Showing papers on "Lambda phage published in 2006"

Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems.

Abstract: During the proteomics period, the growth in the use of recombinant proteins has increased greatly in the recent years. Bacterial systems remain most attractive due to low cost, high productivity, and rapid use. However, the rational choice of the adequate promoter system and host for a specific protein of interest remains difficult. This review gives an overview of the most commonly used systems: As hosts, Bacillus brevis, Bacillusmegaterium, Bacillussubtilis, Caulobacter crescentus, other strains, and, most importantly, Escherichia coli BL21 and E. coli K12 and their derivatives are presented. On the promoter side, the main features of the l-arabinose inducible araBAD promoter (PBAD), the lac promoter, the l-rhamnose inducible rhaPBAD promoter, the T7 RNA polymerase promoter, the trc and tac promoter, the lambda phage promoter pL, and the anhydrotetracycline-inducible tetA promoter/operator are summarized.

An improved recombineering approach by adding RecA to lambda Red recombination.

Abstract: Recombineering is the use of homologous recombination in Escherichia coli for DNA engineering. Of several approaches, use of the lambda phage Red operon is emerging as the most reliable and flexible. The Red operon includes three components: Redalpha, a 5' to 3' exonuclease, Redbeta, an annealing protein, and Redgamma, an inhibitor of the major E. coli exonuclease and recombination complex, RecBCD. Most E. coli cloning hosts are recA deficient to eliminate recombination and therefore enhance the stability of cloned DNAs. However, loss of RecA also impairs general cellular integrity. Here we report that transient RecA co-expression enhances the total number of successful recombinations in bacterial artificial chromosomes (BACs), mostly because the E. coli host is more able to survive the stresses of DNA transformation procedures. We combined this practical improvement with the advantages of a temperature-sensitive version of the low copy pSC101 plasmid to develop a protocol that is convenient and more efficient than any recombineering procedure, for use of either double- or single-stranded DNA, published to date.

Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents

Abstract: Aim: To incorporate into the lambda phage genome, a luxI-based acyl-homoserine lactone (AHL) synthase genetic construct and exploit the autoamplified power of quorum sensing to translate a phage infection event into a chemical signature detectable by a lux-based bioluminescent bioreporter, with focus towards facile detection of microbial pathogens. Methods and Results: The luxI gene from Vibrio fischeri was inserted into the lambda phage genome to construct a model phage-based biosensor system for the general detection of Escherichia coli. The AHL signalling molecules synthesized upon phage infection are detected by an AHL-specific bioluminescent bioreporter based on the luxCDABE gene cassette of V. fischeri. The assay generates target-specific visible light signals with no requisite addition of extraneous substrate. This binary reporter system was able to autonomously respond to lambda phage infection events at target E. coli concentrations ranging from 1 × 108 to 1 CFU ml−1 within 1·5–10·3 h, respectively, in pure culture. When assayed against artificially contaminated lettuce leaf washings, detection within an E. coli inoculum range from 1 × 108 to 130 CFU ml−1 was achieved within 2·6–22·4 h, respectively. Conclusions: The initial feasibility of binary phage-based reporter assays indicates that quorum sensing can be used to translate a phage infection event into an autoamplified chemical signature. Significance and Impact of Study: With further modification, binary phage-based reporter assays may be capable of rapidly and cost effectively detecting pathogenic agents at very low population densities.

Effects of condensing agent and nuclease on the extent of ejection from phage lambda.

Abstract: We have recently demonstrated, that DNA ejection from bacteriophage I can be partially or completely suppressed in vitro by external osmotic pressure. This suggests that DNA ejection from phage is driven by an internal mechanical force consisting of DNA bending and DNA-DNA electrostatic repulsion energies. In the present work we investigate the extent to which DNA ejection is incomplete at zero osmotic external pressure when phage is opened with its receptor in vitro. The DNA fragment remaining in the capsid and the tail that is no longer bent or compressed sand hence for which there is no internal driving force for ejections is shown not to be ejected. We also demonstrate that DNA can be "pulled" out from the capsid by DNase I acting as a DNA binding protein or spermine acting as a DNA condensing agent. In particular, cryo electron microscopy and gel electrophoresis experiments show the following: (i) DNA ejection from bacteriophage I incubated in vitro with its receptor is incomplete at zero external osmotic force, with several persistence lengths of DNA remaining inside the phage capsid, if no nuclease ( DNase I) or DNA condensing agent ( spermine) is present in the host solution; (ii) in the presence of both DNase I and spermine in the host solution, 60% (approximate to 29 kbp) of wild-type lambda DNA (48.5 kbp) remains unejected inside the phage capsid, in the form of an unconstrained toroidal condensate; (iii) with DNase I added, but no spermine, the ejection is complete; (iv) with spermine, but without DNase I added, all the DNA is again ejected, and organized as a toroidal condensate outside. (Less)

