Bifunctional lacZ alpha-ccdB genes for selective cloning of PCR products.
The use of PCR-amplified DNA-fragments is a classical approach to generate recombinant DNA. To facilitate the cloning of PCR products, we have constructed two new pKIL vectors that allow selection of recombinants. The multiple cloning sites (MCS) of these plasmids contain two adjacent Aspel sites and a unique HindII site. Cleavage of these vectors with Aspel produce linearized molecules with a single thymidine nucleotide at the 3' ends allowing TA cloning of Taq-amplified fragments. On the other hand, cleavage with HindII can be used for the cloning of blunt-ended PCR products generated by other DNA polymerases. The LacZ alpha-CcdB fusion protein produced by these plasmids has retained both the CcdB killer activity and the ability to alpha-complement the truncated LacZ delta M15. This bifunctionality allowed us to show that small PCR products (< 1000 bp) that do not disrupt lacZ alpha efficiently do inactivate CcdB, which demonstrates that the CcdB-based selection is well adapted for cloning of PCR products, especially for small size fragments.
Gabant
P
P
Université Libre de Bruxelles, Rhode-Saint-Genèse, Belgium.
Drèze
P L
PL
Van Reeth
T
T
Szpirer
J
J
Szpirer
C
C
D001426:Bacterial Proteins / Q000235:genetics* / Q000494:pharmacology; D001427:Bacterial Toxins / Q000235:genetics* / Q000494:pharmacology; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins; D015183:Restriction Mapping; D019914:Taq Polymerase / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068356:CcdB protein, Plasmid F; D011993:Recombinant Fusion Proteins; D019914:Taq Polymerase; C075914:endodeoxyribonuclease Eam1105I; D015252:Deoxyribonucleases, Type II Site-Specific; C024400:GTYRAC-specific type II deoxyribonucleases; D001616:beta-Galactosidase