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logical stochastic resonance with correlated internal and external noises in a synthetic biological logic block.following the advent of synthetic biology, several gene networks have been engineered to emulate digital devices, with the ability to program cells for different applications. in this work, we adapt the concept of logical stochastic resonance to a synthetic gene network derived from a bacteriophage λ. the intriguing results of this study show that it is possible to build a biological logic block that can emulate or switch from the and to the or gate functionalities through externally tuning the ...201122225395
robert a. weisberg (1937-2011).robert weisberg died suddenly and unexpectedly on 1 september 2011. bob was a major contributor to the study of bacteriophage lambda, and he made seminal contributions to our understanding of site-specific recombination and transcription termination. he was also an exceptional citizen of the microbial community, serving as an editor for both the journal of virology (1983 to 1988) and the journal of bacteriology (1985 to 1995). he will certainly be missed. max gottesman wrote the following tribut ...201121984787
coupling of transcription and replication machineries in λ dna replication initiation: evidence for direct interaction of escherichia coli rna polymerase and the λo protein.transcription proceeding downstream of the λ phage replication origin was previously shown to support initial steps of the λ primosome assembly in vitro and to regulate frequency and directionality of λ dna replication in vivo. in this report, the data are presented indicating that the rna polymerase β subunit makes a direct contact with the λo protein, a replication initiator of λ phage. these results suggest that the role of rna polymerase during the initiation of λ phage dna replication may b ...201120833633
joint effects of topoisomerase alterations and plasmid-mediated quinolone-resistant determinants in salmonella enterica typhimurium.alterations in topoisomerases and plasmid-mediated quinolone-resistant (pmqr) determinants have been identified as main quinolone-resistant mechanisms in salmonella enterica typhimurium. but the joint effects of these mechanisms have not been thoroughly characterized in homologous salmonella typhimurium strains. in this study, isogenic topoisomerase mutants were constructed using phage λ red recombinase system and phage transduction. and the joint effects of topoisomerase mutations (gyra [s83f], ...201120818995
replication of plasmids derived from shiga toxin-converting bacteriophages in starved escherichia coli.the pathogenicity of shiga toxin-producing escherichia coli (stec) depends on the expression of stx genes that are located on lambdoid prophages. effective toxin production occurs only after prophage induction, and one may presume that replication of the phage genome is important for an increase in the dosage of stx genes, positively influencing their expression. we investigated the replication of plasmids derived from shiga toxin (stx)-converting bacteriophages in starved e. coli cells, as star ...201120829283
targeting vector construction through recombineering.gene targeting in mouse embryonic stem cells is an essential, yet still very expensive and highly time-consuming, tool and method to study gene function at the organismal level or to create mouse models of human diseases. conventional cloning-based methods have been largely used for generating targeting vectors, but are hampered by a number of limiting factors, including the variety and location of restriction enzymes in the gene locus of interest, the specific pcr amplification of repetitive dn ...201121080281
comparison of a bacteriophage-delivered dna vaccine and a commercially available recombinant protein vaccine against hepatitis b.a bacteriophage lambda dna vaccine expressing the small surface antigen (hbsag) of hepatitis b was compared with engerix b, a commercially available vaccine based on the homologous recombinant protein (r-hbsag). rabbits (five per group) were vaccinated intramuscularly at weeks 0, 5 and 10. antibody responses against r-hbsag were measured by indirect enzyme-linked immunosorbent assay, by limiting dilutions and by subtyping. specific lymphocyte proliferation in vitro was also measured. after one v ...201121204995
bac-recombineering for studying plant gene regulation: developmental control and cellular localization of snrk1 kinase subunits.recombineering, permitting precise modification of genes within bacterial artificial chromosomes (bacs) through homologous recombination mediated by lambda phage-encoded red proteins, is a widely used powerful tool in mouse, caenorhabditis and drosophila genetics. as agrobacterium-mediated transfer of large dna inserts from binary bacs and tacs into plants occurs at low frequency, recombineering is so far seldom exploited in the analysis of plant gene functions. we have constructed binary plant ...201121235649
prediction of protein-protein interactions between human host and a pathogen and its application to three pathogenic bacteria.molecular understanding of disease processes can be accelerated if all interactions between the host and pathogen are known. the unavailability of experimental methods for large-scale detection of interactions across host and pathogen organisms hinders this process. here we apply a simple method to predict protein-protein interactions across a host and pathogen organisms. we use homology detection approaches against the protein-protein interaction databases, dip and ipfam in order to predict int ...201121310175
dense display of hiv-1 envelope spikes on the lambda phage scaffold does not result in the generation of improved antibody responses to hiv-1 env.the generation of strong, virus-neutralizing antibody responses to the hiv-1 envelope spike (env) is a major goal in hiv-1 vaccine research. to try to enhance the env-specific response, we displayed oligomeric gp140 on a virus-like scaffold provided by the lambda phage capsid. to do this, an in vitro complementation system was used to "decorate" phage particles with glycosylated, mammalian cell-derived envelope oligomers. we compared the immune response to lambda phage particles displaying hiv-1 ...201121310193
effects of macromolecular crowding on an intrinsically disordered protein characterized by small-angle neutron scattering with contrast matching.small-angle neutron scattering was used to examine the effects of molecular crowding on an intrinsically disordered protein, the n protein of bacteriophage ?, in the presence of high concentrations of a small globular protein, bovine pancreatic trypsin inhibitor (bpti). the n protein was labeled with deuterium, and the d(2)o concentration of the solvent was adjusted to eliminate the scattering contrast between the solvent and unlabeled bpti, leaving only the scattering signal from the unfolded p ...201121320458
studies on escherichia coli hflkc suggest the presence of an unidentified ? factor that influences the lysis-lysogeny switch.the lysis-lysogeny decision in the temperate coliphage ? is influenced by a number of phage proteins (cii and ciii) as well as host factors, viz. escherichia coli hflb, hflkc and hfld. prominent among these are the transcription factor cii and hflb, an atp-dependent protease that degrades cii. stabilization of cii promotes lysogeny, while its destabilization induces the lytic mode of development. all other factors that influence the lytic/lysogenic decision are known to act by their effects on t ...201121324212
