Plasmid constructs

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.

Experiment 14 introduced students to some principles and techniques involved in recombinant DNA research. Specifically, the experiment outlined the replication, isolation, and analysis of bacterial plasmid vehicles for molecular cloning experiments. This experiment describes another tool that is essential in hybrid plasmid construction and analysis-restriction enzyme action. The procedures introduced here have also found widespread use in the analysis and characterization of all DNA molecules. 

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

K. Timmis, F. Cabello, and S. N. Cohen, Pwc. Natl. Acad. Scl US, 74 4556-4560 (1974). Utilization of Two Distinct Modes of Replication by a Hybrid Plasmid Constructed in vitro from Separate Replicons. 

Two separated proteins of sRL were expressed with plasmid construct (Fig. lb). The... 

Significant research was conducted with a view to developing and introducing hirudine (an anti-coagulant) as a medicine in Hungary. In hardly more than 30 months, by application of the recombinant DNA technique, and with the use of E coli, Saccharomyces and Streptomyces host organisms, various plasmid constructions were prepared, and from yeast-ferment-broth an HV-1 hirudine component was obtained, which allowed industrial-scale production of hirudine. 

Developed a new high-throughput screening procedure based on a DNA plasmid, constructed to study the expression of in vitro mutated hydrogenases introduced into wild-type algae. 

Plasmid Construction to Examine Promoter Dilution XYLl was ampUfied and fused with the TEFl or TDH3 promoter by PCR as above. This fragment was then inserted into pRS316... 

Plasmid Construction, Yeast Transformation, and Gene Expression... 

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