Recombinant vectors

A vector for in vitro expression of DNA inserts as RNA transcripts can be constructed by putting a highly efficient promoter adjacent to a versatile cloning site. Figure 13.15 depicts such an expression vector. Linearized recombinant vector DNA is transcribed in vitro using SPG RNA polymerase. Large amounts of RNA product can be obtained in this manner if radioactive ribonucleotides are used as substrates, labeled RNA molecules useful as probes are made. 

PEC. The patent includes the production method for the biocatalyst, with a characteristic inhibitory effect on certain enzyme expression. The expression recombinant vector contains a promoter free from manifesting inhibition due to an inorganic sulfur compound or a sulfur-containing amino acid. The recombinant microorganism also contains a gene for desulfurizing the sulfur-containing heterocyclic compound. 

Figure 13.18 Replica plating for the selection of bacteria containing a recombinant vector, (a) The method used to transfer colonies of bacteria between plates, (b) Comparison of the distribution of the colonies between the plates enables identification of the colonies containing the recombinant vector. The dotted circles represent colonies that do not grow on tetracycline.

Once the recombinant vectors have been produced, they are used to transform host cells. In the example of the plasmid pBR322, the host cells are bacteria. Once transformed, the bacteria are plated on selective media so that bacteria transformed with a recombinant plasmid can be easily identified. In the case of plasmid pBR322 shown in Figure 1-6-3, bacteria with recombinant plasmids would be resistant to ampicillin but sensitive to tetracycline. 

Transform competent coli host with recombinant vector and select for recombinants by antibiotic resistance appropriate for the plasmid. 

E. coli host strain AR58 containing defective phage lambda lysogen transformed with a recombinant vector which carries antibiotic resistance for kanamycin (Kan R). 

The recombinant vectors are introduced into E. coli by electroporation... 

Additional notes on the AdEasy system are available at www.coloncancer.org/ adeasy.htm. The most challenging step in the procedure is the generation of successful recombinants. Several factors affected recombination efficiency, including the quality of the preparation of the electrocompetent BJ5183 cells. We found that increasing the time of incubation of the cells on ice from 30 min to 1 to 2 h improved recombination efficiency. We typically isolated at least 10 colonies to ensure identification of a recombinant vector. 

Yan, Z., Zak, R., Luxton, G. W., Ritchie, T. C., Bantel-Schaal, U. and Engel-hardt, J. F. (2002). Ubiquitination of both adeno-associated virus type 2 and 5 capsid proteins affects the transduction efficiency of recombinant vectors. J. Virol. 76, 2043-2053. 

The safety profile of recombinant AAV (rAAV) vectors is attributed to the lack of association with human disease as well as the ability to generate recombinant vectors that contain no viral genes. Similar to adenoviral vectors, AAV can infect both dividing and non-dividing cells, and has a broad tissue tropism. In contrast to adenoviral vectors, rAAV vectors have a limited packaging capacity of about 4.9 kb, limiting their use to diseases in which the deficient gene is relatively small. 

These general approaches of course may be modified or restructured to suit a particular problem. In probably the majority of sequencing undertakings the DNA of interest will have been cloned and amplified in a suitable vector and while in some instances the cloned fragment can be precisely cleaved out from the recombinant vector and isolated by gel electrophoresis, in other cases this may not be possible. The shotgun method described above will yield a complex array of end-labelled fragments and the problem immediately arises of distinguishing which ones... 

Nearly any type of cell (prokaryotic or eukaryotic) can be transformed by the technique of electroporation. Protoplasts are first prepared by enzymatic or chemical disruption of the host-cell membrane polysaccharides. Next, the recombinant vector is introduced to the protoplast suspension residing in a very low ionic strength buffer (or distilled water). This DNA-protoplast suspension is then subjected to one or several 250-V pulses delivered from a cathode and anode placed directly into the solution. This applied voltage gradient will cause a certain population of the cells (—1010 per... 

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