probing dna with micro- and nanocapillaries and optical tweezers.we combine for the first time optical tweezer experiments with the resistive pulse technique based on capillaries. quartz glass capillaries are pulled into a conical shape with tip diameters as small as 27 nm. here, we discuss the translocation of λ-phage dna which is driven by an electrophoretic force through the nanocapillary. the resulting change in ionic current indicates the folding state of single λ-phage dna molecules. our flow cell design allows for the straightforward incorporation of o ...201021339600
recombinant dna-methyltransferase m1.bspaci from bacillus psychrodurans ac: purification and properties.a restriction-modification system from bacillus psychrodurans ac (recognition sequence 5'-ccgc-3') comprises two dna methyltransferases: m1.bspaci and m2.bspaci. the bspacim1 gene was cloned in the pjw2 vector and expressed in escherichia coli cells. high-purity m1.bspaci preparation has been obtained by chromatography on different carriers. m1.bspaci has a temperature optimum of 30°c and demonstrates maximum activity at ph 8.0. m1.bspaci modifies the first cytosine in the recognition sequence 5 ...201021314619
characterisation of methacrylate monoliths for bacteriophage purification.binding of three different bacteriophages (phages), namely t7, lambda and m13 on methacrylate monoliths was investigated. phage m13 exhibited the highest dynamic binding capacity of 4.5×10(13) pfu/ml while t7 and lambda showed capacity of 1×10(13) pfu/ml, all corresponding to values of around 1mg/ml. interestingly, capacity for lambda phage was increased 5-fold by increasing nacl concentration in a loaded sample from 0 to 0.2m while there was a constant capacity decrease for t7 and m13 phages. u ...201021238969
the shear flow processing of controlled dna tethering and stretching for organic molecular electronics.dna has been recently explored as a powerful tool for developing molecular scaffolds for making reproducible and reliable metal contacts to single organic semiconducting molecules. a critical step in the process of exploiting dna-organic molecule-dna (dod) array structures is the controlled tethering and stretching of dna molecules. here we report the development of reproducible surface chemistry for tethering dna molecules at tunable density and demonstrate shear flow processing as a rationally ...201021126082
minimal gene regulatory circuits for a lysis-lysogeny choice in the presence of noise.gene regulatory networks (grns) that make reliable decisions should have design features to cope with random fluctuations in the levels or activities of biological molecules. the phage λ grn makes a lysis-lysogeny decision informed by the number of phages infecting the cell. to analyse the design of decision making grns, we generated random in silico grns comprised of two or three transcriptional regulators and selected those able to perform a λ-like decision in the presence of noise. various tw ...201021188148
evolution of complex gene regulatory circuits by addition of refinements.how do complex gene regulatory circuits evolve? these circuits involve many interacting components, which work together to specify patterns of gene expression. they typically include many subtle mechanistic features, but in most cases it is unclear whether these features are essential for the circuit to work at all, or if instead they make a functional circuit work better. in the latter case, such a feature is here termed 'dispensable', and it is plausible that the feature has been added at a la ...201020833317
the solution structure of the c-terminal ig-like domain of the bacteriophage λ tail tube protein.immunoglobulin (ig)-like domains are found frequently on the surface of tailed double-stranded dna bacteriophages, yet their functional role remains obscure. here, we have investigated the structure and function of the c-terminal ig-like domain of gpv (gpv(c)), the tail tube protein of phage λ. this domain has been predicted through sequence similarity to be a member of the bacterial ig-like domain 2 (big_2) family, which is composed of more than 1300 phage and bacterial sequences. using trypsin ...201020826161
phage wo of wolbachia: lambda of the endosymbiont world.the discovery of an extraordinarily high level of mobile elements in the genome of wolbachia, a widespread arthropod and nematode endosymbiont, suggests that this bacterium could be an excellent model for assessing the evolution and function of mobile dna in specialized bacteria. in this paper, we discuss how studies on the temperate bacteriophage wo of wolbachia have revealed unexpected levels of genomic flux and are challenging previously held views about the clonality of obligate intracellula ...201020083406
icp0 antagonizes icp4-dependent silencing of the herpes simplex virus icp0 gene.icp0 is a regulatory protein that plays a critical role in the replication-latency balance of herpes simplex virus (hsv). absence of icp0 renders hsv prone to establish quiescent infections, and thus cellular repressor(s) are believed to silence hsv mrna synthesis when icp0 fails to accumulate. to date, an icp0-antagonized repressor has not been identified that restricts hsv mrna synthesis by more than 2-fold. we report the unexpected discovery that hsv's major transcriptional regulator, icp4, m ...201020098619
a novel escherichia coli-derived mutation detected with the big blue cii mutant selectable assay.transgenic mouse mutation detection systems allow investigation of the origins and mechanisms of mutation associated with exogenous and endogenous mutagen exposures in individual tissues and cell types. in the past, selection assays for transgenic mutants have been contaminated with nonmurine-derived mutations and assay validation is critical to ensure murine in vivo origins of mutations. this is critical in studies of spontaneous mutations and extrapolation to endogenous mammalian genes. herein ...201020120017
structural energetics of the adenine tract from an intrinsic transcription terminator.intrinsic transcription termination sites generally contain a tract of adenines in the dna template that yields a tract of uracils at the 3' end of the nascent rna. to understand how this base sequence contributes to termination of transcription, we have investigated two nucleic acid structures. the first is the rna-dna hybrid that contains the uracil tract 5'-ruuuuuau-3' from the tr2 intrinsic terminator of bacteriophage lambda. the second is the homologous dna-dna duplex that contains the aden ...201020132823
control of the flow properties of dna by topoisomerase ii and its targeting inhibitor.the flow properties of dna are important for understanding cell division and, indirectly, cancer therapy. dna topology controlling enzymes such as topoisomerase ii are thought to play an essential role. we report experiments showing how double-strand passage facilitated by topoisomerase ii controls dna rheology. for this purpose, we have measured the elastic storage and viscous loss moduli of a model system comprising bacteriophage λ-dna and human topoisomerase iiα using video tracking of the br ...201020858436
stability and instability in the lysogenic state of phage lambda.complex gene regulatory circuits exhibit emergent properties that are difficult to predict from the behavior of the components. one such property is the stability of regulatory states. here we analyze the stability of the lysogenic state of phage λ. in this state, the virus maintains a stable association with the host, and the lytic functions of the virus are repressed by the viral ci repressor. this state readily switches to the lytic pathway when the host sos system is induced. a low level of ...201020870769
