In Benchling, you can model and design constructs using the combinatorial assembly tool. In the combinatorial assembly tool, there are two broad assembly types – cloning and concatenation. 

• Cloning is the process of creating a new construct from multiple smaller sequences using method-specific techniques. In Benchling, you can model cloning of DNA sequences using any of the following methods: Gibson, Golden Gate, and Homology.
• Concatenation is a technique that joins multiple sequences into a single construct end to end. In Benchling, you can model concatenation for DNA sequences, DNA oligos, RNA sequences, RNA oligos, and AA (amino acid) sequences.

Assembly methods

• Gibson assembly: Join up to 15 DNA fragments into a single construct using Gibson primers, regions of homology, or restriction cut sites.
• Golden gate assembly: Join up to 15 DNA fragments into a single construct using Type IIS restriction enzymes and T4 DNA ligase. Optionally, you can use primers to introduce cut sites via PCR.
• Homology assembly: Homology cloning allows you to join up to 10 DNA fragments into a single construct using homology regions or complementary overhangs. This can be used to model yeast recombination cloning, Gibson, HiFi, Gateway cloning*, and/ or In-Fusion cloning**.
• Concatenation assembly: Join multiple fragments together end to end, without any special logic, to form new sequences or oligos. Concatenate DNA sequences, DNA oligos, RNA sequences, RNA oligos, or AA sequences.

*Gateway cloning: where all fragments already contain ATT regions

**In-Fusion cloning: where fragments are modeled as having been PCR-amplified and include homology regions

Assembly overview

All assemblies within Benchling, regardless of cloning method or concatenation, follow the same general steps:

1. Configure the assembly settings and parameters
2. Configure your bins
3. Add fragments to the bins
4. Assemble your constructs

This article covers how to perform each of these steps to create constructs in silico.

Open the assembly set up modal

1. Click on Global create and navigate to the Assembly sub-menu
2. Open the modal for the assembly you wish to use by clicking on one of the following options:
   1. Assemble DNA sequence by cloning: select this option for Gibson, Golden Gate, or Homology assemblies
   2. Assemble sequence and oligos by concatenation: select this option for concatenation

Note: Gibson, Golden Gate, and Homology assemblies can also be created from the sequence view using the Assembly button in the lower right and selecting Combinatorial cloning. 

Set up your assembly

After opening the assembly set up modal, you will configure your assembly settings and parameters. All settings other than the cloning method can be changed within the assembly window.

Define assembly attributes

All assemblies have a set of standard attributes that you must define: 

• Name: Assembly Name
• Location: Project/folder the assembly will be stored in
• Number of fragment bins: Total number of backbone and inserts in the assembly
• Topology of construct: Circular or Linear 

Each assembly type also has a set of unique attributes that you must define. 

Gibson

When you set the cloning method as Gibson, you must also define the following attributes: 

• Fragment Production Method:
  • Add new overlaps using PCR: Use a primer pair to generate new homology regions
    • Min. Tm of binding region (*C)
    • Min. Tm of whole primer (*C)
    • Min. length of homology/binding regions (bp)
    • Max. length of homology/binding regions (bp)
    • Max. Tm difference between primer pairs (*C)
  • Find existing overlaps: Use homology regions that have already been incorporated
    • Min. length of homology/binding regions (bp)
    • Max. length of homology/binding regions (bp)
    • Min. Tm of binding region (*C)
  • Digest with restriction enzyme: Linearize fragments at restriction enzyme cut sites
    • Min. length of homology/binding regions (bp)
    • Max. length of homology/binding regions (bp)
    • Min. Tm of binding region (*C)

Golden Gate

When you set the cloning method as Golden Gate, you must also define the following attributes: 

• Type II Restriction Enzymes: Select any already existing Type IIS Enzyme cut sites
• Fragment Production Method (Golden Gate specific field):
  • Use existing cut sites: Digest fragments at Type IIS enzyme cut sites to expose overhangs
  • Use a primer pair (Optional): Use a primer pair to introduce Type IIS enzyme cut sites and overhangs
    • Pre-recognition site - length (bp)
    • Pre-recognition site - bases
    • Binding region - min. length (bp)
    • Binding region - min. Tm (*C)
    • Max. Tm difference between primer pairs (*C)

Homology

When you set the cloning method as Homology, you must also define the following attributes: 

• Ambiguous construct preferences: In cases where the sequences you wish to assemble could create two distinct constructs, Benchling will create the construct that uses either the larger or smaller fragment in the first bin. This can apply when assembling two circular sequences, since each sequence produces two fragments.

