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Enterobacteria Phage Lambda

Enterobacteria Phage Lambda
Lambda and M13 bacteriophages were among the first vectors developed for molecular cloning and their improvement over time (in comparison with wild type
unmodified strains) reflects the advancements of molecular biology knowledge and
From: Harnessing the Power of Viruses, 2018
Related terms:
Chromosome, Cloning, Bacteriophage, Enzyme, Protein, DNA, Plasmid, DNA Fragment, Escherichia coli, Bacterium
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Phenotypes and Design Principles in
System Design Space
Michael A. Savageau, in Handbook of Systems Biology, 2013
Alternative Growth Modes of Phage Lambda
Bacteriophage lambda can reproduce in two alternative modes of growth. In the
lytic mode, the phage infects a bacterial cell, reproduces many copies of itself, lyses
the host cell, and circulates through the environment to infect another host cell. In
the lysogenic mode, the phage infects a bacterial cell, incorporates its DNA into the
chromosome of the host cell, and remains quiescent, with its DNA being replicated
along with that of the host. Lysogeny is typically a stable state unless the host cell
is compromised in some fashion (e.g., DNA damaged by UV radiation), and then
the phage undergoes an induction process by which it excises its DNA from the host
chromosome and initiates lytic growth. The key regulatory interactions involved in
maintaining the alternative fates of lambda are shown schematically in Figure 15.6.
FIGURE 15.6. Key regulatory circuits maintaining the alternative fates of bacteriophage lambda.Two regulators maintain the lytic mode of replication; the N-gene
product is a positive regulator required for activating transcription of the lytic-specific genes and CRO is a negative regulator required for repressing transcription of the
lysogenic-specific genes. One regulator maintains the lysogenic mode of replication;
the CI gene product is a negative regulator of the lytic transcripts and a positive
regulator of its own transcription.
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Genomic Library
W.C. Nierman, T.V. Feldblyum, in Encyclopedia of Genetics, 2001
Bacteriophage lambda Vectors
Bacteriophage lambda is a virus that infects E. coli. The typical infection cycle results
in the lysis of the E. coli cell and the release of about 100 progeny phage particles,
each capable of infecting another cell. When lambda is plated at low density on a
lawn of E. coli cells on agar medium, the resulting pattern of clearings (plaques) in
the lawn caused by the lysed cells identify the location of individual lambda clones.
Harvesting the phage particles from a plaque (picking a plaque) provides a stock of
the phage clones for subsequent rounds of propagation.
Like the plasmid vectors, wild-type lambda has been extensively engineered for use
as a vector. Genes not essential for the lambda life cycle described above have been
removed to make room for carrying exogenous insert DNA. The early popularity
of lambda as a cloning vector for genomic library construction is a consequence
of the very efficient pathway for getting lambda DNA into E. coli cells. This
was in contrast to the inefficient chemical transformation used for plasmids,
particularly for larger constructs. The bacteriophage lambda DNA or recombinant
lambda DNA-containing inserts is packaged into infectious phage particles using
an efficient in vitro packaging reaction. Once the particles are formed, each one
can inject its DNA into an E. coli cell. The limit on how much exogenous DNA can
be propagated in a lambda vector results from the packaging capacity of the phage
particle, approximately 35–50 kb of DNA. Because of the requirement for lambda
genes for a productive infection, the amount of insert DNA is restricted to 10–20 kb
depending on the specific vector. Figure 2 illustrates the process of constructing a
genomic library in a lambda vector.
Figure 2. Construction of a genomic library in a bacteriophage lambda vector.
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Neurobiology of Steroids
Nancy R. Nichols, ... Caleb E. Finch, in Methods in Neurosciences, 1994
Lambda phage vectors have the advantage of high transformation efficiencies
due to good commercially available packaging extracts; however, the combination of
electroporation of plasmid DNA containing cDNA inserts and certain strains of E. coli
can now rival phage. More importantly, it is our experience that replica plaque lifts
are more reproducible than replica colony lifts; therefore, screening by differential
hybridization may have less inherent errors when using a phage library.
