Bacteriophage vectors

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Bacteriophage vectors Bacteriophage l
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Bacteriophages or phages Basic
aspects "DNA travelling outside the cell " a
nucleic acid genome (DNA or RNA), covered by
protein Lytic development (cycle) - infection
process, after attachment to bacterial cell wall
(at a specific receptor) - replication inside
the cell - assembly of new particles - leaving
the cell, usually, but not always, by lysis of
the cell e.g. filamentous phages Lysogenic
development (by temperate phages) - following
infection, integration into chromosome
("prophage") (site-specific or randomly,
dependent on phage type) - remains silent during
cellular propagation and cell multiplication -
induction by external factors (e.g. DNA damage by
UV) leading to onset of lytic development -
continues as above until cell lysis e.g.
bacteriophage lambda (l) gt while developing
phage vectors besides replication, also the
survival of the phage and its route of
development should be kept into consideration.
The presence of plaques (and their appearance)
is a phenotypic trait allowing visual
identification.

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bacteriophage lambda (l) - 48,502 bp
double-stranded linear DNA - genes clustered
according to their role in a developmental
phase - infection (attachment at LamB) gt
circularization cohesive ends cos site -
early replication (Q), late replication (s
rolling circle), concatenanes - att site, b2
region - immunity region cI gene gt l
repressor (CI) role in lysogeny

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Concatenane DNA cleaved at two cos sites and
packaged in a prehead structure. Role of the
terminase (gene A product) Distance between cos
sites must be more than 37 kb and less than 52
kb. Then attachement of the tail and lysis of
the cell. The switch from early to
late replication requires the gam product.

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About cI Wild-type l produces turbid
plaques because a substantial number of cells
are lysogenized and continu growing. Three
complementation groups of mutants were found that
yield clear plaques the genes were named cI,
cII and cIII. CI is the major l repressor,
which is the only l product present in the
lysogenic state. CI binds to many promoters,
in particular to PL and PR. It also activates
PRM, its own repression maintenance promoter.
Since there is no CI at the time of infection,
another promoter is used to provide the initial
product PRE (repression establishment).
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CI lambda repressor (cI gen) pRM
repression maintenance promoter (or pM) pRE
repression establishment (or pE) pL leftwards
promoter direction of lysogeny (int, xis,
) pR rightwards promoter direction of lysis
(replication, lysis, head tail proteins) N is
an antiterminator CI shuts off PL and PR, and
indirectly also PRE, since this requires CII and
CIII. Environmental conditions have a major
impact on the decision between lysogeny and
lysis.

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If there is sufficient CI (from the competition
for promoters and repression), lysogeny can occur
the cell becomes immune against
superinfection therefore the cI region is
also called imm. Phage 434 is a related
lysogenic phage. The imm regions of l and 434
are exchangeable, but are not cross-reactive.
Hence, a 434 phage can infect a l lysogen, and
vice versa. (imm434 has an EcoRI site, imml
hasnt). An interesting mutant of cI is cI857.
The latter encodes a temperature-sensitive
repressor, which remains active at 32C but is
inactive at 42C. This often allows in vivo
control by a temperature shift. (See also
expression vectors, vector for positive
selection, etc.) Spi the sensitivity to P2
inhibition (or interference) is a phenotype used
for positive selection. Phage l is unable to
grow in an E. coli host which is lysogenic of
phage P2.

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Packaging of phage l DNA. Maturation of
infective particles.

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In vitro packaging of DNA into l particles
Requirements 1) concatemeric DNA with cos at
an appropriate distance fromeach other.
2) all trans factors prepared using 2 amber
mutants strains, lacking an essential
component (D and E, respectively) under
non-suppressing conditions.

Most efficient transformation procedure before
the advent of electroporation.
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Following problems had to be solved before l
vector could be developed and used (a) size
limits. But there is sufficient
possibility (space) for deletions.
(up to more about 25 kb, but the genome
size must be at least 37 kb to be
packageable) (b) there is (was) no unique
restriction cleavage site in 48,5 kb.
The 5 EcoRI sites were eliminated by
random mutagenesis. l cI has no EcoRI 434
cI has one EcoRI cI of phage l and phage
434 appear to be exchangeable (but retain only
their proper immunity.) cI857 is a ts-mutant
active at 32C, inactive at 42C.

