| The novel pETBlue™ System combines the visual identification of recombinants and high plasmid copy number, found in popular blue/white screening vectors, with the tightly controlled high yield protein expression obtained with pET vectors. Blue/white screening is achieved using a weak constitutive E. coli promoter (tet) to drive expression of the lacZ alpha-peptide, whereas expression of target genes is driven by a T7lac promoter in the opposite orientation. Insertion of target sequences into the multiple cloning site (MCS) disrupts expression of the lacZ alpha-peptide and produces a white colony phenotype in strain NovaBlue when plated in the presence of X-gal. Colonies derived from the unmodified vector turn blue. Because T7-driven protein expression requires inserts to be cloned in an antisense orientation relative to the tet promoter, basal expression of target sequences is virtually absent. The high copy number pUC origin of replication present on the pETBlue plasmids greatly increases plasmid yields relative to the pET vectors and provides an advantage for sequencing, mutagenesis and other plasmid manipulations. Target genes in pETBlue vectors can be expressed at high levels, provided that the inserted sequences (1) are in the sense orientation relative to the T7lac promoter, and (2) meet the translation requirements defined by each vector (see below). Protein expression is accomplished in one of two ways; by infection with lambdaCE6 (a phage that expresses T7 RNA polymerase under control of the lambdapL promoter), or by transforming the recombinant pETBlue plasmid into the host strains Tuner™(DE3)pLacI or Origami™(DE3)pLacI followed by induction with IPTG. These hosts carry a chromosomal copy of the T7 RNA polymerase gene under lacUV5 control, and supply sufficient lac repressor via the compatible pLacI plasmid to ensure low level uninduced expression. The lacY status of the Tuner strain allows uniform dose-dependent IPTG induction of the target protein throughout the culture, and Origami strains enhance cytoplasmic disulfide bond formation. pETBlue-1 facilitates the expression of unfused, native protein from inserts that encode an open reading frame at the 5' end. The EcoR V cloning site (GATATC) in this vector is optimally positioned relative to the strong T7 gene 10 ribosome binding site (RBS) as shown below. Inserts that either possess an ATG start codon or an appropriately positioned G-nucleotide at the 5' end will create an optimal E. coli translation initiation site. pETBlue-2 provides a vector-encoded ATG start codon and variety of downstream cloning sites. In-frame cloning of any insert is facilitated by the presence of two sets of three overlapping blunt cutting sites at both the 5' and 3' ends of the MCS. The blunt end left by each of these enzymes terminates in a different position of the codon triplet in the vector-defined reading frame. Therefore, when inserted in the appropriate orientation, any insert can be cloned in-frame by cutting the vector with the appropriate combination of blunt cutting enzymes. Furthermore, any insert that lacks an internal stop codon can be cloned in-frame with the C-terminal HSV•Tag® epitope and His•Tag® sequences. The vector sequences are provided in GenBank format, with significant features indicated. To convert the sequence into other formats, first select the sequence and copy (Command-C on Mac or Ctrl-C on Windows). Next, click here to open the NIH ReadSeq web page in a new window. Paste the sequence into the input field (Command-V on Mac or Ctrl-V on PC), choose your favorite format from the dropdown menu, and hit the "submit" button. You can access sequences, maps, ordering information, newsletter articles and technical bulletins through the following links: |
