PRESENTED BY: SIDRA NOOR. COURSE TITLE: APPLIED GENETICS.

CLONING VECTORSCOSMIDS AND PHASMIDS

CLONING VECTORS

A small piece of DNA, taken from virus or plasmid.

Used for construction of recombinant DNA.

Used for various Cloning purposes.

CosmidsHybrid plasmid that contain lambda phage cos sequence.

Discovered by Collins and Hohn.

Clone DNA insert upto 45kb.

For packaging, specific sequence and cleavage by terminase is require.

Then selection of recombinant DNA is done.

What is Cos sequences?cos sequence contain approx. 200 bases.

It contain 3 sites. Cos N site Cos B site Cos Q site

Antibiotic Selection Marker in Cosmids

Cosmids are predominantly plasmids with a bacterial oriV, an antibiotic selection marker and a cloning site, but they carry one, or more recently two, cos sites derived from bacteriophage lambda.

Loading capacity of cosmids varies depending on the size of the vector itself but usually lies around 40–45 kb.

Cosmids, however, always form colonies and not plaques.

Cosmid library construction

Steps involved in cosmid library construction Cosmid DNA is digested with appropriate endonuclease.

Genomic DNA fragments are generated by partial digestion with MboI or Sau3AI.

Genomic DNA is ligated with vector DNA.

Ligated DNA is mixed with λ bacteriophage and packaged into phage heads.

E.coli strains are infected with phage particles and recombinant cosmid is propogated.

Cosmid library is then screened.

Cosmid library construction

In vitro packaging

The protein that makes head of phage particle are assembled into head, phage DNA is replicated seperately and then inserted into phage head.

Packaging extracts consist of: Empty phage heads. Unattached phage tails. Phage encoded proteins.

Phage proteins: Nu1 and A –codes for terminase to package the cosmid DNA at cos sites. B, C, D, E, and Nu3- codes for synthesis and assembly of phage heads. G, H, I, J, K, L, M, T, U, V, and Z – codes for assembly of phage tails

In vitro packaging

Cosmid rescueIn cosmid rescue, we group together various procedures

in which transforming DNA containing all or some cosmid markers is selectively recovered by in vitro packaging.

Efficiency will be greatest if the genomic transformed DNA can itself be used as packaging substrate.

DNA transformed into a eukaryotic organism may persist there in an integrated state.

Integration into the recipient DNA may occur in homologous or non homologous chromosomes.

Cosmid can also be rescued if a cos containing sequence is rearranged by a physiological process that makes it packageable.

Cosmid rescue

Rhizobium mutant analysis We have modified pRK290 by ligating a BglII

fragment, purified from the cosmid pHC79 and containing the X cos site.

The resulting plasmid, pLAFR1, is an EcoRI cloning vector which is ligated to DNA in the size range of 15-31 kb can be packaged phage heads, infected into E. coli.

In the presence of the helper plasmid pRK2013, a pLAFR1 derivative can be conjugated into a suitable Gram-negative recipient.

The use of pLAFR1 in the genetic analysis of R. meliloti, the nitrogen-fixing endosymbiont of alfalfa.

Phasmids Also called as phagemids, are hybrids formed between small multicopy plasmids and bacteriophages.

A phasmid can be propagated as a plasmid or lytically as a phage.

Lytic functions of phasmid can be switched off by propagation in the appropriate lysogene where the plasmid origin of replication is used for maintenance.

Useful in the analysis of mutations exhibiting non-selectable or lethal phenotypes.

Recombinant DNA formation Phasmids or λ insertion vectors consists of: shortened linear λ genome. containing replication and lytic functions. cohesive ends of phage DNA.

During recombinant DNA construction, one or more copies of plasmid are deleted and DNA insert is integrated into vector.

One copy of the plasmid retained in recombinant DNA.

Both recombinant and unaltered Phasmid are packaged into vector and then ready for infection to E.coli cells.

The plasmid component of a Phasmid vector contains the λ attachment (λattB) site. As a result, the plasmid may integrate into the λ genome that leads to integration of λ genome into the bacterial chromosome; this event is known as lifting the plasmid.

Phasmid Vector λ ZAPHighly developed vector that contain sequences from λ, M13,T3 and T7 genome.

Its genome contain pBluescriptSK- and f1 initiator and terminator sequence from phage.

When phasmid infect E.coli cells and then superinfected by f1 phage the pBlueScript(-) may be recovered.

Phasmid Vector λ ZAP

Features of λ ZAPThe DNA insert is placed within MCS (multiple cloning site)

located in the lacZ gene of pBluescriptSK(-); commonly the unique EcoRI site is used.

It is particularly suitable for cloning of cDNAs using EcoRI linkers.

Integration of DNA insert inactivates lacZ gene; this permits blue/white selection on Xgal plates (white plaque = recombinant phasmid).

DNA insert can be expressed as fusion protein as in the case of phagemid Blue-script.

RNA copies of the DNA insert can be generated.

The recombinant plasmid BluescriptSK(-) is automatically excised in vivo from the phage vector. It can be used in DNA sequencing.

Presence of cl gene allow the phasmid to replicate like plasmid

Absence of cl gene produce lytic repressor and allow phasmid to multiply like phage that produce plagues on bacterial lawn.

If mutant cl gene is present, the temperature sensitive cl gene is produce.

Such vectors replicate like phage at high temperature and replicate like plasmid at low temperature.

cl gene in phasmid

pMYF11 A hybrid of lambda 47.1 vectors and pBR322 plasmid.

It can be used as a replacement vector for BamHI, HindIII, SalGI endonucleases.

The maximum size of fragments to be cloned is 21 kilobase pairs.

A library of Escherichia coli genes is constructed with the help of lambda pMYF11 as a vector molecule.

CI- marker of pMYF11 is replaced with cI+ marker by recombination between the plasmid and prophage 434.

D29 shuttle phasmid Shuttle phasmid constructed by inserting a cosmid into a

non-essential region of the D29 mycobacteriophage which is capable of introducing DNA of interest into the chromosome of mycobacteria.

The present invention further provides a mycobacterial auxotrophic mutant and method of generating auxotrophic mutants.

This invention provides a method of inactivating a mycobacterial virulence gene.

Recombinant mycobacterium which expresses a DNA of interest incorporated into its chromosome by a conditional shuttle plasmid containing the DNA of interest.

D29 shuttle phasmid

P4 and P2 phasmid Plaques of P4 were first isolated from a strain of Escherichia coli lysogenic for temperate bacteriophage P2.

P4 is dependent on P2.

Despite this dependency, both the genomes are unique.

The 11,500-base-pair (bp)-long genome of P4 is about one-third the size of the DNA of P2.

The genomes of the two replicons are essentially heterologous, except for identical 19-base-long single-stranded cohesive ends, and P2 and P4 are heteroimmune.

P4 and P2 phasmid P2 can provide required morphogenic gene products (gp) to P4 during coinfection.

In the absence of P2, P4 is able to replicate its DNA in an E. coli host.

In this situation, vir1 mutation allows for the stable maintenance of P4 in the plasmid state.

Plasmid forming derivative i-e X similar to P4 because they require helper to enter lytic cycle.

They are unlike to P4 coz the required helper genome is homologous or homoimmune.