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[Construction of promoter-probe plasmid vector]
Molekuliarnaia Biologiia
|July 1, 1981
Summary
A novel plasmid vector, pBRS188, facilitates cloning of promoter DNA. This engineered E. coli plasmid enables selection of recombinant DNA by identifying transformants that regain tetracycline resistance.
Area of Science:
- Molecular Biology
- Genetic Engineering
Background:
- The plasmid vector pBR322 is widely used for cloning DNA fragments.
- Efficient cloning requires vectors that allow for easy selection of recombinant DNA.
Purpose of the Study:
- To construct a new plasmid vector, pBRS188, for cloning promoter-containing DNA fragments.
- To develop a method for selecting recombinant DNA based on promoter activity.
Main Methods:
- Construction of pBRS188 by modifying the tetracycline resistance promoter of pBR322.
- Utilizing E. coli DNA polymerase I and nuclease S1 for DNA fragment manipulation.
- Cloning promoter-containing DNA into the unique EcoRI site of pBRS188.
Main Results:
- The modified plasmid, pBRS188, lost its intrinsic tetracycline resistance.
- Cloning of promoter-bearing DNA fragments into pBRS188 restored tetracycline resistance in host cells.
- Regeneration of restriction endonuclease recognition sites at DNA junctions was achieved.
Conclusions:
- pBRS188 is an effective tool for cloning promoter-containing DNA fragments.
- The developed method allows for the selection of recombinant clones by monitoring changes in antibiotic resistance.
- This approach offers a novel strategy for genetic engineering and molecular cloning applications.