Running concatenation assemblies with the combinatorial assembly tool

Sakura
Sakura
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You can model concatenation 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. Then, save a record of the assembly, including the fragments used, as an object in Benchling.

This article explains how to run a concatenation assembly after opening the combinatorial assembly tool.

Learn more about modeling Gibson, Golden Gate, and homology based assemblies in our help center.

Run a concatenation assembly

Running an assembly requires several steps:

  1. Setting up the assembly
  2. Adding sequences
  3. Reviewing and finalizing your constructs

Set up the assembly

To set up the assembly:

  1. 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
    • Sequence type: Select whether you would like to work with DNA, RNA or Amino Acid sequence concatenation 
    • Output Type: Select the sequence type you wish to associate with the finalized constructs
    • Topology of constructs: Circular and linear topology are supported for DNA concatenation assemblies. Linear topology is supported for RNA and Amino Acid concatenation assemblies. 
    • Number of fragments bins: The total number of backbone and inserts in the assembly
  2. Click Save, and a new Assembly tab opens with Overview and Constructs nested tabs where you’ll add sequences.

Add sequences 

In the Assembly tab, create bins or constants to add your sequences to:

  1. Click the + icon next to Bins & Constants to create a new Bin or Constant. 
    • Constants are short residue regions that are unchanged in every construct
  2. Next to a bin, click the + icon and select your method for adding sequences:
    • Add open sequences
    • Add sequences from a worklist. This opens a pop-up window where you can interact with the sequence, linear, or plasmid maps
    • Search for sequences using global search filters
  3. Confirm the start and end sites to use for the assembly and the orientation.
  4. Make adjustments to any other sequences in the bin, then click Add.
  5. [Optional] 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.

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 must 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. 

Finalize and save your construct

To finalize your constructs:

  1. Click Concatenate in the top-right corner. 
  2. In the pop-up window, specify the folder to save your constructs in. Click Next.
    • [Optional] You can add constructs to worklists in this step 
  3. Click Finalize 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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