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Dennis varghese
b.Voc food processing & nutraceuticals
2nd year 4th sem
pims
INTRODUCTION
 Bacteriophage are viruses that are capable of
infecting bacteria.
 In situation, the phage chromosome
integrates into the bacterial chromosome
and multiplies with the latter prophage.
 The prophage may dissociate from the
bacterial chromosome and follow the lytic
cycle
 Several bacteriophages are used as cloning
vectors, the most commonly used E.Coli
phages being (lambda) and M13 phages.
Phage vector
 It is a bacterial virus, or bacteriophage, that infects
the bacterial species Escherichia coli.
 This virus is a temperate phage.
 Temperate phages are basically
 bacteriophages which can choose between a lytic and
lysogenic pathway of development.
Lifecycle
Lysogenic cycle lytic cycle
 The viral DNA exists as a separate molecule
within the bacterial cell.
 Replicates separately from the host bacterial
DNA.
 Viral DNA integrates with bacterial DNA.
 Replicates with the host DNA
Phage vector bacteriophage
Cloning vector
 Two problems had to be solved before lambda
based cloning vectors could be developed:
 (1)The DNA molecule can be increased in size by
only about 5%, representing the addition of only 3
kb of new DNA.
 The total size of the molecule is more than52 kb,
then it cannot be packaged into the head structure
and infective phage particles are not formed.
 This severely limits the size of a DNA fragment
that can be inserted into an unmodified vector
 (2)The lambda genome is so large that it has more
than one recognition sequence for virtually every
restriction endonuclease.
 Restriction cannot be used to cleave the normal
lambda molecule in a way that will allow insertion
of new DNA,
 Because the molecule would be cut into several
small fragments that would be
 very unlikely to re-form a viable lambda genome on
re ligation
Phage vector bacteriophage
Replacement vectors
 A lambda replacement vector has two recognition
sites for the restriction endonuclease used for
cloning.
 These sites flank a segment of DNA that is replaced
by the DNA to be cloned
 Often the replaceable fragment (or “stuffer
fragment”) carries additional restriction sites that
can be used to cut it up into small pieces so that its
own re-insertion during a cloning experiment is
very unlikely.
 Replacement vectors are generally designed to carry
larger pieces of DNA than insertion vectors can
handle.
 Recombinant selection is often on the basis of size,
with non-recombinant vectors being too small to be
packaged into lambda phage heads
Phage vector bacteriophage
Insertion vector
 With an insertion vector a large segment of the non-
essential region has been deleted, and the two arms
ligated together.
 An insertion vector possesses at least one unique
restriction site into which new DNA can be inserted.
 The size of the DNA fragment that an individual vector
can carry depends, of course, on the extent to which
the non-essential region has been deleted.
 Two popular insertion vectors are
Segments of the lambda genome
can be deleted
without impairing viability
 Large segment in the central region of the lambda
DNA molecule can be removed without affecting the
ability of the phage to infect E. coli cells.
 Removal of all or part of this non-essential region,
between positions 20 and 35 on the map decreases the
size of the resulting lambda molecule by up to 15 kb.
 This means that as much as 18 kb of new DNA can now
be added before the cutoff point for packaging is
reached
Phage vector bacteriophage
 This “non-essential” region in fact contains most of the
genes involved in integration and excision of the
prophage from the E. coli chromosome.
 A deleted lambda genome is therefore non-lysogenic
and can follow only the lytic infection cycle.
Natural selection can be used to isolate modified
lambda that lack certain restriction sites
 Even a deleted lambda genome, with the non-essential
region removed, has multiple recognition sites for most
restriction endonucleases.
 If just one or two sites need to be removed, then the
technique of in vitro mutagenesis can be used.
 For example, an EcoRI site, GAATTC, could be changed to
GGATTC, which is not recognized by the enzyme.
 • However, in vitro mutagenesis was in its early stage. when
the first lambda vectors were under development, and even
today would not be an efficient means of changing more
than a few sites in a single molecule
 Instead, natural selection was used to provide strains of
lambda that lack the unwanted restriction sites.
 Natural selection can be brought into play by using as a
host an E. coli strain that produces EcoRI.
 Most lambda DNA molecules that invade the cell are
destroyed by this restriction endonuclease, but a few
survive and produce plaques.
 These are mutant phages, from which one or more EcoRI
sites have been lost spontaneously.
