Navigate to Bulk Assembly
To access the Bulk Assembly tool, navigate to the blue navigation tool bar on the left-hand side of the workspace. Click on the Global create (+ icon), hover over "DNA Sequence" and select "Bulk Assemble DNA."
The Bulk Assembly tool can be used to generate linear constructs or plasmids from various insert fragments and/or backbones, in bulk. Output sequences are created in a combinatorial fashion; where 1 backbones and 2 inserts are matched together to create 2 unique, output plasmids (picture below).
Assembling DNA in bulk involves 5 steps:
- Selecting output sequence topology: Linear or Circular
- Selecting files to work with: Backbone and inserts
- Selecting the direction: Forward or reverse
- Selecting fragment ends: Enzyme, Homology, Concatenation
- Selecting the fragment size: Larger or smaller
1. Select output sequence topology
Before selecting input sequences and assembly methods, you first have the option to designate the desired topology of the output sequences of Bulk Assembly. By default this is set to ˆCircular to enable bulk plasmid creation, but if you are interested in combinatorially producing linear constructs then you can simply toggle this to Linear.
2. Select files to work with
You have multiple options for selecting sequences to work with.
Selecting from folders:
Make sure that your backbone and insert fragments are saved in a folder(s) that you can easily recognize and navigate to. Once you've located your folder, you can designate each fragment(s) as your backbone and/or inserts.
To highlight all of the fragments at once, click on the folder icon on the left hand side of the folder name. To select a specific set of fragments, click on the individual fragments or shift-click for bulk selection of sequences/folders.
Selecting from Worklists:
Worklists allow you to more conveniently add sequences to a Bulk Assembly. To add sequences from a Worklist, first navigate to the Worklist tab, then click the Select Worklist button.
The Select Worklist button will open a modal that lets you search for existing Worklists of sequence entities.
Note: Clicking into the search field will surface recently edited Worklists.
After selecting a Worklist to use, all sequences that are part of that Worklist will automatically be selected as input files for your Bulk Assembly.
3. Select fragment orientation
Select the fragment orientation that you'd like.
4. Select fragment ends by assembly method
Scientists can choose a restriction enzyme that occurs exactly once in the sequence. The neighboring fragment must use a compatible enzyme on one of its ends.
Choose from a dropdown list of restriction enzymes. When selecting fragment ends for your insert, Benchling will automatically remember which restriction enzymes that were selected for the backbone and present them as options under the "Enzyme" list.
Scientists can also use a shared homologous region of neighboring fragments to combine them. Overlaps are used to orient the assembly of the fragments and are not duplicated. Select >7 base pairs as your homology region to assure that a non-specific region of your sequence isn't selected.
Users have 2 ways to input a homology region - manual entry or automatic homology detection. To use automatic homology detection, you must first designate input sequences in at least 2 adjacent insert slots. Once input sequences have been designated and you select Homology as your desired assembly method, you will see a Detect button appear in the Bases field.
Clicking the Detect button will bring up a modal that asks you to specify the expected length of the homology region present. If it is ambiguous which adjacent insert slot you wish to be joining, then you may also see a prompt for an intended Target Slot.
Note: If a homology region is already in use in another slot, then Automatic Homology Detection will not detect it. Instead, Benchling always remembers homology regions that were selected for other insert slots and presents them as options under the Bases input field.
If the homologous regions of two neighboring slots do not match exactly, you will see this error:
Users define the regions of the sequence entered in each slot, and concatenate those sequences together. Users define a region by either:
- searching for bases
- selecting the start/end of the sequence.
5. Select fragment size
Select the fragment piece that you'd like to assemble by choosing between the larger or smaller fragments.
Note: Use the display feature listed under the Fragment summary window to confirm that you’ve selected the correct fragment size and orientation