Detailed methods for growing and constructing a library in gt10 are described
in Davis et al.(8) and Huynh et al.(7). Alternatively, the Uni-ZAP XR vector system
(Stratagene) has also been used in this laboratory for cDNA library construction
and screening by differential hybridization (9). This system combines a phage
vector with Bluescript plasmid rescue, avoiding the extra steps necessary to subclone restriction fragments from recombinant phage DNA into a plasmid vector for
sequencing and making cRNA probes.
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Gustavo Fermin, Paula Tennant, in Viruses, 2018
Viruses Have Allowed Us to Understand How Genes Are Regulated
The bacteriophage lambda was one of the first biological entities whose transcriptional regulation was studied and understood in detail. It was found early on that
lambda, and other phages, possess a temporally controlled pattern of transcription
and gene expression, commonly referred to as immediate early, early, and late
transcription. Gene expression in phage lambda has contributed, almost as equally
as bacterial gene regulation, to an understanding of the many facets of gene
Besides the discovery of lytic and lysogenic cycles in the 1960s, that in itself is
the outcome of specific gene regulatory circuits in action, the study of repressors
(namely, cI and Cro) allowed for the analysis of not only the factors involved in
the expression (activation or repression) of genes but also their kinetics. Beyond
the roles of promoters and operators (which act in cis) as well as the repressors
(which act in trans), phage lambda provided insight into antitermination regulation
(proteins N and Q), the action of genetic switches—as defined by Mark Ptashne—the
intimate relationship between a prophage and its host, the SOS response, and much
more. Since the 1980s Ptashne has focused on applying insights gained from the
study of the lambda bacteriophage to eukaryotic cells, in particular yeast. He wrote
that they “had no way of knowing, at the start, that studying the lambda repressor
and its action would yield a coherent picture of a regulatory switch and even less
an indication that the principles of protein–DNA interaction and gene regulation,
gleaned from the lambda studies, would apply even in eukaryotes.”
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Homology Effects
E. Gerhart H. Wagner, ... Pascale Romby, in Advances in Genetics, 2002
1. Lambda OOP RNA induces facilitated mRNA decay
In the coliphage lambda, CII is a key protein in the establishment of the lysogenic
state. A 77-nt-long antisense RNA, OOP, is encoded between the cII and O gene
sequences, overlapping the 3-end of the cII mRNA. When OOP binds to its target
site, formation of an RNA duplex that extends into the 3 -part of the cII coding
region creates a substrate for RNase III (Figure 12.2). Cleavage by this enzyme
initiates the rapid decay of the cII mRNA segment, carried out by other RNases
(Krinke et al., 1991; Krinke and Wulff, 1990). In support of this, overexpression of
OOP in trans prohibits lysogeny of an incoming lambda phage (Krinke and Wulff,
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Chi Sequences
G.R. Smith, in Encyclopedia of Genetics, 2001
Genetic Properties of Chi
As noted above, in phage lambda crosses Chi stimulates homologous recombination
at or near the site of the mutation. Stimulation is exclusively by the RecBCD pathway
and extends leftward from Chi (with respect to the direction Chi is written here);
stimulation is greatest at Chi and diminishes by a factor of 2 for each 2–3 kb
from Chi (Figure 1A). A Chi site in only one parent shows high activity, even when
the other parent carries a heterology of several kb opposite the Chi site. In this
case recombination is stimulated in the region of homology just to the left of the
heterology. Chi also stimulates E. coli generalized transduction by phage P1 and
transformation by linear DNA (gene targeting). Chi stimulates the formation of high
molecular weight DNA by plasmids that replicate as rolling circles; this stimulation,
like Chi-stimulated homologous recombination, requires RecA protein and may
reflect increased recombination of the plasmids or decreased nuclease activity of
RecBCD enzyme (see below).
Figure 1. (A) Localized stimulation of recombination by Chi in phage lambda crosses.
