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Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

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Chemically Inducible Chromosomal Evolution (CIChE) for Multicopy Metabolic Pathway Engineering.

Aaron M Love¹, Bradley W Biggs², Keith E J Tyo²

¹Manus Bio, Cambridge, MA, USA.

Methods in Molecular Biology (Clifton, N.J.)

|February 22, 2019

Summary

Chemically inducible chromosomal evolution (CIChE) enables stable, multicopy integration of genes into chromosomes. This method uses homologous recombination to rapidly increase gene copy numbers, creating stable strains for research.

Keywords:

Chromosomal integration Escherichia coli Metabolic engineering Molecular cloning Synthetic biology Tools

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Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

Stable integration of heterologous genes into chromosomes is crucial for biotechnology.
Existing methods may lack efficiency or stability for multicopy gene integration.

Purpose of the Study:

To present a generalized workflow for Chemically Inducible Chromosomal Evolution (CIChE).
To enable stable, multicopy chromosomal integration of heterologous genes.

Main Methods:

Utilizing recA-mediated homologous recombination to achieve gene duplication.
Flanking selection markers and genes of interest with homologous regions.
Applying antibiotic selection pressure to drive gene amplification.

Main Results:

Achieved gene copy numbers exceeding 20× within one week.
Demonstrated stability of amplified gene cassettes through recA deletion.
Developed a reproducible workflow for CIChE.

Conclusions:

CIChE is an effective method for generating stable, high-copy number gene integrations.
This technique offers a rapid and scalable approach for genetic engineering.
CIChE has broad applications in synthetic biology and metabolic engineering.