Modeling Gibson assemblies with the combinatorial assembly tool

Gibson assembly overview

Join up to 15 DNA fragments in a single-tube isothermal reaction. Gibson assembly uses primers to create homology regions between adjacent fragments. You can fine-tune the primer parameters below. 

Gibson assembly set up

To open the Combinatorial Assembly tool, see Combinatorial assembly tool: how to open and high through-put limitations.

Within the combinatorial assembly set-up modal, fill out the following fields:

• 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
• Cloning Method: Gibson

• Fragment Production Method (Gibson specific field): 
  • 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)

Click the Save button in the lower right corner of the assembly modal. This will open up a new assembly tab.

Edit name, project folder, topology of the construct, and cloning method specific parameters

All fields other than the Cloning method can be edited:

1. Within your assembly, select the gear icon located next to the assembly name at the top
2. In the assembly 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

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. Spacers can be added next to bins where primers are used to amplify a fragment.

Rename a bin or spacer

Bins and spacers will automatically be named. To rename them: 

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

Add a bin or spacer

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

Remove a bin or spacer

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

Rearrange bins or spacers

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

Add sequences to spacers

1. Within the spacer, click on Enter bases
2. Type or paste the spacer sequence
3. Click out of the spacer or hit enter to save the sequence

Add fragments to bins

Fragment production method

You can change the fragment production method via the dropdown within each bin

• 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

Add fragments directly to bins

1. 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
3. The sequence(s) will populate automatically within the fragments table. In the table, select the desired orientation for each fragment.

Adding up to 1000 sequences to a bin at once

1. Click the + button of the bin you want to add the fragments to
2. Click Search for sequences
3. Click the three lines with dots
4. Within the dropdown, hover over the Results per page
5. Select the desired Results per page; options are 5, 10, 25, 50, 100, 500, 1000
6. Select all the sequences you would like to add from the single page

Add fragments via the Fragments table

Method 1

1. Double click into the Sequence cell
2. Begin typing the name and select the desired entity 
3. In the Bin column, select a Bin for each fragment. This will automatically update the Bins at the top of assembly tab
4. Choose the orientation of the fragment (forward or reverse)

Method 2

Copy and paste from a spreadsheet into the table

Once the Fragment table has been filled out, the following grey-shaded fields will auto-populate:

• Length: length (in base pairs) of the fragment based on the selected start and end points
• Orientation: forward for all fragments except the backbone, which can be changed
• Fragment Production Method: based on the method selected within the fragment bin or the assembly settings
  • If you have selected Add new overlaps using PCR, you are able to enter your preferred 5’ or 3’ primer
  • If you have selected Digest with restriction enzyme, you are able to select the desired 5’ and 3’ enzyme

To learn about Fragment table tools, see Fragment and Construct table tools

Use 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 are able to 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 will warning display in the “Status” cell. 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.

Correct any status issues in the Fragments table

The status column will update automatically as you add fragments to the table. Each status can be defined as:

• Looks good: proceed to use fragment in assembly
• Contains Warning: can proceed to use fragment in assembly, but Benchling has identified a potential error
• Contains Error: can not use fragments in assembly until the error has been resolved

If the status is Contains Warning or Contains Error, an error message will appear within that cell. Prior to beginning to design your constructs, you will need to resolve any errors. Here are some tools to help:

Editing fragments

Without leaving the assembly, you are able to edit the fragments being used via an Edit fragments pop-up modal.

Method 1

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
4. Select Edit fragments from the dropdown
5. To move between fragments in the Edit fragments pop-up modal, click the ⋁ symbol next to the entity chip you want to open

Method 2

Edit one fragment

1. Click on the row containing the fragment you want to edit
2. Select Edit fragment located in the upper right corner of the table

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. Select Edit fragments located in the upper right corner of the table
4. To move between fragments in the Edit fragments pop-up modal, click the ⋁ symbol next to the entity chip you want to open

Tools within the pop-up

• 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

Configure Construct Table

Once the status of everything in the Fragment table is No Errors or Warning, constructs can be assembled via the construct table. You can configure your constructs table using 2 methods:

Create constructs via “Auto-populate”

1. In the upper right hand corner of the Constructs table, click Auto-populate
   1. This will create constructs involving all possible combinations of fragments

Create constructs via Constructs table

Method 1

For every Bin, there will be a corresponding dropdown in the Construct table. Using the dropdown, select your desired sequence from each bin. If you don’t want to use a bin to create your construct, select Skip.

Method 2

Copy and paste from a spreadsheet into the Constructs table

To learn about Construct table tools, see Fragment and Construct table tools

Once you have filled out the Construct 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, these errors will need to be resolved.

Visualize constructs

Once you have resolved all errors within the Constructs table, you are able to view your new constructs. Here are some tools to help users visualize constructs:

View constructs in pop-up modal

Method 1

View single construct

1. Right click on the row containing the construct you want to view
2. Select View construct from the dropdown

View selection of constructs

1. Click on the first construct you want to view
2. Hold shift
3. Click on the last construct you want to view. This should highlight all constructs in between.
4. Right click on any highlighted cell
5. Select View constructs from the dropdown

Method 2

View single construct

1. Click on the row containing the construct you want to view
2. Click the View construct button in the upper right of the table

View selection of constructs

1. Click on the first construct you want to view
2. Hold shift
3. Click on the last construct you want to view. This should highlight all constructs in between.
4. Click the View constructs button in the upper right of the table

Pop-up modal tools

• 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

Constructs tab

You can view all constructs by clicking on the Constructs tab at the top of the assembly window. This tab will show a zoomed out view of your constructs and any associated primers.

Tools within the constructs tab

• Search bar: begin typing the name of a desired construct to quickly find it
• Left/ right arrows: toggle through the pages of constructs
• Results per page (lines with dots next to arrows): change the amount of results that appear on each page; options include– 10, 25, 50, or 100
• Blue entity chip: Click on this chip to open the construct entity in a new tab
• Diagonal arrows (next to blue chips): click these arrows to open the selected construct in the pop-up modal
• View (small blue button next to primers): this will open the pop-up modal to view the primers associated with your construct

Save assembly

To finalize your constructs

1. Ensure all blocking errors are resolved, then click the Assemble button at the top right of the page.
2. Choose to create new sequences as needed:

• • Constructs (required)
  • Primers (optional, recommended if designed during assembly)
  • Fragments (optional, useful for saving fragments instead of PCR amplifying)

3. For each group of sequences (constructs, primers, fragments), select the following options:

• • Add to folder (required)
  • Add to worklist (optional)
    • Since worklists are limited to 500 entities, Benchling will split your new entities into separate worklists. The user can choose the standard name for the worklist. It will be auto-assigned a number in succession (ex. Name 1, Name 2, Name 3) 
  • Add to schema (optional; assigns a schema but does not register new sequences)

4. Access your finalized assembly from the project folder where it was saved.

Finalizing the assembly locks the assembly record, generates new DNA sequences for each construct, creates new primers where relevant, and populates the tables with references to these newly created sequence entities.