The document discusses the characteristics and applications of various cloning vectors, particularly focusing on bacteriophage P1, which can clone DNA fragments up to 100 kb. It describes the life cycle of P1, including its methods of DNA injection, replication, and the creation of transducing particles, as well as its use in genomic libraries and phage therapy. Additionally, it highlights the advantages and limitations of different vectors in cloning processes.

1. P1 DERIVED ARTIFICIAL
CHROMOSOME
Presented by
Nagendra P
16PBT2004 1

2. CLONING VECTORS
Key features of an efficient vector.
 Origin of replication
 Multiple cloning site(MCS)
 Selectable marker
 Easy isolation from host cell
2

3. CLONING VECTORS
Plasmids
Lambda phages
Cosmids
Phasmids and Phagemids
BACs
PACs
YACs
Plant transformation vectors
3

4. CHOICE OF VECTORS
Vector Host Insert size
Lambda phage E.coli 5-25kb
Lambda cosmids E.coli 35-45kb
P1 phage E.coli 70-100kb
PACs E.coli 100-300kb
BACs E.coli ≤ 300kb
YACs S.cerevisiae 200-2000kb
4

5. The major advantage of the cosmid system for cloning is that the DNA insert can be
packaged efficiently into phage lambda particles.
However, the maximum amount of DNA that can be cloned into any one cosmid
vector, 45-48 kilobase pairs (kbp), is limited by the size of the lambda head.
In contrast, YAC cloning vectors can accept and propagate DNA that is as large as
200-800 kbp.
Limitations of the YAC system are
(i) the low efficiency of transformation with vector DNA containing large
inserts
(ii) the decreasing transformation efficiency with increasing insert size
(iii) the need to process transformants individually prior to screening and
(iv) the difficulty in obtaining large amounts of insert DNA from transformed
cells.
5

6. PAC
Bacteriophage P1 was isolated in 1951 by Giuseppe Bertani from the Escherichia coli strain.
Phage P1 is a temperate bacteriophage which has been extensively used for genetic analysis of
Escherichia coli.
It can mediate generalized transduction.
Sternberg and co-workers have developed a P1 vector system.
Has a capacity for DNA fragments as large as 100 kb.
Thus the capacity is about twice that of cosmid clones but less than that of yeast artificial
chromosome (YAC) clones.
6

7. 7

8. STRUCTURE AND GENOME:
P1 has an
icosahedral “head” containing the phage DNA attached to a 220nm long tube,
contractile tail or sheath, and
base plate with six tail fibers.
The P1 capsid is composed of 15 head proteins, 9 tail proteins, and 4 proteins of
undetermined location
In a same virion population, 80% of virions have a head diameter of 85nm, while the
other 20% have a diameter of 65nm.
8

9. GENOME
The genome of P1 Phage is linear double stranded DNA , about 94 Kbp.
The genome is longer, about 120Kbs, than the actual length when in viral particle as it
is created by cutting an appropriately sized fragment from a concatemeric DNA chain
having multiple copies of the genome.
Due to this, the ends of the DNA molecule are identical and are referred to as
being “terminally redundant”. It has large, about 15 kbp terminal redundancy.
In the viral particle it is in the form of a linear double stranded DNA molecule, once
inserted into the host it circularizes because of the its large terminal redundancy and
replicates as a plasmid.
9

10. Two origins of replication,
oriR which replicates it during the lysogenic cycle and
oriL which replicates it during the lytic stage.
The genome has117 genes that are organized in 45 operons.
4 are responsible for the choice between lysis and lysogeny and another 4 are
responsible for plasmid maintenance.
The remaining 37 are invovled in lytic development.
Maintains one copy per host cell.
10

11. 11

12. THE LIFE CYCLE OF P1 PHAGE
1. Adsorption, Injection and protection of the genome
P1 adsorbs to the receptors on the host cells – terminal glucose on the
lipopolysaccharide present on the outer surface of the outer membrane of the host cell.
 A lytic transglycosylase faclitates the puncture of the bacterial cell wall.
P1 contracts its tail and inject its DNA into the cell.
Once inside the cell, P1 DNA circularizes by homologous recombination because
these DNA when packed it is packed by Head full Mechanism. This can be done either
by host or phage recombination system.
12

