Running Golden Gate assemblies with the combinatorial assembly tool

Sakura
Sakura
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Note: This feature is currently in limited availability. If you don’t have access to the new combinatorial assembly tool, contact Customer Support to request access.

 

You can model Golden Gate assemblies and design constructs in bulk using the combinatorial assembly tool. With this tool, you can create visual representations of constructs and their sequence features, like annotations, and focus on relevant information by creating only the needed fragment combinations. Select existing primers for your assembly and carry over annotations from your constructs in the final assembly. Then, save a record of the assembly, including the fragments and primers used, as an object in Benchling.

 

This article explains how to access the assembly tool and how to use it to run a Golden Gate assembly.

 

Running an assembly requires several steps:

  • Set up the assembly
  • Add sequences
  • Review and finalize your constructs

 

Note: The window must stay open and active while the assembly is in progress. In-progress assemblies aren’t saved if you close the window, navigate away from Benchling, or refresh the page. We recommend not leaving your computer during the assembly so the device doesn't sleep or hibernate.

Set up the assembly

In the cloning tool, enter information about your assembly in the modal:

  • Name: The assembly name
  • Location: The project and folder the assembly will be saved to
  • Number of fragment bins: The total number of backbone and inserts in the assembly
  • Topology of construct: Circular or linear  
  • Cloning method: Several cloning methods are supported at this time

Enter additional parameters specific to your assembly type at the bottom of the modal and click Save

 

Add sequences 

A new Assembly tab will open with Overview and Constructs nested tabs. Then create bins or spacers to add your sequences to. To create bins or spacers and add sequences:

  1. Click the + icon next to Bins & Spacers. Spacers can only be added next to bins where primers are used to amplify a fragment.
  2. Next to a bin, click the + icon and select your method for adding sequences. You can add open sequences, search for sequences using global search filters, or add sequences from a worklist. This opens a modal where you can interact with the sequence, linear, or plasmid maps.
  3. In the modal, confirm the start and end site you wish to use for the assembly and the orientation. Select preferred primers, if needed.
  4. Make adjustments to any other sequences in the bin, then click Add.
  5. Repeat the steps above for additional sequences.

 

 

Tip: You can add and edit sequences directly in the Fragments table.  View a sequence in the table by clicking the row number and clicking Edit fragment.

Using existing primer oligos 

If you’re performing an assembly with primers to model PCR amplification, you can use primers you've already created as oligos. You can search for and select oligos in the Fragments table which has two optional columns for 3' and 5' preferred primers. You can specify one forward and reverse primer for a given fragment. 

 

If you add preferred primers, Benchling tries to use them to amplify the fragment and model the cloning reaction. If you don't add any preferred primers, primers are designed for you where relevant.

 

If the preferred primers you selected can’t be used – for example, the primer doesn't bind your fragment, introduce the appropriate cut site, or meet your melting temperature parameters – new primers are designed as part of the assembly and a warning displays. In some cases, your preferred primers might be used in one construct and new primers are designed for another. You can review the status of your primers during confirmation of your constructs.

Review and finalize your constructs

After selecting your fragments, you will:

  1. Specify your constructs
  2. Confirm your construct and resolve errors
  3. Finalize and save your construct

 

Specify your constructs

 

You can specify your constructs two ways:

  • Manually add rows and select your combination of fragments from the Constructs table. 
  • Click Auto-populate in the Constructs table to generate all possible combinations of fragments. 

 

To delete constructs, right-click the row number in the Constructs table and select Delete construct. If any constructs produce a warning or are invalid, error messages display in the Status column. These need to be resolved before finalizing your construct.

 

Confirm your construct and resolve errors

 

After selecting your constructs in the table, click the Constructs tab to preview them. View the construct, its bases, and its annotations in more detail by clicking the diagonal arrows to expand the construct in a modal. If your assembly method involves primers, open the Primers tab to view the primers and their properties designed or used in the assembly. 



Finalize and save your construct

To finalize your constructs:

  1. Click Assemble in the top-right corner. 
  2. In the pop-up window, select a folder to save the constructs and primers associated with the assembly. 
  3. [Optional] Add components of the assembly to worklists. New DNA sequences and primers, where relevant, are created for each construct once finalized.
  4. Click Assemble in the pop-up window to save the files. You can access constructs and primers in the project or folder they were saved in and directly from the assembly, which is maintained as a record in Benchling. 






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