Concatenation

When you choose concatenation as the assembly method, you must also define the following attributes: 

• Sequence Type: DNA, RNA, or AA
• Fragment Types: Automatically chosen based on the sequence type
• Construct Type (set for DNA/RNA only): Sequence or Oligo

Once the assembly settings and parameters are saved, an assembly window will open

Assembly object

After you save your initial assembly settings, Benchling creates an assembly object. This saves your in-progress work, can be searched for later on, and is stored after finalization. 

Edit assembly parameters and settings

Once you've created an assembly object, all settings and parameters, other than the Cloning method, can be edited.

1. In the assembly window, select the gear icon located next to the assembly name
2. In the assembly set up modal, you can make edits to the name, project folder, topology of the construct, and cloning-method specific parameters
3. Click save

Configure bins and spacers

In each assembly type, bins are used to represent a fragment or group of fragments being used to assemble the final construct(s). Bins will automatically be set up based on the “Number of fragment bins” field in the assembly set-up modal. When performing cloning, spacers can be added next to bins where primers are used to amplify a fragment; when concatenating sequences, "constants" can be added next to bins to include short sequence motifs in every construct you create.

Define fragments and add to bins

Within the combinatorial assembly tool, a “fragment” represents the individual parts (promoters, genes, backbones, etc) used to build a final construct.

Fragments can be placed into their respective bins using many methods. Multiple fragments can be placed into one bin. However, fragments in the same bin can not be incorporated into the same construct.

1. To add fragments to a bin:  Click the + within the Bin
2. Select one of the following options to add a sequence: Open sequences, Search for sequences, or Add from worklist
   1. Search for sequences and select one or more fragments to add
   2. Use Worklists to add up to 1000 sequences at once
3. Preview each sequence and optionally change the selected region of the sequence / its orientation
4. Click save to add the sequence(s) to the fragments table with the appropriate bin set

Set fragment production method

Gibson and Golden Gate assembly have multiple fragment production methods that can be set on a bin by bin basis.

Gibson assembly

• Add new overlaps using PCR: Use a primer pair to generate new homology regions
• Find existing overlaps: Use homology regions that have already been incorporated
• Digest with restriction enzyme: Linearize fragments at restriction enzyme cut sites
  • Note: Only one bin can have this fragment production method

Golden gate assembly

• Use existing cut sites: Use existing Type IIS Enzyme Cut Sites
• Use a primer pair: Design or use existing primers to amplify your fragment via PCR, introducing overlaps for assembly

Add spacers/constants and their sequences

Spacers are short DNA sequences used to separate functional elements within a construct. Constant are sequence elements that remain unchanged across constructs.

To add spacers for cloning or constants for concatenation: 

1. Click the + next to the “Bins & Spacers (n)” or "Bins & Constants (n)"
2. Select Add new spacer/constant
3. Within the spacer/constant, click on Enter bases
4. Type or paste the spacer/constant sequence
5. Click out of the spacer/constant or hit enter to save the sequence

Add a bin

1. Near the top of the Assembly page, click the + next to the “Bins & Spacers (n)” or "Bins & Constants (n)"
2. Select Add new bin

Rename a bin

Bins will automatically be named. To rename them: 

1. Click on the current name
2. Enter the new name
3. Click out of that bin or hit enter to save the name

Rearrange bins

1. Click and hold the six dots in the upper right corner of each bin
2. Drag the bin to the new location and drop

Remove a bin

Click the trash can icon in the upper right corner of the bin

Use an existing primer within an assembly

If you have a primer previously created in Benchling as a DNA oligo, you can use that within your Gibson or Golden Gate assembly. This is particularly useful in cases where your assemblies are highly templatized and sequences are designed such that you're often using the same primers for every cloning reaction. To use an existing primer, specify it within the fragments table as a "preferred primer" for a particular fragment. If you do not specify a preferred primer, one will automatically be designed for you by Benchling using the primer parameters defined on your assembly.