I, Ia, etc. are genetic intervals bounded by markers located the indicated distance
from a Chi site in lambda. Solid circles indicate the midpoints of each interval and
the frequency of recombinants per physical length of that interval, normalized to
interval II = 1. (B) Action of purified RecBCD enzyme at Chi. With (ATP) > (Mg2+)
RecBCD enzyme unwinds the DNA substrate, nicks the upper strand about five
nucleotides to the right of Chi, and continues unwinding. With (Mg2+) > (ATP)
RecBCD enzyme degrades the upper strand up to Chi, nicks the lower strand, and
degrades or unwinds it to the left of Chi. Both conditions produce single-stranded
DNA with a 3 end near Chi and extending to its left. RecBCD enzyme loads RecA
protein onto this ‘Chi tail.’ (Reprinted with modification from Smith et al. (1995) with
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Natural Product Biosynthesis by Microorganisms and Plants, Part C
Frank Sainsbury, ... George P. Lomonossoff, in Methods in Enzymology, 2012
3.2.3 Cloning by GATEWAY recombination
Generate the insert with bacteriophage lambda attachment B (attB) sites at
both ends using PCR.
Using BP clonase II, transfer the PCR fragment to a GATEWAY donor vector
via directional recombination. The resultant plasmid is the entry clone.
Using LR clonase II, transfer the gene of interest from the entry vector to the
appropriate pEAQ-HT-DEST vector (see Table 9.3).
Transform competent E. coli and plate onto LB agar plates with kanamycin
(50 μg/mL) selection.
Colonies may be screened by PCR or restriction analysis. Positive clones
are grown overnight, and plasmids are extracted for sequencing (to confirm
insertion) and Agrobacterium transformation.
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Bioseparation Engineering
J. Yin, ... Z.-Y. Shen, in Progress in Biotechnology, 2000
A combined biologi-chemical method using cloned lambda phage lysis genes for cell
disruption and poly- 3 -hydroxybutyrate (PHB) separation was proposed and studied.
The new plasmid named pTU14 with the lysis genes S(-)RRz of DNA (cl857 sam7)
and the PHB biosynthesis genes phbCAB from Alcaligenes eutrophus HI6 was constructed and transformed into E. coli JM109. The recombinant E. coli JM109(pTU14)
cells in stationary phase could be induced to lyse by buffer A (2 mmol/L EDTA in
50 mmol/L Tris buffer, pH = 8) treatment and PHB granules were released completely
with the digestion and dissolving of most of the non-PHB materials. PHB content
could be increased from 66.07% to 90.53%. Further purification of PHB could be
realized by surfactant SDS treatment, and PHB granules of 99.66%) purity were
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Hershey, Alfred
W.C. Summers, in Encyclopedia of Genetics, 2001
In the 1960s Hershey turned his attention to the lysogenic phage lambda and
devised simple yet elegant approaches to study the physical states of the lambda
DNA. He pioneered methods for dealing with large DNA molecules, which are highly
sensitive to breakage by shear forces in solutions. His methods for DNA extraction
(phenol) and zone sedimentation (in sucrose gradients) allowed him to show that
lambda DNA existed in both linear and circular forms, and that it has unpaired
(presumably complementary) cohesive termini. This work was seminal in developing
our current understanding of lysogeny as well as in the applications of lambda
bacteriophage in recombinant DNA technologies.
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Illegitimate Recombination
D. Carroll, in Encyclopedia of Genetics, 2001
An example of site-specific recombination is the integration of the bacteriophage
lambda genome into the host Escherichia coli chromosome. The lambda-encoded
Int protein recognizes the specific attachment sites of both DNAs and, in collaboration with host proteins, brings them together into a preintegration complex.
Recombination proceeds by a topoisomerase-like mechanism, in which hydroxyl
groups on active site tyrosines in the Int protein attack specific phosphodiester bonds
in the target DNA, producing covalent joints between Int and DNA as intermediates
in integration. A subsequent transesterification reaction generates the new DNA
joints and releases the protein.
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