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Sequential Plasmid Curing and Genome Editing in Escherichia coli Nissle 1917
Junhong Chen1, Chernfang Cheng1, Jiacheng Huang1,2
1Innovative Practice Platform for Genetic Engineering Machine Design, State Key Laboratory of Physical Chemistry of Solid Surfaces, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, P. R. China.
ACS Synthetic Biology
|July 1, 2026
Summary
We developed a method to remove cryptic plasmids from the probiotic Escherichia coli Nissle 1917 (EcN). This enhances EcN
Area of Science:
- Microbiology
- Bacterial Genetics
- Synthetic Biology
Background:
- Escherichia coli Nissle 1917 (EcN) is a beneficial probiotic.
- Native cryptic plasmids (pMUT1, pMUT2) in EcN impede genetic manipulation and add metabolic load.
- Efficient genetic engineering of EcN is crucial for its probiotic applications.
Purpose of the Study:
- To establish a workflow for sequentially curing cryptic plasmids from EcN.
- To enable genome editing in EcN using a CRISPR-Cas system.
- To improve EcN's transformability and heterologous protein expression.
Main Methods:
- Sequential plasmid curing using a pEcCas/pEcgRNA system targeting specific genes (HTH domain protein, pMUT2 replicase).
- Introduction of a second pEcgRNA expressing RelB antitoxin for pMUT2 elimination.
- Genome editing via a genome-targeting pEcgRNA.
Main Results:
- Successfully generated a cryptic plasmid-free EcN strain.
- Demonstrated improved exogenous plasmid transformation in the cured strain.
- Achieved efficient heterologous protein expression in the modified EcN.
- Validated the workflow for iterative genome editing.
Conclusions:
- The developed workflow streamlines genetic modification of EcN.
- Cryptic plasmid elimination enhances EcN's utility as a probiotic platform.
- The method is adaptable for curing endogenous plasmids in other bacterial species.
