Paper No. : 04 Genetic engineering and recombinant DNA technology
Module : 20 Phagemids, in vitro packaging, High-cloning capacity vectors
Principal Investigator: Dr Vibha Dhawan, Distinguished Fellow and Sr. Director The Energy and Resouurces Institute (TERI), New Delhi
Co-Principal Investigator: Prof S K Jain, Professor, of Medical Biochemistry Jamia Hamdard University, New Delhi
Paper Coordinator: Dr Mohan Chandra Joshi, Assistant Professor, Jamia Millia Islamia, New Delhi
Content Writer: Dr. Ashutosh Rai, SERB-National Post Doctoral Fellow, ICAR- Indian Institute of Vegetable Research, Varanasi-221305
Content Reviwer: Dr. Sharmistha Barthakur,Principal Scientist, National Research Centre on PlantBiotechnology, New Delhi – 110012, INDIA
Genetic engineering and recombinant DNA technology Biotechnology Phagemids, in vitro packaging, High-cloning capacity vectors
Description of Module
Subject Name Biotechnology
Paper Name Genetic Engineering and Recombinant DNA Technology
Module Name/Title Phagemids, in vitro packaging, High-cloning capacity vectors
Module Id 20
Pre-requisites
Objectives Phagemid vectors and their importance, In vitro packaging mechanisms, High capacity cloning vectors, Types of high capacity cloning vectors and their importance, Summary Keywords Phagemid, in-vitro packaging, Cosmids, BAC, YAC, MAC
Genetic engineering and recombinant DNA technology Biotechnology Phagemids, in vitro packaging, High-cloning capacity vectors
A. Phagemids, in vitro packaging, High-cloning capacity vectors
Cloning of large DNA fragments in bacterial or viral vectors provides easy multiplication of
DNA fragments without any modification, providing an opportunity to store and/ or sequence
these large fragments. This was not possible in past due to unavailability of vectors those
can carry a large DNA fragment of several mega bases. Novel approaches given new
possibilities to create and exploit these high capacity cloning vectors. With initiation of the
human genome project, Bacterial Artificial Chromosomes (BAC) was got familiar to everyone
due its enormous use in human genome project. Bacterial artificial chromosomes and yeast
artificial chromosomes played important role in various sitedirectional cloning activities,
physical mapping and whole genome sequencing projects. To fill the gap between genomic
libraries and sub cloning procedures in sequencing vectors various advanced viral cloning
vectors were used like pEMBL and pBlueScript etc. The functional understanding of these
viral vectors for sub-cloning as well as sequencing is important.
Phagemid Vectors have wide applicability in recombinant DNA technology like.
1. DNA sequencing
2. Mutagenesis study
3. probe generation
4. Phage display systems.
Bacteriophage M13 and its importance in molecular cloning
Bacteriophage M13 phage is filamentous phage that infects E. coli via F-pilus. The genome
is a single stranded circular DNA of size ~6.4kb surrounded by a proteinaceous coat. The
DNA strand present in phage is called plus (+) strand. After entering to E. coli host, it
converts into double stranded DNA molecule called replicative form (RF) by utilizing bacterial
machinery. M13 phage as cloning vector can be obtained in both single stranded as well as
double stranded form. Replicative form double stranded vector are modified and replicated
inside E. coli host similar to a plasmid vector. Single stranded vectors can be isolated by
collecting M13 phage. Phagemid vectors are plasmids having a small segment of a
filamentous phage M-13, fd, or F1 phage capable to carry up to 10 kb passenger DNA.
Genetic engineering and recombinant DNA technology Biotechnology Phagemids, in vitro packaging, High-cloning capacity vectors
Examples: pEMBL series of plasmids pBluescript family plasmids. The M13 origin of
replication allows the packaging of the plasmid in to a M-13 phage when the bacteria is also
infected by M13 helper phage. Phagemids generally predetermine no proteins or may have
only one kind of coat protein. The other structural and functional proteins which are
necessary complete the life cycle of phagemid are typically encoded by helper phage and
usually transcribed by the host.
