Showing papers on "Lambda phage published in 2015"
TL;DR: The major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing are reviewed while highlighting the various applications of recombineered in pushing the boundaries of synthetic biology.
Abstract: The ability to specifically modify bacterial genomes in a precise and efficient manner is highly desired in various fields, ranging from molecular genetics to metabolic engineering and synthetic biology. Much has changed from the initial realization that phage-derived genes may be employed for such tasks to today, where recombineering enables complex genetic edits within a genome or a population. Here, we review the major developments leading to recombineering becoming the method of choice for in situ bacterial genome editing while highlighting the various applications of recombineering in pushing the boundaries of synthetic biology. We also present the current understanding of the mechanism of recombineering. Finally, we discuss in detail issues surrounding recombineering efficiency and future directions for recombineering-based genome editing.
87 citations
TL;DR: The recombinase from native phage or prophage can significantly promote the genetic recombineering frequency in its host, providing an effective genetic tool for constructing genetically engineered strains and investigating bacterial physiology.
Abstract: Bacillus subtilis and its closely related species are important strains for industry, agriculture, and medicine However, it is difficult to perform genetic manipulations using the endogenous recombination machinery In many bacteria, phage recombineering systems have been employed to improve recombineering frequencies To date, an efficient phage recombineering system for B subtilis has not been reported Here, we, for the first time, identified that GP35 from the native phage SPP1 exhibited a high recombination activity in B subtilis On this basis, we developed a high-efficiency GP35-meditated recombineering system Taking single-stranded DNA (ssDNA) as a recombineering substrate, ten recombinases from diverse sources were investigated in B subtilis W168 GP35 showed the highest recombineering frequency (171 ± 015 × 10−1) Besides targeting the purine nucleoside phosphorylase gene (deoD), we also demonstrated the utility of GP35 and Beta from Escherichia coli lambda phage by deleting the alpha-amylase gene (amyE) and uracil phosphoribosyltransferase gene (upp) In all three genetic loci, GP35 exhibited a higher frequency than Beta Moreover, a phylogenetic tree comparing the kinship of different recombinase hosts with B subtilis was constructed, and the relationship between the recombineering frequency and the kinship of the host was further analyzed The results suggested that closer kinship to B subtilis resulted in higher frequency in B subtilis In conclusion, the recombinase from native phage or prophage can significantly promote the genetic recombineering frequency in its host, providing an effective genetic tool for constructing genetically engineered strains and investigating bacterial physiology
51 citations
TL;DR: The natural DNA transfer mechanism from the soil pathogenic bacterium Agrobacterium tumefaciens is adapted to deliver intact large DNA constructs to basidiomycete fungi of the genus Ustilago where they stably integrated into their genome.
Abstract: The genetic transformation of certain organisms, required for gene function analysis or complementation, is often not very efficient, especially when dealing with large gene constructs or genomic fragments. We have adapted the natural DNA transfer mechanism from the soil pathogenic bacterium Agrobacterium tumefaciens, to deliver intact large DNA constructs to basidiomycete fungi of the genus Ustilago where they stably integrated into their genome. To this end, Bacterial Artificial Chromosome (BAC) clones containing large fungal genomic DNA fragments were converted via a Lambda phage-based recombineering step to Agrobacterium transfer-competent binary vectors (BIBACs) with a Ustilago-specific selection marker. The fungal genomic DNA fragment was subsequently successfully delivered as T-DNA through Agrobacterium-mediated transformation into Ustilago species where an intact copy stably integrated into the genome. By modifying the recombineering vector, this method can theoretically be adapted for many different fungi.
5 citations
TL;DR: A bioinformatic study of N15-like phages indicates that cosB(N15) also has an accessory, remote rR2 site, which is proposed to increase packaging efficiency, like R2 and R1 of lambda.
4 citations
TL;DR: It is found that optimal multiplex recombination required long homology lengths in excess of 120 bp, however, efficient multiplexing was possible with only 60 bp of homology, and substantially increased multiplex efficiency using an ExoVII deletion strain.
