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Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
Published on: January 8, 2015
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A Broad Host Range Plasmid-Based Roadmap for ssDNA-Based Recombineering in Gram-Negative Bacteria
Tomás Aparicio1, Víctor de Lorenzo2, Esteban Martínez-García1
1Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Madrid, Spain.
Methods in Molecular Biology (Clifton, N.J.)
|October 5, 2019
Summary
This study presents a new protocol for bacterial genome engineering using recombineering. It enables the identification, cloning, and functional testing of new recombinases in diverse bacterial species.
Area of Science:
- Microbiology
- Molecular Biology
- Synthetic Biology
Background:
- Recombineering, utilizing phage recombination proteins, is crucial for bacterial genome engineering.
- Current single-stranded DNA (ssDNA) recombineering is limited by the availability of suitable recombinases and delivery systems across bacterial species.
Purpose of the Study:
- To develop a standardized workflow for identifying, cloning, and quantifying novel recombinase functions in various microorganisms.
- To overcome limitations in ssDNA recombineering by providing a flexible genetic toolset.
Main Methods:
- A protocol for cloning and expressing selected recombinases in standardized broad-host-range plasmids.
- Functional assessment of recombinase activity using a P. putida EM42 model system.
- Investigating the impact of mutagenic oligonucleotide parameters (length, phosphorothioate protection) on recombineering outcomes.
Main Results:
- Demonstrated a method for testing recombinase function in P. putida EM42, successfully achieving a pyrF gene deletion.
- Showcased the influence of oligonucleotide length and phosphorothioate protection on the efficiency of genome modification.
Conclusions:
- The developed protocol offers a versatile approach for expanding the utility of ssDNA recombineering to new bacterial hosts.
- This method facilitates the discovery and application of novel recombinases for advanced bacterial strain development and genetic studies.
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