Benchling will validate that the preferred primer you added can in fact amplify your fragment and that it meets the primer parameters set on the assembly. 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 will warning display in the “Status” cell. In some cases, your preferred primers might be used in one construct and new primers designed for another. You can review the status of your primers during confirmation of your constructs.

Manage sequences in the fragments and constructs tables 

Each assembly has a fragments table and a constructs table. In these tables, you can configure and interact with all of the sequences that you are using and creating in the assembly.

In the fragments table, you can edit the fragments being used via an Edit fragments pop-up modal.

Edit one fragment

1. Right click on the row containing the fragment you want to edit
2. Select Edit fragment from the dropdown

Edit multiple fragments

1. Click on the first row containing a fragment you want to edit
2. Hold shift and click on the last row containing a fragment you want to edit. This should highlight all rows in between.
3. Right click on any highlighted row and select Edit fragments from the dropdown, or click the Edit fragment(s) button in the upper right corner of the table
4. To move between fragments in the Edit fragment pop-up modal, click the ⋁ symbol next to the entity chip you want to open

Edit fragment tools

• Entity blue chip: Hover over this to see a small pop-up of the entity meta data or click on this to open the entity in another tab
• Orientation: Can only be changed for the Backbone
• View (dropdown): Change the view to one of the following- Sequence map, Plasmid map, Linear map
• Zoom (- to + slider): Change the zoom setting of the sequence you are viewing
• Gear icon (upper right corner): Change what features are present on the sequence you are viewing

Enter split screen

In the top right of the table, “split screen” will open the table in the bottom half of the screen with more rows visible at once.

Enter full screen

In the top right of the table, “full screen” will open the table in the workspace.

Filter table rows by status

1. Click on the Status dropdown on the right side of the table tool bar
2. Select the status you would like to filter for

Sort table columns

Similar to tables within entries, you can sort fragment and construct table columns, making it easier to navigate the table rows. To sort a column, right click on the header and select one of the following sorting methods:

• Text columns: “A to Z” or “Z to A”
• Numerical columns: “lowest to highest” or “highest to lowest”

Delete all rows from the fragments or constructs table

1. Click on the trash can icon in the upper right corner of the table
2. Select Delete all constructs/fragments

Delete all rows with a certain status

1. Following the above instructions, filter for your desired status
2. Click the trash can icon in the right corner of the table
3. Select Delete filtered fragments

Create and view constructs

Create constructs

Once you have configured your fragments and associated settings, you can build out your construct in the construct table. To populate all possible constructs using your fragments, click Autopopulate in the upper right of the constructs table. You can also manually fill out the table to assemble your desired constructs in cases where you do not want every possible combination of fragments.

Once you have filled out the Constructs table, the following grey-shaded columns will auto-populate:

• Name: Assigned name of the new construct (can be changed later)
• Overlap Length: length (in base pairs) of the fragment overlap at each junction
• Status: tells you if the construct has No errors, Contains warning, or Contains error
  • If the status is Contains warning or Contains error, an error message will appear within that cell. Prior to viewing and assembling your constructs, errors will need to be resolved. Warnings can be either resolved or ignored.

View constructs

Once you have resolved all errors within the Constructs table, you are able to view your new constructs. You can do this by clicking into the Constructs tab and clicking View or the diagonal arrow on the individual construct you’d like to view. 

Additionally, you can view your constructs while assembling them in the constructs table by selecting the row containing the construct you want to view then clicking the View constructs button in the upper right of the constructs table or right-clicking and selecting View construct from the dropdown.

Construct visualization settings

• Sequence tab: view the sequence and features of your construct
  • Entity blue chip: hover over this to see a small pop-up of the entity meta data or click on this to open the entity in another tab
  • Orientation: use this to change the orientation to Forward or Reverse
  • View (dropdown): change the view to one of the following- Sequence map, Plasmid map, Linear map
  • Zoom (- to + slider): change the zoom setting of the sequence you are viewing
• Primers tab: view a table containing the primers associated with your construct
• ⋁ (next to blue chip): if viewing multiple constructs, click this symbol to move between constructs

Finalize the assembly

Finalizing an assembly locks the assembly modal and generates new DNA sequences for each newly designed construct. If primers and/or amplified fragments are created as part of the assembly, new DNA oligos can be generated in addition to the construct. Optionally, users can assign an entity schema to the newly created DNA sequences/oligos. Once an entity schema has been selected, entity links can be mapped from the assembly to the entity’s schema fields. Finalized assembly outputs can then be sent to an existing or new registration table within a notebook entry, allowing you to give experimental context to your newly designed constructs. 

