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Chunxiao edited this page Jun 11, 2026 · 8 revisions

Session: Annotating the Four sc2 Circuit Plasmids Using SeqImprove

Session Goal

This session focuses on using SeqImprove in SynBioSuite to annotate the four plasmids from the sc2 genetic circuit. The goal is to import each plasmid FASTA sequence, assign basic metadata, annotate biological parts using a curated SynBioHub resource collection, save the annotated plasmids locally, and upload them to a SynBioHub collection for later review and sharing.

By the end of this session, each plasmid should have:

  • A correct DisplayId and Name matching the FASTA filename
  • A circular plasmid topology
  • A complete DNA sequence
  • Functional annotations generated using SeqImprove
  • A written plasmid description
  • A citation to the source paper
  • A saved local SBOL file
  • An uploaded entry in the MD5_plasmids collection SynBioHub collection
  • A circular VisBOL/SBOL Visual representation

Step 1: Prepare the Input Data

Set up the required files and background information before starting the annotation workflow.

  • Download the PDF for the sc2 circuit design.
  • Read the paper carefully to understand the circuit architecture, plasmid roles, and biological functions.
  • Download the four *.fasta plasmid sequence files from the provided link.
  • Confirm that each FASTA file contains one complete plasmid sequence.
  • Keep the FASTA filenames unchanged, because they will be used as the plasmid DisplayId and Name in SeqImprove.

Step 2: Open SynBioSuite and Start SeqImprove

  1. Go to SynBioSuite:

    https://synbiosuite.org/

  2. Click:

    Use my local file system → Open Folder

  3. Create a new working folder for this session.

  4. Click:

    Select Folder

  5. In SynBioSuite, click:

    Build → Import a plasmid

  6. SeqImprove should open.

  7. Click:

    From Scratch → Submit

Step 3: Enter Plasmid Metadata

In the Overview tab, click the pencil icon to edit the plasmid metadata.

Set the DisplayId and Name to match the FASTA filename.

Example:

DisplayId: plJAI478
Name: plJAI478

Then click the check mark to save.

Use the following metadata fields:

Role: Engineered Plasmid (SO:0000637)
DNA Topology: Circular
Target Organisms: Escherichia coli
References: 10.1038/s41589-024-01730-1

For Target Organisms, type:

E.coli

Then select:

Escherichia coli

For References, enter the DOI:

10.1038/s41589-024-01730-1

Then click the + button to add it.

Step 4: Add the DNA Sequence

  1. Go to the Sequence tab.

  2. Open one FASTA file using any text editor.

  3. Copy only the DNA sequence.

    Do not copy the FASTA header line, for example:

    >plasmid_name

  4. In SeqImprove, click the pencil icon in the sequence text box.

  5. Double-click the space under:

    Enter some text...

  6. Paste the DNA sequence.

  7. Click the green check mark to save.

Step 5: Annotate the Plasmid Sequence

  1. Go to the annotation section.

  2. Under Algorithm, select:

    FlashText

  3. Leave all checkboxes empty.

  4. Click:

    Import Library

  5. Log in using the online SynBioHub database:

    Online database: https://synbiohub.org
Email: your email
Password: your password

  6. Click:

    Submit

  7. Select the resources collection curated in the previous session.

  8. Click:

    Submit

  9. You should see the collection name with a checkbox.

  10. Select the checkbox for the collection.

  11. Click:

Analyze Sequence
  1. Confirm that colored annotated parts appear on the sequence.

Step 6: Add Plasmid Descriptions

Go to the Text tab and add the appropriate description for each plasmid.

Plasmid plJAI_477: Tac → jr7 and jr11 Module

This plasmid carries a kanamycin-resistance backbone and includes attR2/attL2 recombination sites for integration or cassette exchange. It expresses the recombinases jr7 and jr11 from inducible promoter regions, allowing controlled activation of downstream recombination-based genetic logic.