In M13 bacteriophage, DNA is replicated by the rolling circle mechanism. In this mechanism,
one strand is cleaved and the DNA polymerase extends free 3’OH. The 3’ end on the circle
is extended while the rising point rolls around the loop template. The 5’end is displaced and
forms a tail of single stranded DNA. The single stranded tail is converted into double
stranded DNA by synthesis involving RNA primers. DNA replication of Rolling circle type
starts by an initiator protein encoded by the bacteriophage DNA, one strand of double
stranded circular DNA is nicked by initiator protein on the site known as DSO (double-strand
origin). The free 3' hydroxyl group serves as primer and is extended by DNA polymerase III,
while the initiator protein remains bound to the DSO. By this way the intact stand or unnicked
single stranded DNA serves as template strand and the replication proceeds displacing the
nicked single stranded DNA. A host encoded helicase displaces the nicked strand in the
presence of replication initiator protein. Several linearly connected copies circular template
DNA is formed in a conteneous manner joining as head to tail fashion and are called as
concatemer. After nicking of leading strand by initiator protein to stop further synthesis, these
single stranded linear DNA becomes double stranded circular DNA by RNA polymerase and
DNA polymerase III. The RNA primers are removed with the help of DNA polymerase I
replacing it with DNA and is ligated with DNA ligase forming many double stranded circular
DNA.
By this way Phagemid has certain advantages:
1. The carrying capacity of phagemid is higher than phage vectors.
2. Phagemid has higher efficiency in transformation than phage vectors.
3. Phagemids are genetically more stable than recombinant phage vectors.
4. Phagemids can be exploited to generate single stranded DNA template for
sequencing purposes.
Genetic engineering and recombinant DNA technology Biotechnology Phagemids, in vitro packaging, High-cloning capacity vectors
5. Single stranded phagemid vectors inside the phage can be targeted for site-directed
mutagenesis.
6. Single stranded vectors can be used to generate hybridization probes for mRNA or
cDNA.
One of the first hybrid phagmid vectors was pEMBL constructed in 1983. They are
distinguished by the presence of –
1) The bla gene, ampicillin resistance as selectable marker.
2) A alpha-peptide coding short segment of beta-galactosidase (lacZ), containing a MCS and
3) The intragenic (IG) region of phage F1.
These phagemid vectors have been used effectively for DNA sequencing with the Sanger’s
dideoxy method, can serve same functions of M13 derivatives. However, the pEMBL
plasmids have the advantage of being smaller than M13 vectors, and the purification of DNA
is simpler. In addition, long inserts have a higher stability in pEMBL plasmids than M13
vectors. Within bacteriophage such as M-13 the replication process is complex. Phage DNA
molecule generally carry several genes essential for the replication including genes for
components and phage coat protein and phage specific DNA replicative enzymes. Alteration
in any of genes will impair or destroys the replicative ability. So there is less freedom to
modify phage DNA molecule.
Phagemid: in vitro packaging
For the in vitro packaging of phage particles of phagemid vector like pEMBL8. The pEMBL8
was made by transferring 1300 bp fragment of M13 in to pUC8. This piece of M13 in
pEMBL8 contains signal sequences recognized by the enzyme that converts the normal
double stranded M13 molecule in to single stranded DNA before secretion of new phage
particles. The signal sequence is still remains functional even though detached from rest of
the genome of M13. When a normal M13 is used as helper phage, provides necessary
replicative enzymes and phage coat proteins.
Genetic engineering and recombinant DNA technology Biotechnology Phagemids, in vitro packaging, High-cloning capacity vectors
High-cloning capacity vectors
The high capacity cloning vectors, generally utilized for the production of genomic libraries.
These include cosmids, bacterial artificial chromosomes (BACs), P1-derived artificial
chromosomes (PACs) and yeast artificial chromosomes (YACs). These high cloning capacity
vectors have capacity to accommodate DNA fragments much larger than λ replacement
vectors. This reduces the need to screen large number of recombinant clones and these
require lower number of recombinants to be screened for recognition of a gene of interest.
In present table we can see different types of cloning vectors having their compatible
host, insert size, origin of replication and the structural conformation of DNA.