13. 2. P1 DNA Replication and Phage Assembly. (Lytic Cycle)
The P1 plasmid has a separate origin of replication (oriL) that is activated during the
lytic cycle.
Early P1 replication takes place by the theta mode of replication. Later in infection, P1
switch to rolling circle replication.
Rolling circle replication produces concatemers for packaging into phage heads.
At approx 45 minutes after the infection, the cells are filled with concatemers of phage
DNA, assembled phage heads, and assembled phage tails.
Now complete phage must take place. A protein made from phage genome cre
recombinase recognizes a site on the concatemers of phage DNA called the pac site.
The protein cuts the DNA, making a double-stranded end. This end is inserted into a
phage head. The DNA continues to be pushed inside the head until the head is full, a
process called head-full packaging.
After the head is full of DNA, a double stranded cut is made and a tail is attached. Once
the complete virions are assembled, the host cell is lysed, releasing the viral particles.
13

14. 3.The Location of the P1 Prophage in a lysogen (Lysogenic
Cycle)
Prophages can be physically located in one or two places in a lysogen unlike in case of
Lambda, the phage genome is recombined into the bacterial chromosome.
P1 contains an origin for DNA replication and once the phage genome is converted to
circular, double-stranded DNA, it is maintained in the cytoplasm as a stably inherited
extra-chromosomal piece of DNA or plasmid.
Coordinated DNA replication occurs, in order to maintain its population in all daughter
cells. If not, when multiplied by binary fission only one daughter cell will posses the
phage DNA and another daughter cell does not.
14

15. 4. P1 Transducing Particles
Formation of transducing particles or phage particles that contain chromosomal
DNA instead of phage DNA.
E-Coli chromosomes 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.
So the resulting phage carry random pieces of the chromosome in place of phage
genomes.
The ability to package any piece of chromosomal DNA instead of phage DNA
makes P1 a generalized transducing phage.
15

16. CONSTRUCTION OF PAC
P1 vector contains a packaging site (pac) which is necessary for in vitro packaging of
recombinant molecules into phage particles.
The vectors contain two loxP sites. These are the sites recognized by the phage cre
recombinase, the product of the phage cre gene, and which lead to circularization of
the packaged DNA after it has been injected into an E. coli host expressing the
recombinase.
Cre (Causes Recombination) and the recombination site was named loxP (locus of
crossing (x) over, P1)
16

17. 17
CRE RECOMBINASE

18. In strains containing a lacI repressor, the P1 plasmid replicon maintains the DNA at
about one copy per host chromosome.
If IPTG is added to the medium the P1 lytic replicon in the vector is induced and the
copy number of the plasmid increases up to 25 folds in 6 generations.
18

19. USES
PACs are in high demand when it comes to cloning important biomedical sequences,
which are essential for many scientific functions.
This P1 system has been used to construct genomic libraries of mouse, human, fission
yeast, and Drosophila
One of its main uses is the genome analysis and map based cloning of complex plants
and animals, which requires isolation of large pieces of DNA rather than smaller
segments.
Furthermore, PAC based cloning is useful in the study of ‘phage therapy’ and in
scientific studies focusing on how antibiotics act on a particular bacteria.
19

20. REFERENCES
Sternberg N. Bacteriophage P1 cloning system for the isolation, amplification, and recovery of
DNA fragments as large as 100 kilobase pairs. Proceedings of the National Academy of Sciences.
1990 Jan 1;87(1):103-7
Lehnherr H, Yarmolinsky MB, Blattner FR. Genome of bacteriophage P1. JOURNAL OF
BACTERIOLOGY. 2004 Nov:7032-68.
Abremski K, Hoess R. Bacteriophage P1 site-specific recombination. Purification and properties
of the Cre recombinase protein. Journal of Biological Chemistry. 1984 Feb 10;259(3):1509-14.
Nagy A. Cre recombinase: the universal reagent for genome tailoring. genesis. 2000 Feb
1;26(2):99-109.
https://biotechkhan.wordpress.com/2014/07/08/bacteriophage-p1-structure-and-life-cycle/
http://bio.classes.ucsc.edu/bio105l/EXERCISES/P1/introduction.pdf
20

21. 21