Abstract: Recombineering is an in vivo genetic engineering technique involving homologous recombination mediated by phage recombination proteins. The use of recombineering methodology is not limited by size and sequence constraints and therefore has enabled the streamlined construction of bacterial strains and multi-component plasmids. Recombineering applications commonly utilize singleplex strategies and the parameters are extensively tested. However, singleplex recombineering is not suitable for the modification of several loci in genome recoding and strain engineering exercises, which requires a multiplex recombineering design. Defining the main parameters affecting multiplex efficiency especially the insertion of multiple large genes is necessary to enable efficient large-scale modification of the genome. Here, we have tested different recombineering operational parameters of the lambda phage Red recombination system and compared singleplex and multiplex recombineering of large gene sized DNA cassettes. We have found that optimal multiplex recombination required long homology lengths in excess of 120 bp. However, efficient multiplexing was possible with only 60 bp of homology. Multiplex recombination was more limited by lower amounts of DNA than singleplex recombineering and was greatly enhanced by use of phosphorothioate protection of DNA. Exploring the mechanism of multiplexing revealed that efficient recombination required co-selection of an antibiotic marker and the presence of all three Red proteins. Building on these results, we substantially increased multiplex efficiency using an ExoVII deletion strain. Our findings elucidate key differences between singleplex and multiplex recombineering and provide important clues for further improving multiplex recombination efficiency.
1 citations
Patent•
13 Aug 2015
TL;DR: In this paper, a site-directed mutagenetic lambda phase int recombinant protein and a preparation method for its preparation were provided. The recombinant proteins are a lambda phage int-recombinant protein with mutations in G347H and D351H, and are temperature-induced in terms of activity.
Abstract: Provided are a site-directed mutagenetic lambda phase int recombinant protein and a preparation method therefor. The recombinant protein is a lambda phage int recombinant protein with mutations in G347H and D351H, is a recombinant protein that is temperature-induced in terms of activity, where no DNA recombination reaction is started in a low-temperature condition, and a DNA recombination reaction is started at a certain temperature condition, thus allowing for control of the DNA recombination reaction. The recombinant protein is applicable in launching a large sample recombination reaction, thus increasing recombinant sample throughput.
01 Jan 2015
TL;DR: This dissertation systematically investigated the effects on DNA genome ejection of external osmotic pressure controlled by the concentration of osmolyte (PEG 8000) and the presence of polyvalent cations (tetravalent polyamine spermine, Sp4+), and established and quantified the nature of the spontaneous driving force for DNA delivery.
Abstract: Author(s): Jin, Yan | Advisor(s): Gelbart, William M; Knobler, Charles M | Abstract: Because of the unique simplicity of their life cycles, compared to all other evolving organisms, double-stranded (ds) DNA bacteriophages have served as an extremely valuable model for elucidating the basic physics and molecular biology of gene replication and expression. In this dissertation, we present experimental work on two phages, lambda and P22, to study the general genome delivery (ejection) mechanisms of dsDNA bacteriophage. Using lambda phage, we systematically investigated the effects on DNA genome ejection of external osmotic pressure controlled by the concentration of osmolyte (PEG 8000) and the presence of polyvalent cations (tetravalent polyamine spermine, Sp4+). We found that the internal pressure of the capsid decreases from 38 to 17 atm as the [Sp4+] is increased from 0 to 1.5 mM. The existence of Sp4+ can also induce incomplete ejection under zero osmotic pressure when its concentration reaches 0.15 mM or higher; for [Sp4+] below this threshold, the ejection is complete. Further, we observed that the self-attraction induced by Sp4+ affects the configurational dynamics of the encapsidated genome, causing it to get stuck in a broad range of non-equilibrated structures.In order to further study the DNA ejection mechanism from phage capsids, we have systematically determined how DNA transcription in vitro is affected by the presence of different osmolyte and viscogen molecules, so that we can test the transcription-pulling hypothesis of genome delivery in the presence of crowded environments mimicking the cytoplasm of the bacterial cell hosts of phages. We found that at high concentrations of DNA templates, macromolecules can increase the RNA yield due to crowding effects on the initiation step of transcription, while small molecules decrease the yield because of viscosity effects on the elongation step. Experiments carried out at low concentrations show a decrease in yield for large and small molecules, confirming the dominant effect of viscosity effects in the elongation step.Having established and quantified the nature of the spontaneous driving force for DNA delivery, we then studied the ejection behavior of internal proteins from another phage, P22, and their functions in an in vitro osmotic suppression system controlled by PEG 8000. We found that the Outer Membrane Protein A (OmpA) from Salmonella, the natural bacterial host of P22, can significantly enhance the rate of DNA ejection in the presence of the primary receptor, LPS. While the DNA is ejected in the presence of LPS, no ejection of the internal proteins occurs unless OmpA is also present. We also find that their ejection is largely complete before any of the genome is ejected. This finding helps us understand the possible roles that the internal proteins play during infection.