Paper No. : 04 and recombinant DNA technology

Module : 20 , in vitro packaging, High- 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 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, , 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 project, Bacterial Artificial (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. systems.

Bacteriophage M13 and its importance in

Bacteriophage M13 phage is filamentous phage that infects E. coli via F-. 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 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 vector. Single stranded vectors can be isolated by

collecting M13 phage. Phagemid vectors are 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 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 cycle of phagemid are typically encoded by helper phage and

usually transcribed by the host.

In , 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

. 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 , 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 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, and the structural conformation of DNA.

Vector Host Insert Range Introduction on to Origin of Vector structure (KB) host Replication Plasmids E. coli 1-5 colE1 Circular plasmid Cosmids E. coli 5-47 colE1 Circular plasmid M13 E. coli 1-4 Transduction f1 Circular 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 MACs MCC >2000 Transformation Centromeric/ 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 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 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

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 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 .

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 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 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 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 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 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 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