Interaction of bacteriophage lambda with its cell surface receptor: an in vitro study of binding of the viral tail protein gpJ to LamB (Maltoporin).

Abstract: The cell surface receptor for bacteriophage Lambda is LamB (maltoporin). Responsible for phage binding to LamB is the C-terminal part, gpJ, of phage tail protein J. To study the interaction between LamB and gpJ, a chimera protein composed of maltose binding protein (MBP or MalE) connected to the C-terminal part of J (gpJ, amino acids 684-1131) of phage tail protein J of bacteriophage Lambda was expressed in Escherichia coli and purified to homogeneity. The interaction of the MBP-gpJ chimera protein with reconstituted LamB and its mutants LamB Y118G and the loop deletion mutant LamB Delta4+Delta6+Delta9v was studied using planar lipid bilayer membranes on a single-channel and multichannel level. Titration with the MBP-gpJ chimera blocked completely the ion current through reconstituted LamB when it was added to the cis side, the extracellular side of LamB with a half-saturation constant of approximately 6 nM in 1 M KCl. Control experiments with LamB Delta4+Delta6+Delta9v from which all major external loops had been removed showed similar blocking, whereas MBP alone caused no visible effect. Direct conductance measurement with His(6)-gpJ that contained a hexahistidyl tag (His(6) tag) at the N-terminal end of the protein for easy purification revealed no blocking of the ion current, requiring other measurements for the binding constant. However, when maltoporin was preincubated with His-gpJ, MBP-gpJ could not block the channel, which indicated that also His(6)-gpJ bound to the channel. High-molecular mass bands on SDS-PAGE and Western blots, confirming the planar lipid bilayer experiment results, also demonstrated stable complex formation between His(6)-gpJ and LamB or LamB mutants. The results revealed that phage Lambda binding includes not only the extracellular loops.

Swarms and Genes: Exploring λ - Switch Gene Regulation through Swarm Intelligence

Abstract: We demonstrate a 3-dimensional, agent-based model of the lambda-switch gene regulatory system, which simulates the interactions of proteins, promoters and operators during the phases of lysogenic and lytic growth in the phage lambda infected bacterium Escherichia coli. Following a decentralized approach, all regulatory mechanisms result from local interactions of the biomolecular agents within a simulated 3D cytoplasm. We show that our model displays the regulatory lambda switch behavior observed in E. coli, where proteins regulate their own production. Our lambda switch simulations provide a versatile testbed for studying gene regulation in silico, complementing in vitro experiments.

Construction of a large phage display antibody library by in vitro package and in vivo recombination.

Abstract: Capacity and diversity are extremely important to the quality of various phage display libraries. In this work, lambda phage-based in vitro package was applied to construct a filamentous phage display antibody library so as to enlarge its capacity and introduce more sequence diversity in the final library. In vivo recombination via Cre recombinase/lox sites was also exploited to create V(H)/V(L) combination diversity based on multivalent package of lambda phage packaging extracts on phagemid DNA concatemers. The library constructed with 10 microg concatenated phagemid DNA and ten vials of lambda phage packaging extracts was calculated to contain 1.40 x 10(10) independent clones. Higher capacity can be easily achieved when more materials are consumed. This strategy is somewhat more efficient than prior methods.

The impact of phage lambda: from restriction to recombineering

Abstract: Experiments using phage lambda provided early insights into important molecular mechanisms, including genetic recombination and the control of gene expression. Before recombinant DNA technology, the use of lambda, most particularly lambda transducing phages, illustrated the importance of cloning bacterial genes, already providing some insight into how to use cloned genes to advantage. Subsequently, lambda made significant contributions to recombinant DNA technology, including the early generation of genomic and cDNA libraries. More recently, lambda genes associated with recombination have enabled techniques referred to as 'recombineering' to be developed. These techniques permit the refined manipulation, including mutation, of foreign genes in Escherichia coli and their subsequent return to the donor organism.