 Several cycles of infection will eventually result in lambda
molecules that lack all or most of the EcoRI sites.
Phage vector bacteriophage
THANK YOU !

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Phage vector bacteriophage

  • 1. Dennis varghese b.Voc food processing & nutraceuticals 2nd year 4th sem pims
  • 2. INTRODUCTION  Bacteriophage are viruses that are capable of infecting bacteria.  In situation, the phage chromosome integrates into the bacterial chromosome and multiplies with the latter prophage.  The prophage may dissociate from the bacterial chromosome and follow the lytic cycle
  • 3.  Several bacteriophages are used as cloning vectors, the most commonly used E.Coli phages being (lambda) and M13 phages.
  • 4. Phage vector  It is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli.  This virus is a temperate phage.  Temperate phages are basically  bacteriophages which can choose between a lytic and lysogenic pathway of development.
  • 5. Lifecycle Lysogenic cycle lytic cycle  The viral DNA exists as a separate molecule within the bacterial cell.  Replicates separately from the host bacterial DNA.  Viral DNA integrates with bacterial DNA.  Replicates with the host DNA
  • 7. Cloning vector  Two problems had to be solved before lambda based cloning vectors could be developed:  (1)The DNA molecule can be increased in size by only about 5%, representing the addition of only 3 kb of new DNA.  The total size of the molecule is more than52 kb, then it cannot be packaged into the head structure and infective phage particles are not formed.  This severely limits the size of a DNA fragment that can be inserted into an unmodified vector
  • 8.  (2)The lambda genome is so large that it has more than one recognition sequence for virtually every restriction endonuclease.  Restriction cannot be used to cleave the normal lambda molecule in a way that will allow insertion of new DNA,  Because the molecule would be cut into several small fragments that would be  very unlikely to re-form a viable lambda genome on re ligation
  • 10. Replacement vectors  A lambda replacement vector has two recognition sites for the restriction endonuclease used for cloning.  These sites flank a segment of DNA that is replaced by the DNA to be cloned  Often the replaceable fragment (or “stuffer fragment”) carries additional restriction sites that can be used to cut it up into small pieces so that its own re-insertion during a cloning experiment is very unlikely.
  • 11.  Replacement vectors are generally designed to carry larger pieces of DNA than insertion vectors can handle.  Recombinant selection is often on the basis of size, with non-recombinant vectors being too small to be packaged into lambda phage heads
  • 13. Insertion vector  With an insertion vector a large segment of the non- essential region has been deleted, and the two arms ligated together.  An insertion vector possesses at least one unique restriction site into which new DNA can be inserted.  The size of the DNA fragment that an individual vector can carry depends, of course, on the extent to which the non-essential region has been deleted.  Two popular insertion vectors are
  • 14. Segments of the lambda genome can be deleted without impairing viability  Large segment in the central region of the lambda DNA molecule can be removed without affecting the ability of the phage to infect E. coli cells.  Removal of all or part of this non-essential region, between positions 20 and 35 on the map decreases the size of the resulting lambda molecule by up to 15 kb.  This means that as much as 18 kb of new DNA can now be added before the cutoff point for packaging is reached
  • 16.  This “non-essential” region in fact contains most of the genes involved in integration and excision of the prophage from the E. coli chromosome.  A deleted lambda genome is therefore non-lysogenic and can follow only the lytic infection cycle.
  • 17. Natural selection can be used to isolate modified lambda that lack certain restriction sites  Even a deleted lambda genome, with the non-essential region removed, has multiple recognition sites for most restriction endonucleases.  If just one or two sites need to be removed, then the technique of in vitro mutagenesis can be used.  For example, an EcoRI site, GAATTC, could be changed to GGATTC, which is not recognized by the enzyme.  • However, in vitro mutagenesis was in its early stage. when the first lambda vectors were under development, and even today would not be an efficient means of changing more than a few sites in a single molecule
  • 18.  Instead, natural selection was used to provide strains of lambda that lack the unwanted restriction sites.  Natural selection can be brought into play by using as a host an E. coli strain that produces EcoRI.  Most lambda DNA molecules that invade the cell are destroyed by this restriction endonuclease, but a few survive and produce plaques.  These are mutant phages, from which one or more EcoRI sites have been lost spontaneously.  Several cycles of infection will eventually result in lambda molecules that lack all or most of the EcoRI sites.