Bacteriophage vectors

Fig. 4. Construction of recombinant phage in vectors derived from bacteriophage lambda where E represents the enzyme EcoRl. Other terms are defined...

Expression vectors are engineered so that any cloned insert can be transcribed into RNA, and, in many instances, even translated into protein. cDNA expression libraries can be constructed in specially designed vectors derived from either plasmids or bacteriophage A. Proteins encoded by the various cDNA clones within such expression libraries can be synthesized in the host cells, and if suitable assays are available to identify a particular protein, its corresponding cDNA clone can be identified and isolated. Expression vectors designed for RNA expression or protein expression, or both, are available. 

FIGURE 13.15 Expression vectors carrying the promoter recognized by the RNA polymerase of bacteriophage SPG are useful for making RNA transcripts in vitro. SPG RNA polymerase works efficiently in vitro and recognizes its specific promoter with high specificity. 

Yeast (Saeeharomyees eerevisiae) has a genome size of 1.21 X 10 bp. If a genomic library of yeast DNA was constructed in a bacteriophage A vector capable of carrying 16-kbp inserts, how many indi-... 

PAC A high capacity (70-95 kb) cloning vector based upon the lytic E. colt bacteriophage PI that replicates in bacteria as an extrachromosomal element. 

Vector A plasmid or bacteriophage into which foreign DNA can be introduced for the purposes of cloning. 

The bacteriophages or plasmids that have foreign DNA built into them in this manner are called vectors. Usually bacteriophages insert... 

An essential feature of the cloning vector used is that it must be capable of self-replication in the cell into which it is introduced, which is usually E. coli. Two of the most commonly used types of vector in conjunction with E. coli are plasmids and bacteriophage X. Plasmids are circular extra-chromosomal DNA molecules, generally between 5000 and 350 0000 bp in length, that are found naturally in a wide range of bacteria. They generally house several... 

Christensen, A. 2001. Bacteriophage lambda-based expression vectors. Molecular Biotechnology 17, 219-224. 

The first step is the isolation of DNA genes that code for the production of the desired protein. The next stage is the insertion of these genes (foreign DNA) into a vector, or carrier. Common vectors used are the bacteriophage (a virus) and the bacterial plasmids, which are circular bacterial DNA. 

A prerequisite for cRNA transcript synthesis is the presence of the cDNA of interest within a suitable expression vector containing a bacteriophage polymerase promoter (e.g. T3, T7 or SP6). The vector must be linearized downstream of the cDNA insert prior to its use as the template in the in vitro reaction. 

The principles that govern the delivery of recombinant DNA in clonable form to a host cell, and its subsequent amplification in the host, are well illustrated by considering three popular cloning vectors commonly used in experiments with E. coli—plasmids, bacteriophages, and bacterial artificial chromosomes—and a vector used to clone large DNA segments in yeast. 

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