Running homology assemblies with the combinatorial assembly tool

Shelby
Shelby
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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 homology  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.

 

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.

 

Homology details

Use homology modeling  to join fragments using homology regions, or complementary overhangs. This can be used to model, Yeast recombination cloning, Gibson, HiFi, and/or In-Fusion cloning, where fragments are modeled as having been PCR-amplified and include the homology regions. As well as Gateway cloning, where all fragments already contain ATT regions

 

Assembly-wide parameters

The only assembly-wide parameter specific to homology is ambiguous construct preference. This preference determines which fragment in the first bin to use where the sequences being assembled: 

  • Are circular and creating a circular construct, where the reaction can produce one plasmid of interest and a plasmid byproduct. 
  • Could create two distinct constructs

Across all sequences in the first bin, you can choose to use the larger fragment or the smaller fragment. This parameter provides a consistent way to choose the plasmid to create as a sequence.

 

Running an assembly requires several steps:

  • Set up the assembly
  • Configure bins
  • Add fragments 
  • Validate and visualize Constructs 
  • Save the assembly

Set up the assembly

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

  • Name - a name for the assembly object
  • Location - the folder where the assembly object/record will be stored
    • which can be changed/moved only after an assembly is finalized
  • Number of fragment bins - the total number of backbone + inserts in the assembly
  • Cloning method - Homology
    • Note this cannot be changed after the assembly has been saved
  • Ambiguous Construct Preferences - specific parameters

After reviewing the settings, click Save to start the assembly.

Screenshot 2023-09-06 at 11.21.44 AM.png

Configuring bins

An assembly will open in a new tab. At the top, you can add bins, drag bins around, or edit them. For example, you can change the bin names or, in some cases, settings. 

Adding fragments 

Unlike other cloning methods, you don’t need to specify a start and end position for the fragments. The entire sequence is selected by default and that selection cannot be edited. 

There is no start or end column in the fragments table. This is because Benchling scans each fragment to find homology regions that are compatible with its adjacent fragments, which may be:

  • Anywhere in the fragment. They don’t need to be at either end.
  • Different across two constructs at the same junction for the same fragment.

 

Add fragments, or pieces of DNA to be used in the assembly. To do this:

  1. Click + on a fragment bin.
  2. Enter the sequence entity name  into the Fragments table.

From the fragments table, you can:

  • Edit the table cells directly.
  • Access the modal to view and interact with the sequence by right-clicking the row number and then clicking Edit fragment.

If any fragments produce a warning or are invalid, error messages display in the Status column:

  • Looks good  - proceed to use these fragments in their assembly
  • Warning - proceed to use these fragments in their assembly, but we’ve identified a risk For example, the fragment contains >2 cut sites
  • Invalid - cannot use these fragments in their assembly until the error is resolved

Validating constructs

Benchling identifies and takes the longest homology region across two adjacent fragments, which can be anywhere within the selected fragment. The start and end coordinates of each fragment for each construct are reported in their respective columns of the construct tables. Unlike other cloning methods, the start and end of each fragment can vary by construct because Benchling detects junction-specific homology regions. Overlap length at each junction is also reported in the construct table. Start and end coordinates and overlap length can be hidden by clicking in the top-right corner of the constructs table. 

Visualize constructs

On each expanded construct, users can visualize the homology regions on the sequence maps with striped colored lines. These regions can be resolved down to individual bases.

Save the assembly 

When you’re ready to finalize the assembly and have resolved all blocking errors, click Assemble in the top-right corner. You can add constructs and primers, where relevant, to folders and/or worklists. Finalizing an assembly does the following:

  • Locks the record of the assembly
  • Creates new DNA sequences for each of the constructs
  • Creates new primers (DNA oligos) where relevant
  • Populates the tables with chips where newly created sequence entities are referenced

You can access the finalized assembly from the project folder it was saved to.





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