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,