To finalize your assembly:

1. Once all constructs have a status of Ready to assemble, click the Assemble button at the top right of the assembly modal
2. Define your assembly outputs:

• Constructs (required)
• Primers (optional)
• Fragments (optional)

3. Click Next in the bottom right corner of the modal
4. Select the Project folder to associate the assembly outputs with
5. Click Assemble

Create primers and fragments as part of the assembly output

In many cloning workflows, primers and amplified fragments are generated. Primers can be used to insert functional portions of a sequence to support downstream cloning steps. Primers or digestion sites can also be used in an assembly workflow to create amplified DNA fragments that are inserted into the final construct. When finalizing an assembly in Benchling, users have the option to save these primers and/or constructs.

To save primers or constructs:

1. Click Assemble in the upper right corner of the assembly modal
2. When defining your assembly output, toggle on the Primers and/or Amplified Fragments to save these as part of your assembly output

Assign entity schema to assembly outputs and map schema fields

Oftentimes, assembly outputs will be assigned an entity schema and registered. When finalizing your assembly, you can assign an entity schema to each output. Additionally, you can map assembly outputs to entity link fields within entity schemas allowing for more automated information transfer. To do this:

1. For each assembly output type, select an entity schema from the dropdown

• If saving Amplified fragments, you will have the option to assign a schema to all bins or to select one specific fragment bin and its associated schema 

2. Once a schema has been assigned to an assembly output, a table/tables will appear at the bottom of the modal allowing you to map assembly outputs to entity link fields within the entity schema

• Note: At this time, only entity link field types, excluding part links, are supported

Associate outputs with a Study

To associate all of your assembly outputs with an existing study/studies, indicate that within the Studies field located next to the Project Folders field

Save outputs to a worklist

To group your assembly outputs into a Worklist or add them to an existing worklist:

1. Toggle on Add outputs to a worklist at the bottom of the Review and finalize step
2. Select an existing worklist or create a new worklist for each output type 

Send assembly output to Notebook entries

Assemblies are typically done as part of much larger workflows, sometimes involving multiple teams. To provide experimental context to an assembly, you can send assembly outputs directly to existing or new registration tables in a Notebook entry. To do this:

1. Toggle on Add outputs to a Notebook entry near the bottom of the Review and finalize step
2. Begin typing the name of an existing entry and select it from the dropdown
3. In each assembly output category, select an existing registration table from your entry or create a new registration table

• If creating a new registration table, you can specify a table name. If no name is entered, the default table name will be given.
• Note: When adding to an existing registration table, it must be a Register existing entities table type
• Note: The schema type for each output category will be mapped from the previous Define outputs step. To change the selected schema type, click the Back button in the lower right corner of the modal to return to the previous step. Existing configurations will not be lost upon returning to the first step.

4. Click Assemble

• After finalizing your assembly, a green pop-up message will appear in the lower left corner. Clicking on View all will take you to the notebook entry the assembly output was sent to

View assembly history

For every construct assembled in Benchling, Benchling stores how that construct was assembled. "Assembly history" allows you to revist how a sequence was designed when a construct was created via the Assembly Wizard, the new combinatorial cloning tool, and combinatorial concatenation.

To view the visual "assembly history" of your DNA and RNA sequences:

1. Open a DNA or RNA sequence object in your workspace
2. Click Assembly History in the entity tab

Model high-throughput assembly with combinatorial cloning and concatenation

The Benchling combinatorial assembly tool can assemble up to 5000 constructs. Below are some limitations when using the combinatorial assembly tool.

Fragments

• Up to 5000 total fragments
• Up to 100kb per fragment
• 15 fragment bins
  • Exception: only up to 10 fragment bins for Homology-based cloning

Spacers (cloning) / constants (concatenation)

• Up to 20 bases per spacer or constantUp to 15 spacers/constants

Constructs

• Up to 5000 total constructs
• Up to 150kb per construct

Primers

• Up to 100 bases for primers