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• Development and types of bacteriophage l vectors
  
• Insertion vectors
• gt the vector genome size is reduced, within the
  limits of viability (with regard to packaging)
• (the lower limit is gt 37 kb) and allows
  insertion of extra DNA in a unique restriction
  site,
• as long as the upper limit of packaging is not
  exceeded (total amount of DNA lt52 kb).
• gt maximal capacity of cloning between between 10
  and 15 kb (depending on the reduction of
• the genome size)
• gt the target of insertion is a marker providing
  identification and difference between vector and
• recombinants.
• Substitution (or replacement) vectors
• gt more DNA is deleted from the genome (size lt37
  kb), but replaced by an excisable segment,
• that is flanked by identical cleavage sites, so
  that it may be replaced by the DNA
• fragment to be cloned. The excisable segment is
  termed stuffer.
• gt depending on "functional" viability (i.e. not
  on size), inserts up to 25 kb can be cloned.
• (in other words all essential l functions are
  coded in less than 25 kb, although at least

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Plaques
In a plaque part of the cells are lysed, after a
lytic development of the phages. However, a
substantial fraction of the cells continues to
grow (lysogeny), and this causes the turbid
background of the plaque space. Only if all
cells follow the lytic route (i.e. a mutant in
lysogeny), there will be no background and clear
plaques are remain. The first mutants that were
obtained, and which fell into 3 complementation
groups, were therefore named cI, cII and cIII.
The gam gene is required for the switch from Q
to s replication (early to late replication).
However, it is also responsible for the fact that
that l is unable to develop in a strain which is
lysogenized by phage P2. (Sensitivity to phage
P2 inhibition) (or interference). The latter
property is exploited as a positive selection
system for recombinants. (Spi selection). In
such case, the host cell must possess other
properties to enable the conversion of the DNA of
Q-circles to concatemeres. (Hence the use of l
vector - host cell combinations is a rather
specialized field.)
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Phage lambda vectors (l) - packaging
constraints about 37 kb lt DNA size lt about 52
kb - vector types insertion vectors versus
replacement (substitution) vectors - insertion
vector reducing the vector size, but
keeping well above 40 kb insert sizes
possible up to about 10 kb - substitution
vector sum of vectors arms smaller than 35
kb stuffer fragment between the arms to make
vector viable clonable inserts from 5 to 25 kb
- examples (of mostly used) selection /
identification strategies - insertion vectors
- immunity region (turbid gt lt clear)
lgt10 - lacZ (blue gt lt colorless)
lgt11 - substitution vectors - stuffer
fragment - physical selection (restriction
sites) - lacZ or lacZa or lacZam(supF) -
Spi positive selection
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Strategies for identification or selection of
recombinant phages lacZa difference of blue
- colorless plaques cI difference of clear -
turbid plaques Spi phenotype selection a on
P2 lysogenic strain
Including selection based on size (distance
between cos sites)
Strategy - in vitro packaging - ligation
conditions for concatemerisation (high DNA
concentration) - lysates from lysogens
(mutants) - cos site
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Some examples of insertion vectors
cI of phage 434 has a unique EcoRI site. P
a polylinker with SacI, NotI, XbaI, SpeI,
EcoRI, XhoI in lacZa
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Some examples of substitution vectors
Left right arm 29 kb i.e. too small for
packaging. Different strategies to
avoid re-insertion of the stuffer are possible.
- inverted "linker" SalI - BamHI -
EcoRI - BamHI insertion site. Second cleavage
with EcoRI turns stuffer ends into different
extensions than at vector the arms. The
9-nt linker (with 5 bp) is readily removed by
precipitation. (Similarly with other enzyme
combinations and vectors)
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see also notes on next slide.
Summary of important l vectors

