Bacteriophage

Lambda Vectors
 Most suitable cloning vehicles for Eukaryotic
DNA. (Adv. Over plasmids is high).
 Large number of phage plaques –
characterization and screening.
 In vitro packaging system – DNA packaged into
empty phage heads.
 Infectivity of rDNA – high, compared to pure
DNA preparations.
 Gene library – few milli lit. of broth.
 E.coli phages such as Φ80, 21, 82, 434, P2.
 Lytic and Lysogenic pathway.
 100 progeny particles per infected cell.
 Molecular weight – 31 x 106 Da and 48,502 bp
 dsDNA – linear molecule.
 5’ ends - Short complementary sequences -12 nucleotides
long – cos sites.
 Circularisation of DNA afetr infection.
 40 genes – functional clusters.
 Gens (A-J) – Head and Tail proteins. Left side.
 Central region – int, xis, exo etc.,- responsible for
lysogenisation.
 Not essential for lytic growth – deleted to construct suitable
vectors.
 Genes - Right of central region – Six regulatory genes (cI,
cII, cIII, cro, N and Q).
 O and P – essential for DNA replication – lytic growth.
 S and R – lysis of cellular membranes.
 Genes are clustered by function in the lambda
genome

Late control Virus head

Recombination Control region Replication Lysis &tail
gam
int red
att xis cIII N cI cro cII O P Q SR A…J

Pint tL1 PL oL PRM PR tR1 PRE tR2 PR‘ t6S cos

tR3
oR origin

promoter
operator
terminator Not to scale!
General structure - λ vectors
 Regions between J and N – 38 to 68%.
 30 x 485 = 14,500 bp of foreign DNA.
 Also 105% of complementary λ – packaged into
phage heads.
 Some other - Non-essential regions.
 24.6 kb of foreign DNA into λ DNA.
 105% of DNA (52 kb) – upper limit of packagable
DNA.
 75% (38 kb) – lower limit.
 Less than 38 kb – strong selection system.
 Two types of vectors – Insertional vectors and
Replacement vectors.
 Positive Selection for recombinant phage genomes.
Construction of vector
 Wild type λ DNA – many restriction sites.
 Located within essential regions.
 Distribution of sites – altered.
 Point mutations, substitutions, deletions – two step
method.
 First – λ mutants which do not contain restriction site
for particular enzyme.
 Second – Desired cleavage sites introduced by
genetic crosses.
 Comparitively easy – eliminate restriction cleavege
sites completely – growing phages on restricting
hosts.
 Eg., Phage DNA devoid of Eco RI sites will be able
to survive in host cells containing Eco RI rstriction
and modification system.
 Restriction-resistant phages - selected –
crossed subsequently with suitable
susceptible phages – desired combination of
cleavage sites.
 Eco RI sites limited system.

 λb221c1857 - deletion b221 – removes two

Eco RI sites.
 First step of selection – derivative with no
Eco RI restriction sites.
 Recombination with λplac5imm (5 Eco RI
sites) – occurs in limited region near third
Eco RI site from right.
 λ derivatives – different restriction sites.
 Bam HI site – insertion of DNA obtained with a
number of isochizomeric enzymes.
 Strategy change – One Bam HI site occurs in
essential region.
 Cannot be easily eliminated by passages on
restricting host.
 Klein and Murray – mutagenesis of phage lysate
with nitrous acid.
 Mutagenised phage population was amplified,
progeny phages harvested, DNA isolated and
cleaved with Bam HI.
 Small fraction of Bam HI resistant DNA was isolated,
packaged in vitro and propagated in suitable hosts.
 Desired phage derivatives containing only one or
two Bam HI sites – obtained by genetic cross.