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 techniques. From: Harnessing the Power of Viruses, 2018 Related terms: Chromosome, Cloning, Bacteriophage, Enzyme, Protein, DNA, Plasmid, DNA Fragment, Escherichia coli, Bacterium View all Topics 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. > Read full chapter 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. > Read full chapter Neurobiology of Steroids Nancy R. Nichols, ... Caleb E. Finch, in Methods in Neurosciences, 1994 Comments 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. > Read full chapter Introduction 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 expression. 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.” > Read full chapter 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, 1987). > Read full chapter 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 permission. > Read full chapter 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 1. 2. 3. 4. 5. 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. > Read full chapter 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 obtained. > Read full chapter 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. > Read full chapter 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. > Read full chapter ScienceDirect is Elsevier’s leading information solution for researchers. Copyright © 2018 Elsevier B.V. or its licensors or contributors. ScienceDirect ® is a registered trademark of Elsevier B.V. Terms and conditions apply.