Vector Host Insert Range Introduction on to Origin of Vector structure (KB) host cell Replication Plasmids E. coli 1-5 Electroporation colE1 Circular plasmid Cosmids E. coli 5-47 Transduction colE1 Circular plasmid M13 E. coli 1-4 Transduction f1 Circular virus λ- Phage E. coli 20-30 Transduction f1 Linear virus P1- Phage E. coli 70-100 Transduction P1 Linear dsDNA PACs E. coli 100-300 Electroporation P1 Linear dsDNA BACs E. coli 300-350 Electroporation OriS, repE Circular plasmid YACs Yeast 200-2000 Transformation ARS Linear chromosome MACs MCC >2000 Transformation Centromeric/ Linear chromosome telomeric ori
Genetic engineering and recombinant DNA technology Biotechnology Phagemids, in vitro packaging, High-cloning capacity vectors
Bacteriophage P1
The bacteriophage P1 is a temperate phage that
infects E. coli. In its lysogenic cycle the P1 genome
remains as a plasmid in the bacterium. One of its
important feature is that, it hijacks the host machinery
and integrates in to host genome. The viron P1 has
icosahedral head and a tail with six fibres which help it
to anchor the host cell wall. The P1 phage have a
comparatively large genome approx 93 kb, as a linear
double stranded DNA molecule. After insertion in to
host it get circularized and replicates as plasmid. The
phage P1 has two ori, OriR responsible for lysogenic
cycle, where as OriL replicates it during lytic cycle. It can carry a foreign DNA up to 100 kb
and able to replicate it in to the host cytoplasm.
Cosmids
Cosmid vectors provide extra benefit over bacteriophage λ based cloning vector as they
have origin of replication from bacteria. Cosmids are chimeric in nature having region from
both, a bacteria based plasmid and bacteriophage λ. As cosmids have flanking cos sites,
just after their entry into the host cell they adopts circular form. In cosmids, benifit to have
bacterial origin provided efficiency to replicate within bacterial host cells, and can be
maintained in it. The bacterial cells can be selected for transformants on selection media due
to presence of antibiotic resistance gene (tetracycline). Cosmids also have a unique MCS
region into which DNA fragments can be ligated. The packaging of recombinant DNA in to
newly synthesized λ particles or virons are directoly can be used to infect E. coli cells. The
normal infection process just like λ injects the recombinant DNA in to host cells and get
circularized with the help of cos end complementation. In case of cosmids, the selection of
recombinants is made by antibiotic resistant bacterial colonies rather than phage plaques
makes it easy to select and multiply. The cosmids have ability to accomodate between 32 to
47 kbp of DNA due to fact that λ replacement vectors can accept 37 to 51 kbp of DNA while
the size of most cosmids are about 5 kbp. So these have accomodating capacity
Genetic engineering and recombinant DNA technology Biotechnology Phagemids, in vitro packaging, High-cloning capacity vectors
considerably more than λ vectors itself. In cosmids the cloning procedure is same as
discussed earlier in case of λ bacteriophage based cloning vectors.The example of cosmid
vector is pJB-8. Just like plasmid vectors cosmids have both slectable marker system and
an origin of replication. Thus cosmids are simply hybrid vectors of plasmids and cos site of
the λ phage (Collins and Bruning, 1978). Less than 48 kb foreign DNA can be carried by
cosmid and presence of cos sites enables enzymes produced by the actual phage to
package it in to the phage capsids assuming its of correct size. There are some
disadvantages of cosmid vectots that include higher frequency of recombination inside
bacterial host and cosmids are unstable inside E.coli host and thus easy to lose vector.
Artificial chromosomes
Artificial chromosomes are DNA molecules assembled in vitro from defined
constituents that can function like natural chromosomes.
Types of artificial chromosomes:
1. BACs: Bacterial artificial chromosomes
2. YACs: Yeast artificial chromosomes
3. MACs: Mammalian artificial chromosomes
4. HACs: Human artificial chromosomes
5. PACs: P1-derived artificial chromosomes
Bacterial Artificial Chromosome (BAC)
Bacterial artificial chromosomes (BACs) are designed for the cloning of large DNA insert
(typically 100 to 300 kb) in E. coli host. BAC vectors contain a single copy F-plasmid origin of
replication (ori). The F (fertility) plasmid is relatively large and vectors derived from it have a
higher capacity than normal plasmid vectors. F-plasmid has F (fertility) factor which controls
the replication and maintain low copy number. Also conjugation can take place between F+
bacteria (male) and F- bacteria (female) to transfer F-plasmid via pilus.
Common gene components of a bacterial artificial chromosome are:
1. oriS, repE – F responsible for plasmid replication and regulation of copy number.
2. parA and parB for maintaining low copy number and avoiding two F plasmids in a
single cell during cell division.
