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Updated: Jan 4, 2026

Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)

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Published on: March 16, 2012

Split intein-mediated selection of cells containing two plasmids using a single antibiotic.

Navaneethan Palanisamy^1,2,3, Anna Degen^2,4,5, Anna Morath^1,6,7,8

¹Signalling Research Centres BIOSS and CIBSS, University of Freiburg, 79104, Freiburg, Germany.

Nature Communications

|November 2, 2019

Summary

We developed SiMPl, a novel method using split enzymes and intein-mediated protein trans-splicing. This approach simplifies the selection of cells with two plasmids, enhancing production of valuable compounds and improving synthetic biology applications.

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Area of Science:

Synthetic biology
Molecular biology
Biotechnology

Background:

Complex cellular pathways often require multiple plasmids, typically necessitating dual antibiotic selection.
Existing methods for selecting cells with multiple plasmids can be inefficient and cumbersome.

Purpose of the Study:

To introduce SiMPl, a novel method for selecting cells containing two plasmids using a single antibiotic.
To demonstrate the efficacy of SiMPl in both bacterial and mammalian cell systems.
To highlight the advantages of SiMPl over traditional dual-antibiotic selection methods.

Main Methods:

SiMPl utilizes rationally designed split enzymes and intein-mediated protein trans-splicing.
Cells carrying two plasmids are selected based on a single antibiotic resistance marker.
The method was tested for the production of indigoidine and para-amino-L-phenylalanine in bacteria.
SiMPl was applied to obtain double-positive T cells expressing TCRα and TCRβ in a human T cell line.

Main Results:

SiMPl significantly increased the production of indigoidine and para-amino-L-phenylalanine in bacteria compared to traditional methods.
A highly pure population of double-positive T cells (TCRα and TCRβ) was obtained using SiMPl with a single antibiotic.
The method simplifies the selection process, reducing the need for multiple antibiotics.

Conclusions:

SiMPl offers a more efficient and streamlined approach for selecting cells with multiple plasmids.
This method has broad implications for metabolic engineering and the construction of complex synthetic circuits in various cell types.
SiMPl enhances the production of specific biomolecules and facilitates the generation of engineered cell populations.