Site-specific Scission of Lambda Phage Genomic DNA by Ce(IV)/EDTA-based Artificial Restriction DNA Cutter

Abstract: Lambda phage genomic DNA (48502 base pairs) was site-selectively hydrolyzed by artificial restriction DNA cutter (ARCUT), which we have developed by combining Ce(IV)/EDTA and pseudo-complementary P...

Rapid detection of bacteriophage infection and prophage induction using electric biochips

Abstract: Background Bacteria are widely used hosts for production of many biotechnologically important substances. Bacteriophages are viruses that infect bacterial cells. Thus, infection of bacterial cultures by bacteriophages may lead to serious problems, including complete loss of a desired bioproduct and spreading of bacteriophages throughout the whole laboratory. Although phage contamination, and resultant phage infection of bacterial cultures, may cause serious problems in all types of microbiological laboratories, it is especially dangerous when cultivations are performed on a large scale. Moreover, a number of commonly used strains of E. coli contain lambdoid prophages that often bear some regulatory genetic elements useful in the control of the expression of cloned genes. However, under certain conditions a prophage induction occurs that may have similar effects on a bacterial culture as phage infection. Even under standard cultivation conditions, a spontaneous prophage induction occurs with low frequency. However, this rare prophage induction results in appearance of infecting phage particles in amounts ranging from 10-8 to 10-5 pfu (plaque forming units) per bacterial cell. These numbers seem to be low, but when cultivations are performed on a large scale, e.g. reaching 1010 cells per ml, this means from 102 to 105 phages per ml. Considering even a very small bioreactor containing one litre of the culture, this adds up to 108 infecting phage particles. If we consider a 100-litre bioreactor, the number of phages in the medium may reach 1010. The potential (but, in fact, very real) problems described above, indicate a need for rapid detection of phage infection or prophage induction. However, using traditional methods we can detect the presence of phages in a bacterial culture unambiguously after several hours after infection, at best. Unfortunately, this is usually too late to save at least a part of the infected culture, and to avoid phage spreading throughout the laboratory. Therefore, it seems that development of new methods for rapid detection of bacteriophages in bacterial cultures became crucial.

Display of aggregation-prone ligand binding domain of human PPAR gamma on surface of bacteriophage lambda.

Abstract: Aim: To display the aggregation-prone ligand binding domain (LBD) of the human peroxisome proliferator-activated receptor gamma (PPARγ) on the surface of bacteriophages to establish an easy screening assay for the identification of PPARγ ligands. Methods: Plasmids were constructed for the expression of the PPARγ LBD as a fusion to the N-terminus of the g3p protein of filamentous phage or the C-terminus of the capsid protein D (pD) of phage lambda. The fusion proteins were expressed in E coli and solubility characteristics were compared. Polyclonal antibodies against the LBD as well as the pD protein were prepared for Western blot analysis and phage capture assay. Results: The pD-LBD fusion protein was partially soluble, whereas the LBD-g3p fusion protein was detected only in the insoluble fraction. The pD-LBD fusion protein was efficiently incorporated in phage particles. Furthermore, the LBD was shown to be displayed on the surface of bacteriophage lambda. On average, the pD-LBD fusion protein accounted for 28% of the total pD protein in the lambda head capsid. Conclusion: The hydrophobic PPARγ LBD was expressed as a soluble form of fusion protein in E coli and displayed on the surface of bacteriophage lambda when it was fused to the lambda pD protein. The lambda pD fusion system could be used for improving the solubility of proteins that tend to form inclusion bodies when expressed in E coli . The lambda phage particles displaying the LBD of PPARγ may be of great value for the identification of novel PPARγ ligands.

Rapid allelic exchange in enterohemorrhagic Escherichia coli (EHEC) and other E. coli using lambda red recombination.

Abstract: This unit describes an allelic exchange system for enterohemorrhagic E. coli (EHEC), and similar pathogenic species of bacteria. The phage lambda Red recombination system is expressed from a plasmid, inducing a hyper-recombinogenic state where electroporated PCR-generated substrates recombine with the bacterial chromosome at high efficiency. The technique can be used to substitute a drug marker for the gene of interest, or used to generate a clean in-frame deletion of the target gene. Single gene knockouts in EHEC, or deletions of whole pathogenicity islands, can be constructed. A procedure for the preparation of hyper-recombinogenic electrocompetent cells is also described. Besides E. coli K-12 and EHEC, this method has also been used for the construction of gene knockouts in enteropathogenic E. coli (EPEC), enteroaggregative E. coli, and uropathogenic E. coli, as well as Shigella flexneri and Salmonella enterica.

