From Wikipedia, the free encyclopedia
Jump to: navigation, search

In genetics, pBluescript (pBS) or pBluescript II is a commercially available phagemid containing several useful sequences for use in cloning with bacteriophage. The sequences include a multiple cloning site sequence (MCS), antibiotic resistance sequence to ampicillin and an E. coli and f1 helper phage origin of replication. The multiple cloning site sequence is located within a LacZ controlled gene designed to provide a blue coloration when expressed in bacteria. This is usually achieved via X-gal found in agarose growth media used to culture bacteria with pBS. If the gene is disrupted by successful insertion of a DNA sequence, the bacteria exhibit a white coloration in Blue white screening, distinguishing successful recombination from those phagemids which were not altered. These recombinant plasmids can then be used in a variety of molecular techniques. Determining expression and developing genomic libraries are some applications for which the pBS recombinants can be used.

The pBluescript II phagemids (plasmids with a phage origin) are cloning vectors designed to simplify commonly used cloning and sequencing procedures, including the construction of nested deletions for DNA sequencing, generation of RNA transcripts in vitro and site-specific mutagenesis and gene mapping. The pBluescript II phagemids have an extensive multiple cloning site with 21 unique restriction enzyme recognition sites. Flanking the multiple cloning site are T7 and T3 RNA polymerase promoters that can be used to synthesize RNA in vitro. 1, 2

The choice of promoter used to initiate transcription determines which strand of the insert cloned into the multiple cloning site will be transcribed. Circular maps and lists of features for the pBluescript II phagemids are shown in figures 1 and 2. The multiple cloning site and T7 and T3 RNA polymerase promoter sequences are present in the N-terminal portion of a lacZ gene fragment. A total of 131 amino acids of β-galactosidase coding sequence is present in the pBluescript II phagemid, but the coding sequence is interrupted by the large multiple cloning site. (There are 36 amino acids from the initiator Met sequence to the EcoR I site.) pBluescript II phagemids having no inserts in the multiple cloning site will produce blue colonies in the appropriate strains of bacteria (i.e., strains containing lacZΔM15 on an F´ episome, such as XL1-Blue MRF´, among others). pBluescript II phagemids that have inserts will produce white colonies using the same strain, because the inserts disrupt the coding region of the lacZ gene fragment. pBluescript II (+) and (–) are available with two multiple cloning site orientations designated as either KS or SK using the following convention: (1) in the KS orientation, the Kpn I restriction site is nearest the lacZ promoter and the Sac I restriction site is farthest from the lacZ promoter; and (2) in the SK orientation, the Sac I site is the closest restriction site to the lacZ promoter and the Kpn I site is the farthest. Flanking the T3 and T7 promoters are BssH II sites. This rare six-base cutter will allow the insert plus the T phage RNA promoters to be excised and used for gene mapping. pBluescript II phagemids can be rescued as single-stranded (ss) DNA. pBluescript II phagemids contain a 454-bp filamentous f1 phage intergenic region (M13 related), which includes the 307-bp origin of replication. The (+) and (–) orientations of the f1 intergenic region allow the rescue of sense or antisense ssDNA by a helper phage. This ssDNA can be used for dideoxynucleotide sequencing (Sanger method) or site-specific mutagenesis.