Plasmid plJAI_478: Bad → jr11 and yfp Module

This ampicillin-resistance plasmid contains an attL7/attR7-flanked design and expresses jr11 under an arabinose-responsive PBad-associated regulatory region. It also includes yfp, which likely serves as a fluorescent reporter for monitoring successful promoter activation or recombination output.

Plasmid plJAI_515: RiboJ/CH3BS3c → lux Module

This tetracycline-resistance plasmid contains an attR5/attL5 recombination cassette and expresses the lux gene downstream of a RiboJ-insulated promoter region. The lux output provides a luminescent readout, making this plasmid useful for tracking signal processing or recombinase-driven state changes.

Plasmid plJAI_544: RiboJ → phlA-phlC-phlB-phlD Biosynthetic Module

This output plasmid carries the phlA, phlC, phlB, and phlD genes, which together form a biosynthetic pathway module. Its expression is placed under a recombination-linked promoter architecture, suggesting that production of the phl pathway output is activated only after the upstream sensor and recombinase plasmids generate the correct genetic state.

Step 7: Save the Annotated Plasmid Locally

  1. Click:

    Save to Working Directory

  2. Double-click the saved plasmid.

  3. Confirm that the SBOL Visual glyphs are displayed.

  4. Click individual Engineered Region glyphs to explore the annotations.

  5. Use the left and right arrows to move backward or forward through the design.

  6. Add a circular backbone if needed so that the plasmid is displayed as circular.

Step 8: Upload the Plasmid to SynBioHub

Click the dropdown next to:

Upload to SynBioHub

First-Time Upload

Select:

Create New Collection

Use the following collection information:

ID: MD5_plasmids
Name: MD5_plasmids
Description: Subcircuit sc2 is an engineered E. coli strain designed to process two chemical inputs, IPTG and arabinose (Ara), through a genetic logic circuit. The circuit combines these upstream input signals to regulate communication outputs, producing DAPG and OC6 as downstream signaling molecules.
Citation: 10.1038/s41589-024-01730-1 (39317847)

Later Uploads

For the remaining plasmids, use the existing collection:

Use Existing Collection → Root Collection → Select your Build collection → Submit

For example, select:

plasmid_collection

or the appropriate existing build collection containing the sc2 plasmid entries.

Step 9: Verify the Uploaded Plasmid in SynBioHub

  1. Go to the dev2 SynBioHub instance.

  2. Log in.

  3. Open:

    Manage your collection

  4. Open the relevant Build collection.

  5. Go to the Members tab.

  6. Filter by:

    sbol2:ComponentDefinition

  7. Explore the uploaded plasmid entries.

  8. Filter by:

    engineered plasmid

  9. Open each plasmid entry and check:

    • Description
    • Citation
    • Sequence annotation
    • Plasmid metadata
    • VisBOL/SBOL Visual display
    • Circular plasmid backbone

  10. Confirm that the plasmid visualization is circular.

Step 10: Repeat for All Four Plasmids

Repeat Steps 3–9 for each of the four sc2 plasmids.

Use the same general workflow for every plasmid:

Import plasmid → Add metadata → Add sequence → Annotate → Add description → Save locally → Upload to SynBioHub → Verify

Final Checklist

Before finishing the session, confirm that all four plasmids have been completed.

  • All four FASTA sequences were imported.
  • Each plasmid has the correct DisplayId and Name.
  • Each plasmid is labeled as an engineered plasmid.
  • Each plasmid is marked as circular.
  • Escherichia coli is added as the target organism.
  • DOI citation 10.1038/s41589-024-01730-1 is included.
  • SeqImprove annotations were generated using the curated resource collection.
  • Each plasmid has a written description in the Text tab.
  • Each plasmid was saved to the working directory.
  • Each plasmid was uploaded to `MD5_plasmids' collection.
  • Each uploaded plasmid was checked in SynBioHub.
  • Each plasmid has a circular SBOL Visual/VisBOL representation.

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