing by the following operations: right-click a bin (or any parts of the bin) → Insert → Insert Direct Synthesis Firewall. 

Finally, you can modify the assembly method by selecting the method from the drop-down menu in the “Assembly Reaction Details” (click on the third-down icon from the right-side panel)

That is about as fancy as we need to get for this design. If you navigate to the green "Submit for Assembly" button at the upper right corner of the design editor,  you can run the assembler and build your library. Cool. We will cover interpreting the output from the assembler in part 2 of this tutorial.

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