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A single step three-strain in vivo Gateway reaction
Aaron Nicholas Gillman1, Alexandra Helleux2, Sören Abel3
1Department of Pharmacy, Faculty of Health Sciences, UiT - The Arctic University of Norway, 9037 Tromsø, Norway; Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, University Park, PA 16802, USA.
Plasmid
|November 21, 2021
Summary
We created a streamlined, single-step method for DNA recombination and vector conjugation, simplifying molecular cloning and enabling efficient screening of protein-protein interactions using bacterial two-hybrid systems.
Area of Science:
- Molecular Biology
- Synthetic Biology
- Microbiology
Background:
- Traditional molecular cloning methods can be time-consuming and labor-intensive.
- Efficient DNA recombination and vector construction are crucial for genetic engineering and high-throughput screening.
- Existing bacterial two-hybrid systems often require complex vector construction and selection steps.
Purpose of the Study:
- To develop a simplified, highly efficient, single-step method for in vivo DNA recombination and vector conjugation.
- To create modular and minimal destination vectors for flexible genetic construct design.
- To demonstrate the utility of the system for bacterial two-hybrid (B2H) screening.
Main Methods:
- A novel Gateway reaction was developed for in vivo recombination of target DNA into destination plasmids.
- A conjugation system was employed to transfer the final vector into a recipient strain in a single step.
- Modular destination vectors with modified Gateway inserts were designed for easy insertion of tags, promoters, or conjugation elements.
- Split adenylate cyclase tags were incorporated into destination vectors for bacterial two-hybrid studies.
Main Results:
- The developed protocol simplifies DNA recombination and vector conjugation into a single, efficient step.
- The system is robust, cost-effective, and compatible with 96-well plate formats across various temperatures.
- Freely chosen recipient strains sensitive to ccdB counterselection and RP4α conjugation can be used.
- The system was successfully applied to screen a library of diguanylate cyclases for protein-protein interactions using B2H.
Conclusions:
- This novel single-step protocol significantly streamlines molecular cloning and genetic manipulation.
- The modular destination vectors offer flexibility for diverse applications, including B2H assays.
- The system provides a powerful and efficient tool for high-throughput screening of protein-protein interactions in bacteria.