[recombinant dna-methyltransferase m1.bst19i from bacillus stearothermophilus 19: purification, propeties and amino acids sequence analysis].the m1.bst19i dna-methyltransferase gene from restriction-modification system bst19i (recognition sequence 5'-gcatc-3') in bacillus stearothermophilus 19 has been cloned in the expressing vector pjw, that carries a tandem of thermo inducible promoters p(r)/p(l) from phage lambda. highly purified enzyme has been isolated by chromatography on various resins from e. coli cells where it accumulates in a soluble form. study of m1.bst19i properties has revealed that enzyme has a temperature optimum 50 ...201020873229
kinetic paths, time scale, and underlying landscapes: a path integral framework to study global natures of nonequilibrium systems and networks.we developed a general framework to quantify three key ingredients for dynamics of nonequilibrium systems through path integrals in length space. first, we identify dominant kinetic paths as the ones with optimal weights, leading to effective reduction of dimensionality or degrees of freedom from exponential to polynomial so large systems can be treated. second, we uncover the underlying nonequilibrium potential landscapes from the explorations of the state space through kinetic paths. we apply ...201020886967
functional characterization of a new holin-like antibacterial protein coding gene tmp1 from goat skin surface metagenome.we have identified a holin-like gene from a goat skin surface metagenome. the orf designated tmp1 coding for 34 amino acids shared sequence similarity with putative holin-like toxin genes. to analyze the antibacterial activity of tmp1 encoded protein, this orf was cloned and expressed in escherichia coli bl21(de3). the expressed gene product tmp1 exhibited antibacterial activity against gram-positive bacteria but not to gram-negative bacteria. a single transmembrane domain (tmd) was identified w ...201020927512
probability landscape of heritable and robust epigenetic state of lysogeny in phage lambda.computational studies of biological networks can help to identify components and wirings responsible for observed phenotypes. however, studying stochastic networks controlling many biological processes is challenging. similar to schrödinger's equation in quantum mechanics, the chemical master equation (cme) provides a basic framework for understanding stochastic networks. however, except for simple problems, the cme cannot be solved analytically. here we use a method called discrete chemical mas ...201020937911
physical interaction between the herpes simplex virus type 1 exonuclease, ul12, and the dna double-strand break-sensing mrn complex.the herpes simplex virus type 1 (hsv-1) alkaline nuclease, encoded by the ul12 gene, plays an important role in hsv-1 replication, as a ul12 null mutant displays a severe growth defect. the hsv-1 alkaline exonuclease ul12 interacts with the viral single-stranded dna binding protein icp8 and promotes strand exchange in vitro in conjunction with icp8. we proposed that ul12 and icp8 form a two-subunit recombinase reminiscent of the phage lambda red α/β recombination system and that the viral and ce ...201020943970
dna hybridization sensor based on pentacene thin film transistor.a dna hybridization sensor using pentacene thin film transistors (tfts) is an excellent candidate for disposable sensor applications due to their low-cost fabrication process and fast detection. we fabricated pentacene tfts on glass substrate for the sensing of dna hybridization. the ss-dna (polya/polyt) or ds-dna (polya/polyt hybrid) were immobilized directly on the surface of the pentacene, producing a dramatic change in the electrical properties of the devices. the electrical characteristics ...201020952180
protection of mice by a λ-based therapeutic vaccine against cancer associated with human papillomavirus type 16.human papillomavirus (hpv) oncoproteins (i.e. e6 and e7) are constitutively expressed in cervical cancer cells. the proteins are ideal targets to be used for developing therapeutic vaccines against existing hpv-associated carcinomas. to date, whole bacteriophage ('phage')-λ particles, rather than purified 'naked' dna, have been described as highly efficient delivery vehicles for a dna vaccine.201020956855
an active intracellular device to prevent lethal disease outcomes in virus-infected bacterial cells.synthetic biology includes an effort to logically control cellular behavior. one long-term goal is to implement medical interventions inside living cells, creating intracellular "disease fighters"; one may imagine a system that detects viral infection and responds to halt the spread of the virus. here, we explore a system designed to display some of the qualitative features that such disease prevention systems should have, while not claiming that the system itself has any medical application. an ...201020967799
quality control for unfolded proteins at the plasma membrane.cellular protein homeostasis profoundly depends on the disposal of terminally damaged polypeptides. to demonstrate the operation and elucidate the molecular basis of quality control of conformationally impaired plasma membrane (pm) proteins, we constructed cd4 chimeras containing the wild type or a temperature-sensitive bacteriophage λ domain in their cytoplasmic region. using proteomic, biochemical, and genetic approaches, we showed that thermal unfolding of the λ domain at the pm provoked the ...201020974815
conformational distributions at the n-peptide/boxb rna interface studied using site-directed spin labeling.in bacteriophage λ, interactions between a 22-amino acid peptide (called the n-peptide) and a stem-loop rna element (called boxb) play a critical role in transcription anti-termination. the n-peptide/boxb complex has been extensively studied, and serves as a paradigm for understanding mechanisms of protein/rna recognition. particularly, ultrafast spectroscopy techniques have been applied to monitor picosecond fluorescence decay behaviors of 2-aminopurines embedded at various positions of the box ...201020980674
effects of salts on internal dna pressure and mechanical properties of phage capsids.based on atomic force microscopy nanoindentation measurements of phage λ, we previously proposed a minimal model describing the effect of water hydrating dna that strengthens viral capsids against external deformation at wild-type dna packing density. here, we report proof of this model by testing the prediction that dna hydration forces can be dramatically decreased by addition of multivalent ions (mg(2+) and sp(4+)). these results are explained using a dna hydration model without adjustable pa ...201021035458
structure of the bacteriophage t4 long tail fiber receptor-binding tip.bacteriophages are the most numerous organisms in the biosphere. in spite of their biological significance and the spectrum of potential applications, little high-resolution structural detail is available on their receptor-binding fibers. here we present the crystal structure of the receptor-binding tip of the bacteriophage t4 long tail fiber, which is highly homologous to the tip of the bacteriophage lambda side tail fibers. this structure reveals an unusual elongated six-stranded antiparallel ...201021041684
structural analysis of soft multicomponent nanoparticle clusters.quantitative techniques are essential to analyze the structure of soft multicomponent nanobioclusters. here, we combine electrospray differential mobility analysis (es-dma), which rapidly measures the size of the entire cluster, with transmission electron microscopy (tem), which detects the hard components, to determine the presence and composition of the softer components. coupling analysis of tem and es-dma derived data requires the creation and use of analytical models to determine the size a ...201021049904
