Bacteriophages recombinant

Table 2.1 Efficiency of cloning PCR fragments by bacteriophage recombination...

Feher, T., Karcagi, I., Blattner, F.R., and Posfai, G. (2012) Bacteriophage recombineering in the lytic state using the lambda red recombinases. Microb Biotechnol 5,466-476. 

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

Cre is a bacterial recombinase (cre=causes recombination), which recognizes loxP sites of bacteriophage P. If two loxP (loxP= locus of x-ing over of bacteriophage P) sites have a parallel orientation, the DNA segment between these sites will be deleted by the action of the Cre recombinase. 

A standard curve is defined by light emission from the standards containing known concentrations of recombinant bacteriophage. A quadratic equation is used to fit the curve to the RLU of the four standards. A maximum of two points from different standards may be eliminated by the data management software in order to achieve the best curve fit. The concentration of the target nucleic acid in the sample is determined from this standard curve. An example of the output from the data management software for the second-generation HCV assay is shown in Fig. 6. 

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... 

Sauer B, Henderson N (1988) Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage PI. Proc Natl Acad Sci USA 85 5166-5170... 

Similar methods were used soon after in the laboratory of Paul Berg to join DNA segments from simian virus 40 (SV40) to DNA derived from bacteriophage A, thereby creating the first recombinant DNA molecule with DNA segments from different species. 

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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