Lambda vectors

Lambda vectors

 Lambda was first discovered at the Pasteur Institute by Andre

Lwoff when he observed strains of E. Coli.

 He showed that the cells of these bacterial strains carried

bacteriophage in a dormant form (prophage).

 Phage can alternate between lysogenic (non-productive) and

lytic (productive) growth cycles.
Lambda vectors
 Lambda Bacteriophage

 Double stranded DNA molecule

 5' twelve-base-pair sticky ends (cos sites)

 It is used as a cloning vector, accommodating fragments of DNA up to 15

kilobase pairs long. For larger pieces, the cosmid or YAC’s are used.

 Will accept foreign DNA and still complete their life cycle.

 Distinguish cells that have foreign and non foreign DNA.

 Should replicate in host

 Gene of interest can be identified and grown in large amounts.

 Non essential genes can be removed and replaced by foreign gene.

 Lambda Bacteriophage

 Should carry one or more selectable markers that

identify the parent and recombinant vectors

 Should have restriction sites in non-essential regions of

DNA into which foreign DNA can be inserted

 easy to make and maintain library

 Enzymes needed:

1. Restriction enzymes: cuts the DNA at specific sequences to generate

a set of fragments

2. DNA ligase: inserts DNA restriction fragments into replicating DNA

molecules to produce recombinant DNA
 Lambda vectors

 1) Insertion vectors
cos cos

EcoRI

 2) Replacement vectors

cos cos

20Kb
EcoRI EcoRI
 Limitations

 size of DNA to be introduced into the host cell

 Problem: When making genomic libarary of large size (plants and

mammals) only a portion of those fragments will be represented. If gene of
interest is located in a large fragment, then you won’t be able to isolate that
gene from the library.

 Solution: use a vector that can accept large fragments of DNA

 What determines choice of vector?

1) Insert size
2) Vector size
3) Restriction sites
4) Cloning efficiency
 Lambda vectors

1) Central 1/3 is the “stuffer” fragment.

2) Segments of the lambda DNA are removed and a stuffer fragment is put in, this
keeps the vector at a correct size.
Origin of replication

Ori is a DNA segment recognized by the cellular

DNA-replication enzymes. Without replication
origin, DNA cannot be replicated in the cell.
Selective marker

Selective marker is required for

maintenance of plasmid in the cell.
Because of the presence of the
selective marker the plasmid
becomes useful for the cell.

Under the selective conditions, only

cells that contain plasmids with
selectable marker can survive.
 Many cloning vectors contain a
multiple cloning site (DNA
segment with several unique sites
for restriction nucleases located
next to each other)
Gene to be cloned can be
introduced into the cloning vector
at one of the restriction sites
present in the cloning site.
Lambda vectors
 steps in cloning with l :
 Isolate vector DNA and gene of interest
 Cut both with restriction enzyme(EcoRI)
 Connect two fragments of foreign DNA with DNA ligase.
(recombinant DNA)
 Package DNA by adding cell extracts containing head
and tail proteins
 Transfer recombinant molecules into host cell (transform)
 Grow/select transformants: check recombinant phage for
the presence of desired foreign DNA sequence by
observing its genetic properties.

Brock Biology of Microorganisms, 9th Edition (2000)

Prentice Hall, Madigan, Martinko, Parker
Molecular Biology of the Cell, 3rd Edition, Garland Publishing, Inc. 1983
-PL ( promoter) for transcription for the left side of l with N and cIII
-PR (promoter) for right, including cro, cII and the genes encoding
the structural proteins.
-OL and OR is short non-coding region of genome, they control the
promoters.
-cI (repressor) protein of 236 a.a. which binds to OR and OL,
preventing transcription of cro and N, but allowing transcription
of OL, and the other genes in the left hand end.
-cII and cIII encode activator proteins which bind to the genome.
-Cro (66 aa) protein which binds to OR and OL, blocking binding of
the repressor to this site to prevent lysogeny.
-N codes an antiterminator protein and allows transcription from PL
and PR. It also allows RNA polymerase to transcribe a number
of phage genes, including those responsible for DNA
recombination and integration of the prophage, as well as cII
and cIII.
-Q is an antiterminator similar to N, but only permits extended
transcription from PR
-Two Termination sites- One between N and CIII and other
between cro and CII.


http://www-micro.msb.le.ac.uk/224/Phages.html#Lambda
How do cells leave lysogeny cycle and go to lytic cycle?

 By stress
 Ultraviolet irradiation of cells, this causes induction of a host cell protein, RecA
whose normal function is to induce the expression of cellular genes which permit
the cell to adapt to and survive in altered environmental conditions.

 RecA cleaves the cI repressor protein.

 Which proteins determine the cycle?

 Lysogenic cycle: cI proteins

predominate

 Lytic cycle: cro proteins

predominate
 DNA lambda replication

 Initation of replication at the lambda origin requires “activation” by transcription

starting from PR.
 DNA replication is between O and P gene proteins.
 Oril –Origin of phage l (with 4 binding sites adjacent to AT rich region)
 O protein binds to lambda origin causing a structural change in the
origin.

 P protein interacts with O protein


 Lambda proteins O and P form a complex with DnaB at the lambda
origin (complex is inactive) This forms a spherical structure called an
“O-Some” (~100bp of DNA)

 P protein (lambda’s) brings dnaB to the origin making the duplex

larger (~160bp)

 The AT rich region becomes susceptible to nuclease attack

(recognizes unpaired DNA), melting the DNA duplex.

 Shock proteins (dnaK, dnaJ and grpE gene) dissociate the oril
O.P.dnaB complex to liberate dnaB

 dnaB initiates unwinding of duplex.

 Primase starts chain initations and polII starts elongation.

DNA Replication, W.H. Freeman and Co. (1992) Kornberg,A.
 l circles multiply by theta form(q) and
continues for 5-15 minutes after infection.

 Rolling circles predominates after 15

minutes and produce linear concatemers
(genomes linked end to end).

 Packaging requires THF (termination

host factor) provided by the host cell.

DNA Replication, W.H. Freeman and Co. (1992) Kornberg,A.

 References

 http://www.uic.edu/classes/phar/phar331/lecture6/
 http://www.sh.lsuhsc.edu/gradcore/IDSP117/16
 http://www.gmu.edu/departments/biology/385-Ch04c-rDNA
 Brock Biology of Microorganisms, 9th Edition (2000)
Prentice Hall, Madigan, Martinko, Parker
 Recombinant DNA: A short course, W.H. Freeman and Co.(1983) Watson, Tooze, Kurtz
 http://www-micro.msb.le.ac.uk/224/Phages.html#Lambda
 DNA Replication, W.H. Freeman and Co. (1992) Kornberg,A.
 Genes VII, Oxford Unine Press. (2000), Lewin Benjamin
 http://www.biocan.com/pdf/FAQ%20TrueBlue%20Vectors.pdf
 http://www.cc.ndsu.nodak.edu/instruct/mcclean/plsc731/cloning/cloning1.htm
 http://www.biochem.arizona.edu/classes/bioc461/Chapter6Powerpoint.pdf