Bacteriophage biology and Lambda Phage Library Invitro Packaging

1. “Bacteriophage biology and Lambda Phage Library Invitro
Packaging.”
Instructor
Dr. Shamboo Prasad
Asstt. Professor
Deptt. of PMB&GE
Submitted by
Ajit Tiwari
PhD. Scholar
A-5020/09

2. Lambda phage
• Order Caudovirales
• Family Siphoviridae
• Genus λ-like viruses
• Species Enterobacteria
phage λ

3. Morphology
• Head: icosahedral
symmetry
• Tail: Helical Symmetry
• one tail fiber
• Capsid not enveloped
• Linear dsDNA (phage)
• Infected E.coli cell

4. Genome
• genome contains about 50,000 nucleotide pairs
• encodes 50-60 different proteins
• Genome is 54% of virion by weight

5. • The ends of the genome have sticky ends 12 bp long
=> cos sites (cohesive ends)
• The ends are joined by bacterial enzyme, Ligase
• Closed circle dsDNA (E.coli)

6. linear genome becomes circularized

7. Cloning Vectors
• plasmids
• viruses
• bacteriophage
• lambda ()
• filamentous
(ssDNA)
• combination
• large phage that infects E. coli
• phage attaches to bacteria and
injects DNA
• complex genetics and life
cycle with two phases:
• lytic
• lysogeny
Phage 

8. Infected in E.colicell

9. Life cycle of lambda phage

11.  as a Cloning Vector
• infectious  can be assembled in vitro
• foreign DNA can be incorporated into
the  genome
• non-essential genes removed
• phage assembly can occur with 40-52 kb of DNA
(wild-type   50kb)
• 13 kb ‘stuffer fragment’ (lysogeny
genes) discarded
• accommodates 11-20 kb foreign DNA
Insertion Vector Replacement Vector
• accommodates up to 7-10 kb
foreign DNA (depending on
vector)

12. I1 Genomic libraries
I2 cDNA libraries
I3 Screeningprocedures
Gene libraries and screening

13. The Central Dogma
DNA
mRNA
Protein
Double-stranded
Precursor
RNA
exon
intron
AAAAAAAAAAn
AAAAAAAAAAn Single-stranded
AAAAAAAAAAn
double-stranded
Reverse transcription
cDNA

14. DNA libraries - a collections of DNA sequences
• DNA libraries, like conventional libraries, are used to collect and
store information.
• In DNA libraries, the information is stored as a set of DNA
molecules.
• All DNA libraries are collections of DNA fragments that represent a
particular biological system of interest.
• The two most common uses for these DNA collections are DNA
sequencing and gene cloning.

15. Genomic library cDNA library
Genomic DNA mRNASource
Species or strains Species or strains
Tissues
Developmental stages
Variation
12k -- 20k 0.2k -- 6kInsert size
Equal Correlate with
expression level
Representation
Gene structure
Infer protein identity
Encoded protein
Infer protein identity
Purpose

16. Genomic libraries
• extract genomic DNA
• cut with a restriction enzyme (want only
partial cutting*)
• mix with an excess of plasmid cut with the
same enzyme
• ligate
• transfer (transform) into bacteria.

19. cDNA
A cDNA or complementary DNA, is a DNA copy of an RNA, usually mRNA
cDNA mRNA
Double stranded Single stranded
Stable unstable
Easy to manipulate More difficult to manipulate
Need to be transcribed into
RNA to make a protein
Can be directly used to make
a protein

20. I 2 cDNA libraries
• mRNA isolation, purification and fractionation
mRNA can be readily isolated from lysed eukaryotic cells
by adding magnetic beads which have oligo(dT) covalently
attached. The mRNA binds to the oligo(dT) via its poly(A) tail
and thus be isolated from the solution. The integrity of an
mRNA preparation can be checked by translation in a wheat
germ extract or reticulocyte lysate and then visualizing the
translation products by polyacrylaminde gel electrophoresis.
Integrity can also be studied using gel electrophoresis, which
allows the mRNA to be size fractionated by recovering chosen
regions of the gel lane. Specific sequences can be removed from
the mRNA by hybridization.
Gene libraries and screening

21. cDNA synthesis
TTTTTTTTT
First strand
Nick translation
cDNA library

22. AAAAAA AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
cDNA synthesis
Infect cells
cDNA library
+
phage vector

23. • Synthesis of cDNA: In the first synthesis, reverse transcriptase is used to
make a cDNA copy of the mRNA by exetending a primer, usually
oligo(dT), by the addition of deoxyribounucletides to 3’-end. Synthesis can
be detected by trace labeling. 3’-Tailing of the first strand cDNA using
terminal transferase makes full-length second strand synthesis easier.
Reverse transcriptase or Klenow enzyme can extend a primer
[e.g.oligo(dG)] annealed to a homopolymeric tail [e.g.oligo(dC)] to
synthesize second strand cDNA.
• Treatment of cDNA ends: To avoid blunt end ligation of cDNA to vector,
linkers are usually added to the cDNA after the ends have been
repaired(blunted) using a single strand-specific nuclease followed by
Klenow enzyme. The cDNA may also be methylated to keep it from being
digested when the added linkers are cleaved by a restriction enzyme.
Adaper molecules can be used as an alternative to linkers.
• Ligation to vector: The vector is usually dephosphorylated using alkaline
phosphatase to prevent self-ligation, and so promote the formation of
recombinant molecules. Plasmid or phage vectors can be used to make
cDNA libraries, but the phage λgt11 is preferred for the cnstruction of
expression libraries.

24. I3 Screening procedures
• Screening: Screening to isolate one particular clone from a gene library
routinely involves using a nucleic acid probe for hybridization. The probe
will bind to its complementary sequence allowing the required clone to be
identified.
• Clony and plaque hybridization: A copy of the position of colonies or
plaques on a petri is made on the surface of a membrane, which is then
incubated in a solution of labeled probe. After hybridization and washing,
the location of the bound label is determined. The group of
colonies/plaques to which the label has bound is diluted and re-plated in
subsequent rounds of screening until an individual clone is obtained.

25. Fig. 1. Screening by plaque hybridization

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