characterization of the relationship between integrase, excisionase and antirepressor activities associated with a superinfecting shiga toxin encoding bacteriophage.shigatoxigenic escherichia coli emerged as new food borne pathogens in the early 1980s, primarily driven by the dispersal of shiga toxin-encoding lambdoid bacteriophages. at least some of these stx phages display superinfection phenotypes, which differ significantly from lambda phage itself, driving through in situ recombination further phage evolution, increasing host range and potentially increasing the host's pathogenic profile. here, increasing levels of stx phage φ24(b) integrase expression ...201021062824
the aribo tag: a reliable tool for affinity purification of rnas under native conditions.although rna-based biological processes and therapeutics have gained increasing interest, purification of in vitro transcribed rna generally relies on gel-based methods that are time-consuming, tedious and denature the rna. here, we present a reliable procedure for affinity batch purification of rna, which exploits the high-affinity interaction between the boxb rna and the n-peptide from bacteriophage λ. the rna of interest is synthesized with an aribo tag, which consists of an activatable riboz ...201021071425
identification of antisense rna stem-loops that inhibit rna-protein interactions using a bacterial reporter system.many well-characterized examples of antisense rnas from prokaryotic systems involve hybridization of the looped regions of stem-loop rnas, presumably due to the high thermodynamic stability of the resulting loop-loop and loop-linear interactions. in this study, the identification of rna stem-loops that inhibit u1a protein binding to the hpii rna through rna-rna interactions was attempted using a bacterial reporter system based on phage lambda n-mediated antitermination. as a result, loop sequenc ...201020156995
microfluidic assays for dna manipulation based on a block copolymer immobilization strategy.methods to manipulate and visualize isolated dna and oligonucleotide strands are important for investigation of their biophysics as well as their interactions with proteins. herein, we report such a method by combining a block copolymer surface functionalization strategy with microfluidics. the copolymer poly(l-lysine-graft-polyethylene glycol) (pll-g-peg) coated one surface of the microfluidic channels, rendering it passive to adsorption and thus minimizing any noise arising from nontargeted ad ...201020158193
an antimicrobial peptide that targets dna repair intermediates in vitro inhibits salmonella growth within murine macrophages.the hexapeptide wrwycr was previously identified on the basis of its ability to inhibit bacteriophage lambda integrase-mediated recombination by trapping and preventing resolution of the holliday junction intermediate. this peptide inhibits several unrelated dna repair enzymes that bind to and process holliday junctions and branched dna substrates. wrwycr and its d stereoisomer, wrwycr, are bactericidal against both gram-positive and gram-negative bacteria, causing the accumulation of dna breaks ...201020176906
emergence of acrab-mediated tigecycline resistance in a clinical isolate of enterobacter cloacae during ciprofloxacin treatment.tigecycline resistance remains rare amongst enterobacteriaceae in the uk, as elsewhere, but has been associated with upregulation of the acrab efflux system. using isolates of an enterobacter cloacae strain that developed tigecycline resistance in vivo during ciprofloxacin therapy as well as laboratory-selected mutants, we investigated the role of this pump and the global regulator rama in tigecycline resistance. laboratory mutants were selected from a susceptible clinical isolate in vitro by ex ...201020189357
missa is a highly efficient in vivo dna assembly method for plant multiple-gene transformation.we describe a highly efficient in vivo dna assembly method, multiple-round in vivo site-specific assembly (missa), which facilitates plant multiple-gene transformation. missa is based on conjugational transfer, which is driven by donor strains, and two in vivo site-specific recombination events, which are mediated by inducible cre recombinase and phage lambda site-specific recombination proteins in recipient strains, to enable in vivo transfer and in vivo assembly of multiple transgenic dna. the ...201020200068
cbf gene copy number variation at frost resistance-2 is associated with levels of freezing tolerance in temperate-climate cereals.frost resistance-1 (fr-1) and fr-2 are two loci affecting freezing tolerance and winter hardiness of the temperate-climate cereals. fr-1 is hypothesized to be due to the pleiotropic effects of vrn-1. fr-2 spans a cluster of c-repeat binding factor (cbf) genes. these loci are genetically and functionally linked. recent studies indicate cbf transcripts are downregulated by the vrn-1 encoded mads-box protein or a factor in the vrn-1 pathway. here, we report that barley genotypes 'dicktoo' and 'nure ...201020213518
the 2009 lindau nobel laureate meeting: werner arber, physiology or medicine 1978.swiss microbial geneticist, werner arber shared the 1978 nobel prize in physiology or medicine with hamilton smith and daniel nathans for their discovery of restriction endonucleases. werner arber was born in granichen, switzerland in 1929. following a public school education, he entered the swiss polytechnical school in zurich in 1949, working toward a diploma in natural sciences. there, his first research experience involved isolating and characterizing an isomer of chlorine. following graduat ...201020220745
production of recombinant proteins in e. coli by the heat inducible expression system based on the phage lambda pl and/or pr promoters.the temperature inducible expression system, based on the pl and/or pr phage lambda promoters regulated by the thermolabile ci857 repressor has been widely use to produce recombinant proteins in prokaryotic cells. in this expression system, induction of heterologous protein is achieved by increasing the culture temperature, generally above 37 degrees c. concomitant to the overexpression of heterologous protein, the increase in temperature also causes a variety of complex stress responses. many s ...201020298615
backbone 1h, 13c, and 15n resonance assignments for lysozyme from bacteriophage lambda.lysozyme from lambda bacteriophage (lambda lysozyme) is an 18 kda globular protein displaying some of the structural features common to all lysozymes; in particular, lambda lysozyme consists of two structural domains connected by a helix, and has its catalytic residues located at the interface between these two domains. an interesting feature of lambda lysozyme, when compared to the well-characterised hen egg-white lysozyme, is its lack of disulfide bridges; this makes lambda lysozyme an interes ...201020300891
integration of stable extracellular dna released from escherichia coli into the bacillus subtilis genome vector by culture mix method.the stable cloning of giant dna is a necessary process in the production of recombinant/synthetic genomes. handling dna molecules in test tubes becomes increasingly difficult as their size increases, particularly above 100 kb. the need to prepare such large dna molecules in a regular manner has limited giant dna cloning to certain laboratories. recently, we found stable plasmid dna of up to 100 kb in escherichia coli culture medium during the infection and propagation of lambda phage. the extrac ...201020308163