Construction of a Positive Selection Marker by a Lethal Gene with the Amber Stop Codon(s) Regulator

Abstract: A novel positive selection marker for Escherichia coli transformation was developed. The marker consisted of a DNA fragment encoding the C-terminal ribonuclease domain (CRD) of colicin E3 (colE3) and one or more amber stop codons between the initiation codon and the E3-CRD coding sequence. The toxicity of the marker was controlled by the suppressor activity the host cells possessed. This allowed both effective selection and propagation of the vector possessing the maker by selecting appropriate hosts from among those widely distributed: sup+ strains for selection and sup0 strains for propagation respectively. The insert DNA fragment was introduced onto the vector by replacing the marker DNA. The transformants harboring the vector with an insert grew, but those without an insert were effectively removed by the killing activity of E3-CRD encoded on the marker DNA. The marker was also successfully applied to lambda phage display vector.

The interaction between lambda phage and its bacterial host

Abstract: Our interest in the adsorption of lambda phages onto bacterial cells was triggered by the controversial results Schwartz obtained in the 70's (Schwartz 1976) Lambda phages bind to specific receptors, named LamB, on the cell's surface during the infection process Phage adsorption onto the cell wall is a diffusion-limited process One of the controversies is the rate of adsorption, which in some cases appears to exceed the theoretical limit imposed by the physical law of random diffusion We revisited this problem by carrying out experiments along with new theoretical analyses Our measurements show that the population of unbound phages decreases with time in a double-exponential fashion Using fluorescence microscopy we quantified the number of receptors per cell This dissertation describes the adsorption of lambda phages onto their host cells and a kinetics model, which allows the calculation of adsorption, desorption, and irreversible binding rates from a single measurement The long-term interaction between lambda phage and its bacterial host in a co-habitual environment is approached as well A complex mathematical model describing the dynamics of the two populations (bacteria and phages) is presented along with experimental results Surprising phenomena of bacterial persistence against phage infection are also reported

OOP RNA : A regulatory pivot in temperate lambdoid phage development

Abstract: The first studies on OOP RNA suggested that OOP was involved in λ DNA replication initiation or repressor establishment transcription. Once it was demonstrated that OOP RNA was capable of binding to cII mRNA and regulating its translation, the early hypotheses were abandoned. It has since been demonstrated that OOP RNA over-expression from a plasmid is capable of inhibiting the Rex Exclusion phenotype of a resident prophage, but the mechanism of this inhibition remains unknown. Recently, new evidence has been obtained which re-opens the early hypothesis that OOP RNA may indeed play a role in λ replication. The expression of OOP RNA is regulated by numerous factors. Transcription of OOP RNA is induced by factors involved in λ replication initiation, but not by actual DNA synthesis. The oop gene is an SOS gene, repressed by LexA. OOP is regulated post-transcriptionally by the process of polyadenylation, which shortens its intracellular half-life. Sequence analysis of various lambdoid phage genomes shows that the oop gene and its upstream regulatory region are conserved, suggesting an essential role in the phage lifecycle.

Simultaneous effects of shaking and temperature on verotoxin1 phage induction from verotoxigenic escherichia coli strains

Abstract: Induction of lambda phage carring verotoxin1 gene from a verotoxigenic strains of Escherichia coli and released verotoxin1 were studied under environmental factors of shaking and termperature. Verotoxin1 phage in Escherichia coli PA 101 and transductants was confirmed by bacteriophage detection assay. Shaking of culture media and increasing temperature until 42 oC increased phage particles in supernatants of Escherichia coli PA 101. Our results indicate that environmental factors such as shaking movements in natural inhabitates of bacteria such as river or sewage streams and temperature rise in summer season could be factors in induce and release free verotoxin1 – producing phage particles in nature that in turn could be the source of phage spreading to other related bacteria , and responsible for increased outbreaks of food borne diseases with verotoxigenic Escherichia coli in warm monthes of year in tropical areas.