Vector plasmids, construction

Figure 4.4. Schematic illustration of directional topoisomerase cloning of PCR products into the pUNI vector. The PCR product to be cloned has the sequence 5 -CACC appended at the 5 end to direct the orientation of cloning. The Vaccinia virus topoisomerase I enzyme forms a covalent adduct with the cloning vector to create a cloning competent plasmid construct. The loxP site is 5 to the insertion site. The vector and PCR product are designed to fuse the ORF in-frame with loxP.

For other production hosts (yeast, insect, and mammalian cells), standard promoter formats have been used in combination with FITP cloning methods to produce vectors for expression screening (see Section 2.3.2). A particularly interesting development is the use of multipromoter plasmids for expression in two or more hosts from a single vector. The construction of a dual E.coli (T7 promoter) and baculovirus transfer vector (polH promoter) for expression in insect cells has been described (Chambers et al., 2004). A three-promoter vector (T7, plO, and hCMV or CAG promoter) is available from Novagen (pTrlEX ) and its use reported for comparing protein expression in E. coli and insect cells (Xu and Jones, 2004). 

A. Plasmid Construction and Establishment of Host-Vector System in S. fradiae... 

Fig. 2.4. Vector characterization. (A). SDS acrylamide gel electrophoresis of a rAAV-UF-11 vector. Approximately 2.5 x 1011 drp were loaded on a 10% SDS-polyacrylamide gel. The three capsid protein bands in the correct stoichiometry are visualized by silver staining. (B) Infectious center assay of a rAAV-CB-hAAT stock when probed for the transgene each spot represents a vector infected cell. (C) Dot blot analysis of the rAAV stock to determine the titer of DNase-resistant particles of rAAV. Amounts (ng) refer to a serial dilution of the packaged rAAV vector plasmid used to construct the standard curve. The stock has a calculated titer of 4.3 x 1013 drp/ml.

Dertzbaugh MT, Macrina FL (1989) Plasmid vectors for constructing translational fusions to the B subunit of cholera toxin. In Gene 82 335-342. 

Figure 2. Plasmid constructions of the Synechocystis sp. PCC6803 woxA gene. A. pRBl, woxA cloned into a Bluescript vector ...

Construction of a Vector Plasmid pTRZ90 and Subcloning of a-amylase Gene of B. stearothermophilus... 

The upper growth temperature (59 C) of B. stearothermophilus (pTB90 (6.7 MDal, Km Tc )) could be elevated to 65 C when pTB90 was replaced by a vector plasmid, pTRZ90 (7.9 MDal, Km. ). The new vector plasmid was constructed by a series of steps including mutagenesis of pLPll (9.5 MDal ... 

Yoon K-H, Lee J-K, Kim BH (1991) Construction of a Clostridium acetobutylicum-Escherichia coli shuttle vector. Biotechnol Lett 13 1-6 Yoshino S, Yoshino T, Hara S, Ogata S, Ha) shida S (1990) Construction of shuttle vector plasmid between Clostridium acetobutylicum and Escherichia... 

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