substrate specificity and biochemical properties of m3.bstf5i dna methyltransferase from the bstf5i restriction-modification system.optimal conditions for dna methylation by the m3.bstf5i enzyme from bacillus stearothermophilus and kinetic parameters of lambda phage dna modification and that of a number of oligonucleotide substrates are established. comparison of m1.bstf5i and m3.bstf5i kinetic parameters revealed that with similar temperature optima and affinity for dna, m3.bstf5i has nearly fourfold lower turnover number (0.24 min(-1)) and modifies the hemimethylated recognition site with lower efficiency under optimal con ...201020331425
selenomethionine or methylseleninic acid inhibits mutagenesis of a reporter gene in mouse bone marrow.recent laboratory and clinical studies have utilized selenium in the form of pure seleno-l-methionine (semet) in combination with dna-damaging cancer chemotherapy drugs. in mice, the selenium protected bone marrow and other tissues from dose-limiting toxicity. in fact, because of the protection from dose-limiting toxicity, a doubling or even tripling of the maximum tolerated dose (mtd) was enabled. previously we showed that semet protects bone marrow by a dna repair mechanism that requires the x ...201020332431
myth and reality: practical test system for the measurement of anti-dna antibodies in the diagnosis of systemic lupus erythematosus (sle).the myth persists that only the labor intensive farr radioimmunoassay and crithidia luciliae immunofluorescence (cl-ifa) are systemic lupus erythematosus (sle)-specific tests. we compared them to elisa with bacteriophage lambda dna (el-dsdna) and denatured calf thymus dna (el-ssdna). by percentile ranking, the specificity cut-off level was set both out of clinical context (socc) on 100 blood bank donors, and in clinical context (sicc) on 100 patients with either rheumatoid arthritis or scleroder ...201020333761
escherichia coli mw005: lambda red-mediated recombineering and copy-number induction of oriv-equipped constructs in a single host.escherichia coli strain el350 contains chromosomally integrated phage lambda red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. bac and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the dna is difficult to isolate in high yield and purity. to overcome this limitation vectors, e.g. pcc1fos, have been constructed that contain the additional replica ...201020350301
fluorogenic peptide substrates for serine and threonine phosphatases.a new fluorescent assay for ser/thr protein phosphatases has been developed. hydrolysis of a phosphoser residue liberates the ser hydroxyl group, which induces a cyclization reaction on the n-terminal carbamate and releases a fluorescent reporter. sequence selectivity is observed using several peptide substrates against alkaline phosphatase (alp), bacteriophage lambda protein phosphatase (lambda-ppase), and vaccinia h1 related phosphatase (vhr). these studies suggest that the assay could be a us ...201020359238
bacteriophage lambda: a paradigm revisited.bacteriophage lambda has an archetypal immunity system, which prevents the superinfection of its escherichia coli lysogens. it is now known that superinfection can occur with toxigenic lambda-like phages at a high frequency, and here we demonstrate that the superinfection of a lambda lysogen can lead to the acquisition of additional lambda genomes, which was confirmed by southern hybridization and quantitative pcr. as many as eight integration events were observed but at a very low frequency (6. ...201020375161
regions and residues of an asymmetric operator dna interacting with the monomeric repressor of temperate mycobacteriophage l1.previously, the repressor protein of mycobacteriophage l1 bound to two operator dnas with dissimilar affinity. surprisingly, the putative operator consensus sequence, 5'ggtgga/ctgtcaag, lacks the dyad symmetry reported for the repressor binding operators of lambda and related phages. to gain insight into the structure of the l1 repressor-asymmetric operator dna complex, we have performed various in vitro experiments. a dimethyl sulfate protection assay revealed that five guanine bases, mostly di ...201020377203
fadd-calmodulin interaction: a novel player in cell cycle regulation.analyses of knockout and mutant transgenic mice as well as in vitro studies demonstrated a complex role of fadd in the regulation of cell fate. fadd is involved in death receptor induced apoptosis, cell cycle progression and cell proliferation. in a search for mechanisms that might regulate fadd functions, we identified, upon the screening of a lambda-phage cdna library, calmodulin (cam) as a novel fadd interacting protein. cam is a key mediator of signals by the secondary messenger calcium and ...201020420860
the tripartite capsid gene of salmonella phage gifsy-2 yields a capsid assembly pathway engaging features from hk97 and lambda.phage gifsy-2, a lambdoid phage infecting salmonella, has an unusually large composite gene coding for its major capsid protein (mcp) at the c-terminal end, a clpp-like protease at the n-terminus, and a approximately 200 residue central domain of unknown function but which may have a scaffolding role. this combination of functions on a single coding region is more extensive than those observed in other phages such as hk97 (scaffold-capsid fusion) and lambda (protease-scaffold fusion). to study t ...201020427067
cloning and sequencing the first hla gene.this perspectives article recounts the isolation and sequencing of the first human histocompatibility gene (hla) in 1980-1981. at the time, general knowledge of the molecules of the immune system was already fairly extensive, and gene rearrangements in the immunoglobulin complex (discovered in 1976) had generated much excitement: hla was quite obviously the next frontier. the author was able to use a homologous murine h-2 cdna to identify putative human hla genomic clones in a lambda-phage libra ...201020457890
recombinant lambda-phage nanobioparticles for tumor therapy in mice models.abstract: lambda phages have considerable potential as gene delivery vehicles due to their genetic tractability, low cost, safety and physical characteristics in comparison to other nanocarriers and gene porters. little is known concerning lambda phage-mediated gene transfer and expression in mammalian hosts. we therefore performed experiments to evaluate lambda-zap bacteriophage-mediated gene transfer and expression in vitro. for this purpose, we constructed recombinant lambda-phage nanobiopart ...201020459865
decision making at a subcellular level determines the outcome of bacteriophage infection.when the process of cell-fate determination is examined at single-cell resolution, it is often observed that individual cells undergo different fates even when subject to identical conditions. this "noisy" phenotype is usually attributed to the inherent stochasticity of chemical reactions in the cell. here we demonstrate how the observed single-cell heterogeneity can be explained by a cascade of decisions occurring at the subcellular level. we follow the postinfection decision in bacteriophage l ...201020478257
enhancement of bacteriophage λ stability using a λq-s- mutant in the continuous culture of escherichia coli.in this study, we used a bacteriophage λq⁻s⁻ mutant that increased the stability of recombinant escherichia coli during continuous culture. the operation was conducted in two stages: the first stage was carried out to promote cell growth, and the second stage was performed for product formation. the productivity of recombinant proteins depends on the substrate concentration of the fresh medium supplied to the second stage (s₃) and dilution rate of the second stage (d₂). with the optimal value of ...201020499104
