Oct 05, 2025

Public workspaceAdd Arabinose

DOI
https://dx.doi.org/10.17504/protocols.io.dm6gpm558gzp/v1
  • igemnthuth 1
  • 1iGEM_NTHU_Taiwan
  • iGEM NTHU
igemnthuth
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gpm558gzp/v1
Protocol Citation: igemnthuth 2025. Add Arabinose. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpm558gzp/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: 228985
Keywords: synthetic replication operon, expression of the synthetic replication operon, sgrna repression system, orthogonal replication system, orthogonal replication, improving orthogonal replicon transformation efficiency, orthogonal replicon transformation efficiency, downstream operon component expression, arabinose, induced dcas9
Abstract
To control expression of the synthetic replication operon in the orthogonal replication system, this step uses an arabinose-induced dCas9–sgRNA repression system to reduce downstream operon component expression, thereby improving orthogonal replicon transformation efficiency.
Materials
  • LB broth
  • 10% glycerol solution (pre-chilled)
  • Arabinose
  • ddH₂O
  • Pre-chilled electroporation cuvettes (2 mm gap)
  • Pre-chilled centrifuge tubes (50 mL × 2; 1.5 mL × 1)
  • Shaking incubator
  • Ice-water bath and ice bucket
  • Spectrophotometer (for OD600 measurement)
  • Refrigerated centrifuge
  • Electroporator
Troubleshooting
Pre-culture
Pick colonies that have successfully taken up the following plasmids:

  • EcOrep operon plasmid
  • dCas9 + sgRNA expression plasmid (e.g., pRT17/19/21)
  • Mutant DNAP plasmid
Inoculate into 15 mL LB broth supplemented with the corresponding antibiotics for all three plasmids, and incubate overnight at 37 °C, 220 rpm (~12 h).
Main Culture and Arabinose Induction
The next day, transfer 5 mL of the overnight culture into 200 mL fresh LB broth (1:40 dilution) in a 2 L shake flask; incubate at 37 °C, 220 rpm.
When OD600 ≈ 0.2, add arabinose to a final concentration of 10 mM (e.g., add 2 mL of 1 M stock to 200 mL culture) and continue incubation.
Grow to OD600 ≈ 0.4, then immediately proceed to the following steps for electroporation pre-treatment.
Preparation of Competent Cells
Place the culture on ice to cool for 10 minutes (gentle swirling optional).
Centrifuge at 4 °C, 4500 × g for 3 minutes; carefully discard the supernatant.
Wash the cell pellet with 50 mL pre-chilled 10% glycerol; repeat the wash twice (total 2 washes).
Finally, resuspend the pellet in 1 mL pre-chilled 10% glycerol to obtain the competent cells.
Aliquot into several 100 μL tubes (for subsequent O-replicon electroporation). This completes the arabinose treatment stage.