Oct 05, 2025
Tag and Catcher Assembly
- igemnthuth 1
- 1iGEM_NTHU_Taiwan
- iGEM NTHU
Protocol Citation: igemnthuth 2025. Tag and Catcher Assembly. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvrw7rblmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 03, 2025
Last Modified: October 05, 2025
Protocol Integer ID: 228982
Keywords: spytag protein, spytag, spycatcher, catcher assembly, conjugation, protein
Abstract
Induce the strain to secrete SpyCatcher and SpyTag proteins for in vitro conjugation.
Materials
- 1 M IPTG
- PBG
- Centrifuge
- 0.22 μm filter membrane
- Ultrasonic cell disruptor (sonicator)
Troubleshooting
Cell Disruption by Sonication
Resuspend cells in PBS, aiming for approximately 1–5 × 10⁹ cells/mL per mL suspension for effective lysis.
Place the cell suspension in an ice bath to prevent overheating.
Disrupt cells using a sonicator:
- Power setting: 20–40% amplitude
- Pulse duration: 30 seconds to 1 minute total, with cycles of 10 s on / 10 s off to avoid overheating.
- Total sonication time: 3–5 minutes, adjusted according to sample volume and lysis efficiency.
Collecting Supernatant (SpyCatcher/SpyTag Proteins)
Centrifuge at 4500 × g, 4 °C, 5 min.
Collect the supernatant and filter through a 0.22 μm membrane.
Preparing Reaction Conditions
Adjust protein concentration to fall within 1–10 μM.
Prepare PBS buffer for the reaction.
SpyCatcher/SpyTag Covalent Ligation
Mix SpyCatcher and SpyTag proteins at a 1:1 molar ratio.
- Example: if both are at 1 μM, add 1 μL of each into a 50 μL reaction volume.
Incubate at room temperature for 1 hour.
Stop the reaction by placing the mixture on ice.