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Lambda [λ]-Phage :
➢ Discovered by American Microbiologist, Esther Miriam Zimmer
Lederberg (December 18, 1922 – November 11, 2006).

➢ A Temperate phage having icosahedral Head, Tail & Tail Fiber (gpJ)

➢ Head contains Linear Double Stranded DNA of 48,490 bp

➢ Mixing purified empty head, tail assembly and λ-DNA produce infective Phage

gpJ
(magnification x100,000) provided by Prof.
Hendrix to Genetic Switch, Mark Ptashne
➢ Both ends have single-stranded 5' extension of 12 bases
➢ These ends complementary to each other, called as cohesive
or cos
12nt Linear Double Stranded DNA of 48,490 12nt
G
GGGCGGCGACCTC CCCCGCCCGCTGGA
G

5’------G GGGCGGCGACCTC------3’
3’------CCCCGCCCGCTGGA G------5’
GGCGGCG A
GGCCCGCCGTG C CT
Virus Terminase (RE like) RE cut cos site
& produce cohisive ends
C GA C
Circular G
5’------G GGGCGGCGACCTC------3’ Double Stranded
3’------CCCCGCCCGCTGGA G------5’ DNA of 48,502
Entry to Bacteria ( E.coli, K12 Strain, a natural Host )
➢ Entry from Right 5' extension (cos R), followed by the entire genome, enters the host cell.
➢ host ligase closes gaps to form covalently closed circular DNA. Host Gyrase forms supercoiled.
➢ Size of circular DNA in host is 48,502 bp. Replication & Transcription from this Circular DNA.

gp J protein of tail fibre Lamb B Receptor of Bacteria (Maltose)

λ – Phage linear DNA
outer membrane

Peptidoglycan Periplasmic space

Inner membrane

λ – Phage DNA inside Bacteria

Bacterial Cytoplasm
Bacterial Ligase

Genomic DNA
λ – Phage Circularized DNA
After Entry to Bacteria
1) Lytic Cycle ( lysis of bacteria, releases ~100 phage particles)
2) Lysogenic Cycle (integration host chromosome as prophage divides along with
host DNA)
3) Depending upon nutritional status the prophage DNA could be excised from
chromosome and follow the lytic life cycle

λ- Phage (with phage Linear DNA) Lysis &

Lytic Cycle

λ- Phage Progeny
Phage Attachement
Rolling Circle Replication

Infection

E.Coli phage Circularized DNA

Integration
Bacteria
host Bacterial Genome Lysogeny
Constructing λ-Phage as Vector

1) About 20 kb of this phage DNA is important for intergration and excision, So it could be
removed Lytic Cycle ( releases ~100 phage particles)

2) Lysogenic Cycle (integration host chromosome as prophage)

Two types of λ-Vectors
1) Insertion λ Vector
2) Replacement λ Vector

Insertion λ-Vectors e.g. λgt10, λzapII

➢ Simplest of λ phage vectors

➢ Contained 75% of the genomes of wild type λ phage

➢ Insertion possibility 0-10kb

➢ ~25% of the non-essential part of phage could be removed

➢ Insertion vectors must contain restriction site for insertion of
GOI Inserted

➢ Its problem as wild type λ phage has many restriction sites

Insertion λ-Vectors e.g. λgt10, λzapII contd....

➢ Natural selection was used to get rid of the

Restriction endonuclease ( only available alternative at that time)

➢ Alternatively carry out

site-directed mutagenesis
➢ λ Vector with no Eco RI was used to generate
insertion vector λ gt10

➢ This λgt10 Vector was now created by inserting

sites EcoRI site within specific region of λ phage

➢ This specific site is cI lambda repressor gene which

shuts off the transcription

➢ Selection is between lytic or lysogenic cycle

➢ if c1 is functional the turbid plaques (no lysis)

if c1 is nonfunctional due to insertion of GOI clear
plaque mean recombinant DNA.
➢ Insertion size ~8kb
➢ With K12 wild type E coli RI strain , 99.9% lytic & 0.1%
lysogenic ( probability does exists , we do not want it )

➢ So to be more selective, HFl (High Frequency Lysogeny)

strain of bacteria was used (99.9% lysogenic & 0.1%
lytic)

➢ Infecting Hfl E coli RI strain with a mixture of

recombinant and non-recombinant insertion phage
(where insertion inactivates cI - the lambda repressor)
results in progeny viruses that are exclusively
recombinants. All non-recombinant phage go lysogenic
and produce no viral progeny.
Generation of λgt10 vector
Wild Type

Cut cI site
Remove part of non-
essential region

Re-ligate & add Eco RI RE

site with in cI

λgt10 Vector

Eco RI
 λgt10 Vector

Eco RI site in frame of cI gene

Eco RI

GOI Ligase

Empty Head and

Tail assembly
Recombinant λgt10 Phage
 λZAPII Vector

lacZ’
Resriction
Endonuclease
sites

λZAPII Vector, Gene of interest size increases

to ~10 kb
Replacement λ-Vectors e.g. λEMBL4
Head &tail assembly non-essential Lysogeny Lytic

Cut Cut

Head &tail assembly Lytic

 EcoRI BamHI SalI
GAATTCGGATCCGTCGAC GTCGACGGATCCGAATTC
CTTAAGCCTAGGCAGCTG CAGCTGCCTAGGCTTAAG
Cut with Bam HI
Sal I BamHI EcoRI
Remove

Insert Gene of
Interest Cut T4 DNA Ligase
with Bam HI

GATCC G GATCCGAATTC
G CCTAGGCTTAAG

Purified Head
and Recombinant λEMBL4
Tail Assembly
Cosmids pLFR-5
➢ λ cos sites fragments
having Sca I site
➢ Plasmid region fragments
▪ oriC
▪ BamHI region out side cos
site
▪ Tet R outside other cos site
▪ Insert Size 45 kb