mutations altering a structurally conserved loop-helix-loop region of a viral packaging motor change dna translocation velocity and processivity.many double-stranded dna viruses employ atp-driven motors to translocate their genomes into small, preformed viral capsids against large forces resisting confinement. here, we show via direct single-molecule measurements that a mutation t194m downstream of the walker b motif in the phage lambda gpa packaging motor causes an 8-fold reduction in translocation velocity without substantially changing processivity or force dependence, whereas the mutation g212s in the putative c (coupling) motif caus ...201020525695
acridine-n peptide conjugates display enhanced affinity and specificity for boxb rna targets.arginine-rich peptides and small-molecule intercalating agents utilize distinct molecular mechanisms for rna recognition. here, we combined these distinct binding modules in an effort to create conjugate ligands with enhanced affinity and specificity using the bacteriophage lambda n peptide-boxb interaction as a model system. we first designed and synthesized a series of peptide-acridine conjugates using portions of the rna-binding domain of n protein (11- and 22- residue peptide segments) and t ...201020527807
cylinders vs. spheres: biofluid shear thinning in driven nanoparticle transport.increasingly, the research community applies magnetophoresis to micro and nanoscale particles for drug delivery applications and the nanoscale rheological characterization of complex biological materials. of particular interest is the design and transport of these magnetic particles through entangled polymeric fluids commonly found in biological systems. we report the magnetophoretic transport of spherical and rod-shaped particles through viscoelastic, entangled solutions using lambda-phage dna ...201020571853
assembly and maturation of the bacteriophage lambda procapsid: gpc is the viral protease.viral capsids are robust structures designed to protect the genome from environmental insults and deliver it to the host cell. the developmental pathway for complex double-stranded dna viruses is generally conserved in the prokaryotic and eukaryotic groups and includes a genome packaging step where viral dna is inserted into a pre-formed procapsid shell. the procapsids self-assemble from monomeric precursors to afford a mature icosahedron that contains a single "portal" structure at a unique ver ...201020620152
transcription, processing and function of crispr cassettes in escherichia coli.crispr/cas, bacterial and archaeal systems of interference with foreign genetic elements such as viruses or plasmids, consist of dna loci called crispr cassettes (a set of variable spacers regularly separated by palindromic repeats) and associated cas genes. when a crispr spacer sequence exactly matches a sequence in a viral genome, the cell can become resistant to the virus. the crispr/cas systems function through small rnas originating from longer crispr cassette transcripts. while laboratory ...201020624226
a forward-genetic screen and dynamic analysis of lambda phage host-dependencies reveals an extensive interaction network and a new anti-viral strategy.latently infecting viruses are an important class of virus that plays a key role in viral evolution and human health. here we report a genome-scale forward-genetics screen for host-dependencies of the latently-infecting bacteriophage lambda. this screen identified 57 escherichia coli (e. coli) genes--over half of which have not been previously associated with infection--that when knocked out inhibited lambda phage's ability to replicate. our results demonstrate a highly integrated network betwee ...201020628568
intramolecular fluorescence correlation spectroscopy in a feedback tracking microscope.we derive the statistics of the signals generated by shape fluctuations of large molecules studied by feedback tracking microscopy. we account for the influence of intramolecular dynamics on the response of the tracking system and derive a general expression for the fluorescence autocorrelation function that applies when those dynamics are linear. we show that in comparison to traditional fluorescence correlation spectroscopy, tracking provides enhanced sensitivity to translational diffusion, mo ...201020655860
h-ns-mediated repression of crispr-based immunity in escherichia coli k12 can be relieved by the transcription activator leuo.the recently discovered prokaryotic crispr/cas defence system provides immunity against viral infections and plasmid conjugation. it has been demonstrated that in escherichia coli transcription of the cascade genes (casabcde) and to some extent the crispr array is repressed by heat-stable nucleoid-structuring (h-ns) protein, a global transcriptional repressor. here we elaborate on the control of the e. coli crispr/cas system, and study the effect on crispr-based anti-viral immunity. transformati ...201020659289
inhibition of superinfection and the evolution of viral latency.latent viruses generally defend their host cell against superinfection by nonlatent virulent mutants that could destroy the host cell. superinfection inhibition thus seems to be a prerequisite for the maintenance of viral latency. yet viral latency can break down when resistance to superinfection inhibition, known as ultravirulence, occurs. to understand the evolution of viral latency, we have developed a model that analyzes the epidemiology of latent infection in the face of ultravirulence. we ...201020660193
protein damage and death by radiation in escherichia coli and deinococcus radiodurans.deinococcus radiodurans is among a small number of bacterial species that are extremely resistant to ionizing radiation, uv light, toxic chemicals, and desiccation. we measured proteome oxidation (i.e., protein carbonylation, pc) in d. radiodurans as well as in standard and evolved resistant strains of escherichia coli exposed to ionizing radiation or uvc light and found a consistent correlation with cell killing. the unique quantitative relationship between incurred pc and cell death holds over ...201020660760
phages have adapted the same protein fold to fulfill multiple functions in virion assembly.evolutionary relationships may exist among very diverse groups of proteins even though they perform different functions and display little sequence similarity. the tailed bacteriophages present a uniquely amenable system for identifying such groups because of their huge diversity yet conserved genome structures. in this work, we used structural, functional, and genomic context comparisons to conclude that the head-tail connector protein and tail tube protein of bacteriophage lambda diverged from ...201020660769
single-stranded heteroduplex intermediates in lambda red homologous recombination.the red proteins of lambda phage mediate probably the simplest and most efficient homologous recombination reactions yet described. however the mechanism of dsdna recombination remains undefined.201020670401
the polypeptide core of microcin e492 stably associates with the mannose permease and interferes with mannose metabolism.microcin e492 (mcce492) is an antibacterial protein whose activity on target cells requires manyz, the inner membrane component of the mannose permease. we show here that mcea, the polypeptide core of mcce492, stably associates with manyz both in the presence and in the absence of mceb, the mcce492 immunity protein. the two known physiological activities of the mannose permease were assayed in cells co-expressing mcea and mceb. under these conditions, growth on mannose as the sole carbon source ...201020674740
