Related Experiment Videos
Plasmid vectors derived from phage Mu allow direct selection of transformants containing cloned HindIII and PstI
Abstract:
The vector plasmids pKN001 and pKN80 both contain the EcoRi.C fragment of E.coli phage Mu DNA which codes for a killing function that is efficiently expressed upon transformation into Mu-sensitive bacteria. By in vitro insertion of HindIII fragments at the single HindIII site of pKN80 or of PstI fragments at the single PstI site of pKN001 the killing function is inactivated. The resulting plasmids have a selective advantage over the religated vector when transformed into Mu-sensitive bacteria. More than 90% of the transformations contain hybrid plasmids. These results show the usefulness of Mu DNA containing plasmids pKN001 and pKN80 as vectors that allow the direct selection for recombinant plasmids.
Insights
New plasmids derived from E. coli phage Mu DNA enable direct selection of recombinant plasmids. Insertion of DNA fragments inactivates a killing function, providing a selective advantage for desired hybrid plasmids in Mu-sensitive bacteria.
Area of Science:
- Molecular Biology
- Bacteriophage Genetics
- Recombinant DNA Technology
Background:
- The EcoRI.C fragment of E. coli phage Mu DNA encodes a potent killing function expressed in Mu-sensitive bacteria.
- Existing vector systems may lack efficient methods for direct selection of recombinant molecules.
Purpose of the Study:
- To develop novel vector plasmids for efficient direct selection of recombinant DNA molecules.
- To utilize the killing function of E. coli phage Mu DNA for selection purposes.
Main Methods:
- Construction of vector plasmids pKN001 and pKN80 containing the EcoRI.C fragment of E. coli phage Mu DNA.
- In vitro insertion of HindIII fragments into pKN80 and PstI fragments into pKN001 to inactivate the killing function.
- Transformation of Mu-sensitive bacteria with modified and religated vectors.
Main Results:
- Insertion of DNA fragments at specific sites inactivated the phage Mu killing function.
- The resulting hybrid plasmids demonstrated a selective advantage over religated vectors in Mu-sensitive bacteria.
- Over 90% of transformations yielded hybrid plasmids, indicating high efficiency of selection.
Conclusions:
- Plasmids pKN001 and pKN80 containing the EcoRI.C fragment of E. coli phage Mu DNA are effective vectors.
- These vectors facilitate the direct selection of recombinant plasmids by leveraging a selectable killing function.
- The developed system offers a valuable tool for molecular cloning and genetic engineering applications.