Genetic engineering and recombinant DNA technology Biotechnology Phagemids, in vitro packaging, High-cloning capacity vectors
3. A selectable marker for antibiotic resistance; fue BACs also have blue/white selection
having lacZ gene at the cloning site.
4. T7 and Sp6 phage based promoters examining transcription of genes of interest.
Yeast Artificial Chromosome (YAC)
Yeast Artificial Chromosomes were described first time by Murray and Szostak in 1983, a
YAC has sequences to exist inside E. coli as a circular plasmid and contains sequences to
maintain as linear nuclear chromosome in yeast. As YAC vectors can accommodate 100-
500 kb of insert DNA. The number of clones in a genomic library can be greatly reduced.
YAC vectors have following elements:
1. coli origin of replication
2. Yeast origin of replication
3. Elements of eukaryotic yeast chromosome (centromere and telomere region)
4. Selection markers for both the hosts (Bacterial as well as Yeast)
YAC is a vector used to clone DNA fragments larger than 100 kb and up to 3,000 kb. YACs
are useful for the physical mapping of complex genomes and for the cloning of large genes.
Yeast artificial chromosomes are created artificially joining centromere (CEN), telomeres
(TEL), and an origin of replication as autonomous replicating sequence (ARS) elemets
necessory for replication and conservation of YAC in host yeast. A circular plasmid is used
to create YAC by cleaving it in to two linear fragments by appropriate restriction
endonucleases. These two linear fragments ligated with desired foreighn DNA using DNA
ligase enzyme forming single large linear DNA molecule. TRP1 and URA3 genes are
integrated in the YAC vector to provide a selection system for identifying transformed yeast
cells that include YAC by complementing recessive alleles trp1 and ura3 in yeast host cell.
Mammalian Artificial Chromosome (MAC)
MACs or mammalian artificial chromosomes, like YACs, rely on the presence of centromeric
and telomeric sequences and origin of DNA replication. The MAC have ability to replicate
autonomously and segregate in mammalian cells. These MAC can be modified for
expression studies of not only large genes and their coding regions but also the control
elements found DNA.
There are two basic procedures for preparation of MACs.
Genetic engineering and recombinant DNA technology Biotechnology Phagemids, in vitro packaging, High-cloning capacity vectors
1) In first method, telomere-directed fragmentation of natural chromosomes is used. For
example, a human artificial chromosome (HAC) has been derived from chromosome 21
using this method.
2) Another method involves de novo assembly of cloned centromeric, telomeric, and
replication origins in vitro.
MAC vectors are difficult to assemble as compared to YAC vectors. Mammalian DNA has
higher degree of repetition and larger centromere and telomere regions. Also the sequences
necessary for chromosome replication in mammalian system are not well defined till now.
MAC vectors have application in the field of gene therapy and eukaryotic protein expression
and production.
B. Summary
The novel sequencing tools and eager to know in deep about functional mechanism of complex organism opened a rush toward whole genome sequencing projects. The cDNA libraries and development of genomic sequence based markers also made it necessary to have cloning vectors that can accommodate large DNA segments. The gap was filled by several artificial chromosome vectors those replicate independent of their corresponding hosts. Bacterial artificial chromosomes and Yeast artificial chromosomes played a crucial role in genomic library construction. For the sub cloning and sequencing purposes M13 based phagemids were developed. These phagemids are easy to handle and are reliable for storage for longer periods. In recent few years plant artificial chromosomes are being developed to create novel tools for transgenic developments.
Bibliography:
Cooke H. 2001. Mammalian artificial chromosomes as vectors: progress and prospects; Cloning Stem Cells, 3(4): 243-249. Dente L., Cesareni G., Cortese R. 1983. pEMBL: a new family of single stranded plasmids; Nuc. Acid Res. 11 (6) 1645-1655. Hall B.G. 2004. Predicting the evolution of antibiotic resistance genes. Nat Rev Microb 2 (5): 430–435. http://bioinfo2010.wordpress.com/2009/07/08/vector-bacteriophage-lambda-and-m13-7th- april/ Kim et al . 1992. Stable propagation of cosmid-sized human DNA inserts in an F-factor based vector . Nucleic Acids Res. 20 (5): 1083–1085.
Genetic engineering and recombinant DNA technology Biotechnology Phagemids, in vitro packaging, High-cloning capacity vectors