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Notes 1) With replacement vectors, physical
selection as shown in EMBL3 and 4 is often
combined with positive selection by the use of
the gam gene on the stuffer. The host strain
should have the required characteristics. 2) In
l-ZAP vectors, such as l-ZAP II in previous
table, the target region is a fasmid DNA,
linearized in the Ff-ori region as described
before. These ZAP vectors are insertion vectors
after cloning an insert, the fasmid can be
excised in vivo by infection of an E. coli
strain, harbouring an Ff phage (in particular
M13K07 which is only inefficiently packaged) and
infective Ff particles containing a
single-strand of the insert DNA will be produced.
This is an efficient way to subclone the l
inserts and to reduce the DNA size range of
vector sequences from around 40-45 kb to about
3-5 kb. 3) Similar to other vector systems,
and as shown in l-GEM-11 and l-ZAPII,
polylinkers and phage RNA promoter sequences are
often extra elements for specific applications.
In l-GEM-11 SfiI sites are flanking the
polylinker to facilitate (often, since it has an
octamer recognition site) the intact excision of
the inserts.
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Cosmid vectors
Summary - cos fragment of l cloned in plasmids
- ligation of 2 linearised cosmid vector to a
large DNA fragment may bring the cos sites at
an appropriate distance for packaging in to l-
heads (within the size limits described
above) - in vitro packaging facilitates
introduction of the DNA into the cell gt
circularization at the 12-nt sticky ends gt
replication as a plasmid (ori) gt selection
marker required to obtain colonies (no plaques,
no virus) - DNA insertion strategies, and
problems - problem aspects -
multimerisation of vector DNA - scrambling
- ligation strategies choice of
dephosphorylation or no dephosphorylation -
dephosphorylation of insert fragments -
partial fill-in of e.g. BamHI and SalI ends
(2-nt) gt no circularisation nor concatenation
of those fragments - sizing on agarose gel or
sucrose gradient (losses !) - ligation
requires high DNA concentration
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Cosmid vector A cosmid is a plasmid, into which
a cos locus has been inserted. This locus
contains all cis elements to package DNA in a l
head structure. The required substrates are
long DNA chains formed by concatenation that have
(at least) 2 cos regions at a sufficient
distance from each other, so that l-terminase
can cleave and incorporate the DNA in a
(pre)head structure and ultimately form an
infective particle. (Hence, a distance of
between slightly less than 40 kb and slightly
more than 50 kb is necessary). One of the first
cosmid vectors was pHC79 (B. Hohn J. Collins),
derived from pBR322 by inserting a 1,78 kb kb l
BglII fragment surrounding the cos site.
Insertion was done by partial cleavage with
Sau3AI, excising the segment between positions
1461 to 1668. Neither the ori, nor the
resistance genes were touched. Size of the
vector 6,4 kb. The same fragment was cloned
into pAT153 (which resembles pBR327), but in the
BamHI site of tetA. (Loss of TcR.) This vector
is HomerI. Later is became clear that much
smaller cos-regions are sufficient. (Down to
about 250 bp.)

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• Principle of cosmid cloning.
• Ligation conditions must favour
• concatemerisation high DNA concentration.
• Avoiding multi-vector concatenanes
• these also may have two cos sites at the
• appropriate distance for packaging, in spite
• of the extra number of sites in between.
• Avoiding the scrambling of a diversity of
• multiple smaller insert fragments up to the
• required size. The insert should (ordinarely)
• be a single, contiguous sequence from
• a genome DNA.
• gt hence, much attention has been given,
• over the years, to the procedures of DNA

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Original (theoretical) concept
Successive presence of a unique genomic DNA
fragment and a linearized cosmid vector DNA.
Other combinations of ligation (c, d, e, f, g)
are not packagable. Following packaging, the
DNA is transduced into the cell by infection.
The DNA circularizes via the cos ends() and
replicates as a plasmid. Transformants
(recombinants) are selected by the antibiotic,
against which there is a resistance gene encoded
by the vector.
() the cohered l cohesive ends
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pJB8 cosmid vector Tinkering with restriction
enzymes and alkaline phosphatase at linearized
vector DNA, so that single addition of vector
arms at the target DNA is feasible. HindIII
and SalI flank the cos region. BamHI is the
insertion site. BamHI and Sau3AI make GATC 5
protruding ends. The DNA to be inserted
is dephosphorylated so that it cannot scramble
with other fragments.
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Cloning in pJB8 as in previous figure. A
more general scheme.
Selection or screening of recombinant colonies
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c2XB vector with 2 cos regions
Between the two cos regions there is a
restriction site which is not readily
ligated. (e.g. blunt ends, or partially
filled-in sticky ends). Insertion is done in
the other part of the vector (here in BamHI).

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pWE cosmid vectors Specific properties
NotI sites for efficient excision of the
inserts RNA promoters flanking the site of
insertion very closely, so that transcripts
probes are exclusively from the insert sequences.
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More examples of insertion strategies in the
chapter on Genomic Libraries.
Today, many 'multipurpose vector's, in
particular plasmid vectors, contain many modular
properties (aspects) allowing a range of
applications. Apart from the essential ori,
there are among the latter ABr, lacZa, F1-ori,
PT7, PT3, PSP6, cos, etc. In more specialized
vectors ("special-purpose vectors"), still other
characteristics are incorporated e.g. for
conjugation/mobilisation, protein expression,
. Vectors for cloning of larger genomic
fragments P1-derived, BAC, PAC, YAC are dealt
with in a separate chapter.
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