dual expression system for assembling phage lambda display particle (ldp) vaccine to porcine circovirus 2 (pcv2).the bacteriophage lambda small capsid protein d forms trimers on the phage head. d-fusion polypeptides can be expressed from plasmids in e. coli and remain soluble without aggregation. we report a dual expression system for the display of four immunodominant regions of porcine circovirus 2 (pcv2) capsid protein (cap) as d-cap fusions on lambda display particles (ldp). the ldp-d-cap preparation proved an effective vaccine in pigs, eliciting both cellular and humoral immune responses and pcv2 neut ...201020674873
mutagenesis of the repeat regions of herpesviruses cloned as bacterial artificial chromosomes.cloning of infectious and pathogenic herpesvirus genomes in a bacterial artificial chromosome (bac) vector greatly facilitates genetic manipulation of their genomes. bac-based mutagenesis strategies of viruses can advance our understanding of the viral gene functions and determinants of pathogenicity, and can ultimately help to develop molecularly defined improved vaccines against virus diseases. unlike the virus stocks, where continuous passage in tissue culture can lead to phenotypic alteratio ...201020676975
structural and functional views of salt-bridge interactions of λ integrase in the higher order recombinogenic complexes visualized by genetic method.the integrase protein encoded by bacteriophage λ (int) catalyzes site a specific dna recombination by which the viral chromosome is inserted into and excised out of the host genome through the formation of higher order recombinogenic nucleoprotein complexes. genetic and biochemical studies on the int carried out by isolating "multimer-specific" mutants had revealed informative functional characteristics of specific electrostatic interactions occurring among the functional domains of int. the λ i ...201020708599
reca-independent single-stranded dna oligonucleotide-mediated mutagenesis.the expression of beta, the single-stranded annealing protein (ssap) of bacteriophage lambda in escherichia coli promotes high levels of oligonucleotide (oligo)-mediated mutagenesis and offers a quick way to create single or multiple base pair insertions, deletions, or substitutions in the bacterial chromosome. high rates of mutagenesis can be obtained by the use of mismatch repair (mmr)-resistant mismatches or mmr-deficient hosts, which allow for the isolation of unselected mutations. it has re ...201020711416
ion-dependent dynamics of dna ejections for bacteriophage lambda.we studied the control parameters that govern the dynamics of in vitro dna ejection in bacteriophage lambda. previous work demonstrated that bacteriophage dna is highly pressurized, and this pressure has been hypothesized to help drive dna ejection. ions influence this process by screening charges on dna; however, a systematic variation of salt concentrations to explore these effects has not been undertaken. to study the nature of the forces driving dna ejection, we performed in vitro measuremen ...201020712993
rapid collapse into a molten globule is followed by simple two-state kinetics in the folding of lysozyme from bacteriophage λ.stopped-flow fluorescence and circular dichroism spectroscopy have been used in combination with quenched-flow hydrogen exchange labeling, monitored by two-dimensional nmr and electrospray ionization mass spectrometry, to investigate the folding kinetics of lysozyme from bacteriophage λ (λ lysozyme) at ph 5.6, 20 °c. the first step in the folding of λ lysozyme occurs very rapidly (τ < 1 ms) after refolding is initiated and involves both hydrophobic collapse and formation of a high content of sec ...201020806781
lambda red recombineering in escherichia coli occurs through a fully single-stranded intermediate.the phage lambda-derived red recombination system is a powerful tool for making targeted genetic changes in escherichia coli, providing a simple and versatile method for generating insertion, deletion, and point mutations on chromosomal, plasmid, or bac targets. however, despite the common use of this system, the detailed mechanism by which lambda red mediates double-stranded dna recombination remains uncertain. current mechanisms posit a recombination intermediate in which both 5' ends of doubl ...201020813883
expression, purification, and characterization of a novel ca(2+)- and phospholipid-binding protein annexin b2.annexin b2 (anxb2) is a novel member of the annexin family of ca(2+)- and phospholipid-binding proteins from cysticercus cellulosae. to obtain highly pure anxb2 with an easy and inexpensive purification approach, its cdna was cloned into the prokaryotic expression vector pjla503 and the translation initiation codon was immediately under the control of the inducible bacteriophage lambda promoters p(r) and p(l). after induction by shifting temperature, large amounts of non-fusion protein were prod ...201019455404
generation of a recombinant full-length human antibody binding to botulinum neurotoxin a.in order to develop a recombinant full-length human anti-botulinum neurotoxin a (bont/a) antibody, human peripheral blood mononuclear cells (pbmc) were collected from three healthy volunteers and induced for bont/a-specific immune response by in vitro immunization. the genes encoding human fd fragment, consisting of antibody heavy chain variable region and constant region 1 with the genes encoding antibody light chain, were cloned from the immunized pbmc. afterwards, one combinatory human antige ...201019466383
specific hydrophobic residues in the alpha4 helix of lambdacii are crucial for maintaining its tetrameric structure and directing the lysogenic choice.the cii protein of the temperate bacteriophage lambda is the decision-making factor that determines the viral lytic/lysogenic choice. it is a homotetrameric transcription activator that recognizes and binds specific direct repeat sequences ttgcn(6)ttgc in the lambda genome. the quaternary structure of cii is held by a four-helix bundle. it is known that the tetrameric organization of cii is necessary for its activity, but the molecular mechanism behind this requirement is not known. by specific ...201019776236
dna packaging by lambda-like bacteriophages: mutations broadening the packaging specificity of terminase, the lambda-packaging enzyme.the dna-packaging specificities of phages lambda and 21 depend on the specific dna interactions of the small terminase subunits, which have support helix-turn-recognition helix-wing dna-binding motifs. lambda-terminase with the recognition helix of 21 preferentially packages 21 dna. this chimeric terminase's ability to package lambdadna is reduced approximately 20-fold. phage lambda with the chimeric terminase is unable to form plaques, but pseudorevertants are readily obtained. some pseudorever ...201019841094
hfld, an escherichia coli protein involved in the lambda lysis-lysogeny switch, impairs transcription activation by lambdacii.the cii protein of bacteriophage lambda is the key regulator for the lytic-lysogenic choice of the viral lifecycle. an unstable homotetrameric transcription activator of the three phage promoters p(e), p(i) and p(aq), lambdacii is stabilized by lambdaciii and destabilized by the host protease, escherichia coli hflb (ftsh). in addition, other e. coli proteins hflk, hflc and hfld also influence lysogeny by acting upon cii. among these, hfld (22.9kda), a peripheral membrane protein that is exposed ...201019853572
