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A bacteriophage T7-based expression vector, pBT7, with color selection for the recombinant
I N Maruyama1, Y G Mikawa, H I Maruyama
1Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037.
Gene
|September 6, 1993
Summary
A new bacterial plasmid, pBT7, enables easy identification of DNA inserts using color selection. This method allows for high-level protein production, demonstrated with a Caenorhabditis elegans gene.
Area of Science:
- Molecular Biology
- Bacteriophage Systems
- Recombinant DNA Technology
Background:
- Bacterial expression plasmids are crucial for protein production.
- Efficient selection methods for recombinant clones are needed.
- The bacteriophage T7 RNA polymerase/promoter system is widely used.
Purpose of the Study:
- To develop a bacterial expression plasmid with positive color selection for DNA inserts.
- To facilitate the identification of recombinant clones.
- To enable high-level protein overproduction.
Main Methods:
- Construction of the pBT7 plasmid by amplifying and cloning the T7 gene 10 promoter into pUC18.
- Utilizing the lacZ alpha-peptide for blue-white screening.
- Overproducing a fragment of the Caenorhabditis elegans unc-13 gene product.
Main Results:
- The pBT7 plasmid provides positive color selection: blue colonies for non-recombinant, white for recombinant.
- Insertion of the T7 promoter fragment did not inactivate lacZ alpha-peptide activity.
- High-level overproduction (up to 50% of total protein) of the target gene was achieved.
Conclusions:
- The pBT7 plasmid is an effective tool for positive color selection of recombinant clones.
- This system simplifies screening and enhances protein expression efficiency.
- pBT7 facilitates high-yield production of specific proteins, such as from C. elegans.