Bacteriophage cloning

Plasmid cloning. Anneal frag- Bacteriophage cloning. Insert Cosmid cloning. Cleave 35 to... 

Southard-Smith M, Pierce JC, MacDonald RJ. Physical mapping of the rat tissue kallikrein family in two gene clusters by analysis of PI bacteriophage clones. Genomics 1994 22 404-417. 

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. 

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. 

An alternative to repeated cloning of PCR products is a recombination-based approach developed by Liu et al. (1998) to permit the cloning of a PCR product into a plasmid and the rapid conversion of the plasmid to a number of different expression systems without the necessity of cloning the PCR product multiple, independent times. The method, termed the univector plasmid-fusion system (UPS), involves the insertion of the PCR product into a particular type of plasmid, called the univector, which can then be placed under the control of a variety of promoters or fused in-frame to various tag sequences. The system is based upon plasmid fusion using the Cre-lox site-specific recombination system of bacteriophage PI (Sternberg et al., 1981). The Cre enzyme is a site-specific recombinase that catalyzes recombination between two 34 base pair (bp) loxP sequences and is involved in the resolution of dimers formed during replication of the... 

Figure 5.8. Schematic illustration of T7 bacteriophage display system. The gene encoding the protein of interest is fused to the gene 10 protein that encodes a coat protein in the head of the phage. Cloning is to the C-terminus of coat protein 10. Figure adapted from Sche et al. (1999).

Figure 5.15 The filamentous single-stranded DNA bacteriophage fd. Orientation of the proteins and genes in the virion. Note the intergenic space which contains the origin of DNA synthesis. Gene cloning is done in this intergenic space.

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... 

NPN43C9 was shown to give a rate acceleration for hydrolysis of [50] of approximately 1.5 X 105, and its values of Km and Fmax were approximately the same as those for its Fab fragment, whose RNA sequence was subsequently used in cloning and expression of Fabs in a bacteriophage A system (Huse et al., 1989). Such an enterprise is capable of giving a greatly... 

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