fine tuning of the e. coli nusb:nuse complex affinity to boxa rna is required for processive antitermination.phage lambda propagation in escherichia coli host cells requires transcription antitermination on the lambda chromosome mediated by lambdan protein and four host nus factors, nusa, b, e (ribosomal s10) and g. interaction of e. coli nusb:nuse heterodimer with the single stranded boxa motif of lambdanutl or lambdanutr rna is crucial for this reaction. similarly, binding of nusb:nuse to a boxa motif is essential to suppress transcription termination in the ribosomal rna (rrn) operons. we used fluor ...201019854945
tuning a genetic switch: experimental evolution and natural variation of prophage induction.genetic switches allow organisms to modulate their phenotype in response to environmental changes. understanding the evolutionary processes by which switches are tuned is central to understanding how phenotypic variation is realized. prophage induction by phage lambda is the classic example of a genetic switch and allows lambda to move between two different modes of transmission: as a lysogen it reproduces vertically as a component of the host genome; as a free phage it reproduces horizontally b ...201019891623
phisite: database of gene regulation in bacteriophages.we have developed phisite, database of gene regulation in bacteriophages. to date it contains detailed information about more than 700 experimentally confirmed or predicted regulatory elements (promoters, operators, terminators and attachment sites) from 32 bacteriophages belonging to siphoviridae, myoviridae and podoviridae families. the database is manually curated, the data are collected mainly form scientific papers, cross-referenced with other database resources (embl, uniprot, ncbi taxonom ...201019900969
evaluation of humoral and cellular immune responses against hsv-1 using genetic immunization by filamentous phage particles: a comparative approach to conventional dna vaccine.phage display is based on expressing peptides as a fusion to one of the phage coat proteins. to date, many vaccine researches have been conducted to display immunogenic peptides or mimotopes of various pathogens and tumors on the surface of filamentous bacteriophages. in recent years as a new approach to application of phages, recombinant bacteriophage lambda particles were used as dna delivery vehicles to mammalian cells. in this study, recombinant filamentous phage whole particles were used fo ...201019903497
quantification of cyprinid herpesvirus 3 in environmental water by using an external standard virus.cyprinid herpesvirus 3 (cyhv-3), a lethal dna virus that spreads in natural lakes and rivers, infects common carp and koi. we established a quantification method for cyhv-3 that includes a viral concentration method and quantitative pcr combined with an external standard virus. viral concentration methods were compared using the cation-coated filter and ultrafiltration methods. the recovery of virus-like particles was similar for the two methods (cation-coated filter method, 44%+/-19%, n=3; ultr ...201019915032
single-molecule and fret fluorescence correlation spectroscopy analyses of phage dna packaging: colocalization of packaged phage t4 dna ends within the capsid.linear dnas of any sequence can be packaged into empty viral procapsids by the phage t4 terminase with high efficiency in vitro. packaging substrates of 5 kbp and 50 kbp, terminated by energy transfer dye pairs, were constructed from plasmid and lambda phage dnas. nuclease and fluorescence correlation spectroscopy (fcs) assays showed that approximately 20% of the substrate dna was packaged and that the dna dye ends of the packaged dna were protected from nuclease digestion. upon packaging, both ...201019962991
selection of bacteriophage lambda integrases with altered recombination specificity by in vitro compartmentalization.in vitro compartmentalization (ivc) was employed for the first time to select for novel bacteriophage lambda integrase variants displaying significantly enhanced recombination activity on a non-cognate target dna sequence. these variants displayed up to 9-fold increased recombination activity over the parental enzyme, and one mutant recombined the chosen non-cognate substrate more efficiently than the parental enzyme recombined the wild-type dna substrate. the in vitro specificity phenotype exte ...201019966270
dna heats up: energetics of genome ejection from phage revealed by isothermal titration calorimetry.most bacteriophages are known to inject their double-stranded dna into bacteria upon receptor binding in an essentially spontaneous way. this downhill thermodynamic process from the intact virion to the empty viral capsid plus released dna is made possible by the energy stored during active packaging of the genome into the capsid. only indirect measurements of this energy have been available until now, using either single-molecule or osmotic suppression techniques. in this work, we describe for ...201019969001
efficacy of bacteriophage therapy in a model of burkholderia cenocepacia pulmonary infection.the therapeutic potential of bacteriophages (phages) in a mouse model of acute burkholderia cenocepacia pulmonary infection was assessed. phage treatment was administered by either intranasal inhalation or intraperitoneal injection. bacterial density, macrophage inflammatory protein 2 (mip-2), and tumor necrosis factor alpha (tnf-alpha) levels were significantly reduced in lungs of mice treated with intraperitoneal phages (p < .05). no significant differences in lung bacterial density or mip-2 l ...201020001604
identification of adamts13 peptide sequences binding to von willebrand factor.adamts13 cleaves multimeric von willebrand factor (vwf) to regulate vwf-mediated thrombus formation. to search adamts13 peptide sequences binding to vwf, a lambda-phage library expressing various peptides of adamts13 on the surface was screened using vwf either immobilized or in solution under static condition. by the first screening, peptides sharing the c-terminus of spacer domain from arg(670) to gln(684) (epitope-a) were selected. to explore additional sites, peptide sequences from the first ...201019944670
vaccinia virus particles mix inefficiently, and in a way that would restrict viral recombination, in coinfected cells.it is well established that poxviruses are subjected to genetic recombination, but attempts to map vaccinia virus genes using classical genetic crosses were historically confounded by high levels of experimental noise and a poor correlation between physical and genetic map distances. these virus-by-virus crosses also never produced the 50% recombinant progeny that should be seen in experiments involving distant markers. poxviruses replicate in membrane-wrapped cytoplasmic structures called viros ...201020032178
homologous overexpression of a lipase from burkholderia cepacia using the lambda red recombinase system.red recombinase system of the lambda phage is widely used for recombination of short linear dna fragments and genome. using this system, we obtained t7 rna polymerase (rnap) substitution mutants in burkholderia cepacia. to test the expression abilities of the t7 mutants, four different lipase expression vectors were transformed and the lipase activity of these recombinants was evaluated. our results suggest that 500 nt homology between the unit and the genome is sufficient to generate mutations ...201020033831
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