APPLICATION OF PHAGES IN BIOTECHNOLOGY TRANSDUCTION CRE LOX P SYSTEM LECTURE 12: APPLICATION OF PHAGES IN BIOTECHNOLOGY TRANSDUCTION CRE LOX P SYSTEM Viro102: Bacteriophages & Phage Therapy 3 Credit hours NUST Centre of Virology & Immunology

P1 Phages Genus: P1 like Viruses Family: MYOVIRIDAE A temperate phage that infects E. coli It is the largest of the coli phages Consists of double stranded DNA, terminally redundant, & circularly permuted In lysogeny, P1 can exist within a bacterial cell as a circular DNA in that it exists by replicating as if it were a plasmid & does not cause cell death Alternatively, in its lytic phase, P1 can promote cell lysis during growth resulting in host cell death

Electron micrograph of a bacteriophage P1 particle Electron micrograph of a bacteriophage P1 particle. The black bar represents 100 nm

TRANSDUCTION It’s the process of moving a piece of chromosomal DNA from one cell to an other using bacteriophage to carry DNA Transduction was describe first time by Zinder and Lederberg in 1952 as way to move heritable trait from one strain of Salmonella enterica serovar typhimurium to another They notice that agent responsible for chromosomal DNA transfer is not filterable

This process allows bacteria to acquire segment of DNA approximately 5o to 100 kb It increase the pace of evolution by restoring mutated section of chromosome Or allow acquisition of block of new genes ( Evolution) Transduction is by product of phage life cycle having no benefit to it

P1 has two characteristics that make it suited to fanction as transducing particle: It encodes nuclease that degrade host DNA, so when DNA packaging begins, Host DNA would present at large amount. P1 packaging system is not very much fastidious. So the size of DNA is not exactly the same in all particles

GENERALIZED TRANSDUCTION SPECIALIZED TRANSDUCTION

In generalized, any part of the bacterial chromosome may be transduced. Examples of generalized transducing phage are bacteriophage P22 in Salmonella and bacteriophage P1 in E. coli. In specialized, only certain special pieces of the bacterial chromosome can be transduced. Bacteriophage l is the classic example of a specialized transducing phage.

The E. coli chromosome contains many pseudopac sites or sites that can be used to initiate packaging of host chromosomal DNA into maturing phage. These pseudopac sites are used much less frequently than the phage pac sites but they are used. The resulting phage carry random pieces of the chromosome in place of phage genomes.

P1 & Cre LoxP System A unique system of site specific recombination was observed Discovered by Professor Brian Sauer. Patented by US chemical company DuPont. Great potential to be used as a biotechnology tool. It has been successfully applied in yeasts, plants, mammalian cell cultures, and mice.

Cre Lox P System This system has two major players: Cre (Cyclization Recombination) is a recombinase enzyme found in Bacteriophage P1 38 kDa virus encoded enzyme Responsible for symetric recombination that near the loxP site

Cont’d The second player is: LoxP (locus of X-over P1) is a site on the phage P1 Consists of a 34 bp The core sequence (8 bp) is flanked by two inverted repeats (13 bp) ATAACTTCGTATA GCATACAT TATACGAAGTTAT INVERTED REPEATS INVERTED REPEATS

Role in P1 Life Cycle The first role involves circularization of P1 DNA The P1 DNA in the phage head is a linear double stranded molecule and must be circularized when it enters the host The second role involves the breakdown of dimer P1 DNA molecules that may form as a result of replication or homologous recombination

Cont’d The most obvious role of P1’s Cre LoxP system is in the evolution of the phage particle. This takes place by the site specific recombination between the host cell genome & phage or this phage & other phages infecting the same cell.

SITE SPECIFIC RECOMBINATION Process of exchange of DNA between segments that possess some degree of homology. SITE SPECIFIC RECOMBINATION AGCTCCTTAGGAAA TCGAGGAATCCTTT SEQUENCE 1: Site-specific recombinases perform rearrangements of DNA segments by recognizing and binding to short DNA sequences (sites), at which they cleave the DNA backbone, exchange the two DNA helices involved and rejoin the DNA strands DNA sequences with limited degree of homology that are recognized by Recombinases RECOMBINASE AGCTCCTTAGGAAA TCGAGGAATCCTTT SEQUENCE 2: AGCTCCTTAGGAAA TCGAGGAATCCTTT SEQUENCE 1: SEQUENCE 2:

Cre LoxP System Cre brings the two loxP sites close together whether they lie in direct or inverse orientation. The efficiency of the enzyme, Cre is not disturbed whether the DNA is super coiled, linear or relaxed circle unlike all the other recombinase enzymes.

WHEN LOX-P SITE ORIENTED IN SAME DIRECTION GENE CRE GENE If the loxP sites are oriented in the same direction on a chromosome segment (cis arrangement), Cre recombinase mediates a deletion of the floxed segment.

WHEN LOX-P SITE ORIENTED IN OPPOSITE DIRECTION GENE CRE GENE If the loxP sites are oriented in opposite directions, Cre recombinase mediates the inversion of the floxed segment.

WHEN LOX-P SITE IS IN DIFFERENT CHROMOSOMES GENE CRE GENE If the loxP sites are located on different chromosomes (trans arrangement), Cre recombinase mediates a chromosomal translocation or exchange of fragments.

Video for Cre-Lox recombination mechanism http://www. ytpak. com/watch

Thank You!