Showing papers on "Lambda phage published in 2019"
TL;DR: Quantitation of the transcriptional output of the resulting expression device with GFP reporter technology in various gene dosages identified conditions of unprecedented induced/uninduced ratios (>300 folds) and very high total transcriptional capacity in this bacterial host.
Abstract: The higher intracellular ATP levels of genome-edited strains of P. putida that result from deleting various energy-consuming functions has been exploited for expanding the window of thermal tolerance of this bacterium. Unlike instant growth halt and eventual death of the naturally occurring strain P. putida KT2440 at 42 °C, the EM42 variant maintained growth and viability of most of the population at the higher temperature for at least 6 h. The authors took advantage of this quality for implementing a robust thermo-inducible heterologous expression device in this species. To this end, the cI857/PL pair of the lambda phage of Escherichia coli was reshaped as a functional cargo that followed the SEVA (Standard European Vector Architecture) format. Quantitation of the transcriptional output of the resulting expression device with GFP reporter technology in various gene dosages identified conditions of unprecedented induced/uninduced ratios (>300 folds) and very high total transcriptional capacity in this bacterial host. The broad-host range nature of the cognate replication origins makes expression vectors pSEVA2214 (low plasmid copy number), pSEVA2314 (medium), and pSEVA2514 (high) to cover a wide range of heterologous expression needs in P. putida and possibly other Gram-negative species.
25 citations
TL;DR: It is found that the side tail fibers interfere with phage DNA ejection process, most likely through the binding with their receptors, OmpC, leading to a more frequent failed infection, however, the side Tail fibers do not seem to affect the lysis-lysogeny decision-making or lysis time.
9 citations
TL;DR: A novel modeling mechanism for the lysis-lysogeny switch is presented, by transferring the interactions of the main proteins, the lambda right operator and promoter regions and the ribonucleic acid (RNA) polymerase, to a finite state machine (FSM), to determine cell fate.
Abstract: The lysis-lysogeny switch in E. coli due to infection from lambda phage has been extensively studied and explained by scientists of molecular biology. The bacterium either survives with the viral strand of deoxyribonucleic acid (DNA) or dies producing hundreds of viruses for propagation of infection. Many proteins transcribed after infection by $\lambda $ phage take part in determining the fate of the bacterium, but two proteins that play a key role in this regard are the cI and cro dimers, which are transcribed off the viral DNA. This paper presents a novel modeling mechanism for the lysis-lysogeny switch, by transferring the interactions of the main proteins, the lambda right operator and promoter regions and the ribonucleic acid (RNA) polymerase, to a finite state machine (FSM), to determine cell fate. The FSM, and thus derived is implemented in field-programmable gate array (FPGA), and simulations have been run in random conditions. A Markov model has been created for the same mechanism. Steady state analysis has been conducted for the transition matrix of the Markov model, and the results have been generated to show the steady state probability of lysis with various model values. In this paper, it is hoped to lay down guidelines to convert biological processes into computing machines.
7 citations
TL;DR: These data show that using long homology arms and a newly developed vector, the first time nearly a full size BAC insert is isolated with a frequency of correct clones not previously reported.
Abstract: DNA polymers can comprise millions of base pairs and encode thousands of structural and regulatory genetic elements. Thus, the precise isolation of specific DNA segments is required for accurate gene dissection. Although polymerase chain reaction (PCR) is a standard tool for this purpose, increasing DNA template size leads to the accumulation of polymerase errors, hindering the precise isolation of large-size DNA fragments. Unlike PCR amplification, DNA gap repair (DGR) is a virtually error-free process. However, the maximal size of bacterial artificial chromosome (BAC) insert isolated so far by recombination-mediated genetic engineering (recombineering) is <90 Kilobase pairs (Kbp) in length. Here, we developed a compact bacteriophage P1 artificial chromosome (PAC) vector, and we used it to retrieve a DNA segment of 203 Kbp in length from a human BAC by DGR in Escherichia coli (E. coli). We analyzed the efficiency of DGR with repressed (recombineering-) and derepressed lambda phage red genes (recombineering+). We showed that both DGR efficiency and the percentage of PAC clones containing the expected 203 Kbp BAC insert improved with increasing size of homology arms. In recombineering+ E. coli cells and with an efficiency of electroporation of 8x109/1 microgram pUC plasmid DNA, DGR efficiency and the percentage of correct PAC clones were about 5x10-6 and 1% for 30 bp; 6x10-6 and 30% for 40 bp; and 1.5x10-5 and 80% for 80 bp homology arms, respectively. These data show that using long homology arms and a newly developed vector, we isolated for the first time nearly a full size BAC insert with a frequency of correct clones not previously reported.
1 citations
12 Aug 2019
TL;DR: In this paper, the nucleotide sequence of the suicidal plasmid pSS1129 has been determined and the main genetic determinants encoded in it have been annotated, including resistance to ampicillin and gentamicin and sensitivity to streptomycin.
Abstract: The in vitro transfer of genetic material to Bordetella pertussis, the etiological agent of whooping cough, is carried out by bacterial conjugation from Escherichia coli strains. The suicidal plasmid pSS1129 has been widely used to construct mutants of B. pertussis. This plasmid, in addition to having a replicative origin of E. coli, has another of transfer, which allows its mobilization from a conjugative strain of E. coli to B. pertussis where it cannot replicate and is only conserved by integration into the chromosome by homologous recombination. Additionally, it contains markers that confer resistance to ampicillin and gentamicin, as well as sensitivity to streptomycin, useful for determining the site of bacterial integration and exchange of genetic material. The nucleotide sequence of this plasmid is unknown. The objective of this study was to establish the nucleotide sequence of pSS1129, as well as to annotate the main genetic determinants encoded in it. The assembly of the resulting sequences from Macrogen, Korea, generated a total sequence of 9690 bp. By means of homology search in international databases, bla, traJ, aacC, rpsL and gcuP genes, replication ColE1 origin and RK2 plasmid transfer origin, as well as, the cos site of lambda phage were identified. Several unique restriction sites useful for cloning genes in this plasmid were also located. The nucloetide sequenece of plasmidio pSS1129 determined in this work, will facilitate its use in the design and obtaining new generations of mutants of B. pertussis for vaccine purposes.