Combinatorial MAB-Based Joint Channel and Spreading Factor Selection for LoRa Devices.

Long-Range (LoRa) devices have been deployed in many Internet of Things (IoT) applications due to their ability to communicate over long distances with low power consumption. The scalability and communication performance of the LoRa systems are highly dependent on the spreading factor (SF) and channel allocations. In particular, it is important to set the SF appropriately according to the distance between the LoRa device and the gateway since the signal reception sensitivity and bit rate depend on the used SF, which are in a trade-off relationship.

Evolution of carrying capacity in evolution experiments focusing on a single locus on the Escherichia coli chromosome.

We performed a series of evolution experiments, the results of which illustrated the relationship between mutations and increased carrying capacity (K). Performing an evolution experiment with repeated cycles of mutation by PCR and selection makes it possible to obtain results over shorter culture durations than in methods reported previously relying on spontaneous mutation and selection. We constructed random mutant populations of Escherichia coli in which members differed only in part of the genomic copy of the glutamine synthetase gene and performed daily serial transfer culture where the populations were in K-selected environments.

Quantitative analysis of the bacteriophage Qbeta infection cycle.

In this study, the infection cycle of bacteriophage Qbeta was investigated. Adsorption of bacteriophage Qbeta to Escherichia coli is explained in terms of a collision reaction, the rate constant of which was estimated to be 4x10(-10) ml/cells/min. In infected cells, approximately 130 molecules of beta-subunit and 2x10(5) molecules of coat protein were translated in 15 min.

Replication of genetic information with self-encoded replicase in liposomes.

In all living systems, the genome is replicated by proteins that are encoded within the genome itself. This universal reaction is essential to allow the system to evolve. Here, we have constructed a simplified system involving encapsulated macromolecules termed a "self-encoding system", in which the genetic information is replicated by self-encoded replicase in liposomes.

Reconstitution of DNA strand exchange mediated by Rhp51 recombinase and two mediators.

In the fission yeast Schizosaccharomyces pombe, genetic evidence suggests that two mediators, Rad22 (the S. pombe Rad52 homolog) and the Swi5-Sfr1 complex, participate in a common pathway of Rhp51 (the S. pombe Rad51 homolog)-mediated homologous recombination (HR) and HR repair.

Emergence of polyproline II-like structure at early stages of experimental evolution from random polypeptides.

To examine whether a primordial functional protein at the early stages of evolution has structural features, we carried out experimental evolution consisting of 25 cycles (generations) of mutation and selection toward DNA-binding function using a random-sequence polypeptide of 139 amino acid residues with no secondary structure as the initial sequence. In each generation, 16 clones were sampled arbitrarily for sequence analysis, and a phylogenetic tree was constructed. Polypeptide evolution proceeded from the initial point on branch I in 2 main directions of branches II and III.

A novel sequence-specific RNA quantification method using nicking endonuclease, dual-labeled fluorescent DNA probe, and conformation-interchangeable oligo-DNA.

We have developed a novel, single-step, isothermal, signal-amplified, and sequence-specific RNA quantification method (L-assay). The L-assay consists of nicking endonuclease, a dual-labeled fluorescent DNA probe (DL-probe), and conformation-interchangeable oligo-DNA (L-DNA). This signal-amplified assay can quantify target RNA concentration in a sequence-specific manner with a coefficient of variation (Cv) of 5% and a lower limit of detection of 0.

Phenotypic plasticity of Escherichia coli at initial stage of symbiosis with Dictyostelium discoideum.

We observed the change in the physiological state of Escherichia coli cells at the initial stage for establishing a new symbiotic relationship with Dictyostelium discoideum cells. For the physiological state, we monitored green fluorescence intensity due to a green fluorescent protein (GFP) gene integrated into the chromosome by flow cytometry (FCM). On co-cultivation of the two species, a new population of E.

Insight into the sequence specificity of a probe on an Affymetrix GeneChip by titration experiments using only one oligonucleotide.

High-density oligonucleotide arrays are powerful tools for the analysis of genome-wide expression of genes and for genome-wide screens of genetic variation in living organisms. One of the critical problems in high-density oligonucleotide arrays is how to identify the actual amounts of a transcript due to noise and cross-hybridization involved in the observed signal intensities. Although mismatch (MM) probes are spotted on Affymetrix GeneChips to evaluate the noise and cross-hybridization embedded in perfect match (PM) probes, the behavior of probe-level signal intensities remains unclear.

Effective selection system for experimental evolution of random polypeptides towards DNA-binding protein.

An experimental evolution with selection based on binding affinity to DNA was carried out on a library of phage-displayed random polypeptides of about 140 amino acid residues. First, we constructed a system to artificially evolve phage-displayed random polypeptides toward binding to a target DNA containing a restriction enzyme site, in which random polypeptides capable of binding the DNA were recovered as complexes with the target DNA by digestion with the restriction enzyme. The experimental evolution cycle, including the above selection system and random mutagenesis for generating the next mutant library, was repeated until the fourth generation.

Extracting characteristic properties of fitness landscape from in vitro molecular evolution: a case study on infectivity of fd phage to E.coli.

We have developed a methodology for extracting characteristic properties of a fitness landscape of interest by analyzing fitness data on an in vitro molecular evolution. The in vitro evolution is required to be conducted as the following "adaptive walk": a single parent sequence generates N mutant sequences as its offsprings, and the fittest individual among the N offsprings will become a new parent in the next generation. N is the library size of mutants to be screened in a single generation.

Experimental rugged fitness landscape in protein sequence space.

The fitness landscape in sequence space determines the process of biomolecular evolution. To plot the fitness landscape of protein function, we carried out in vitro molecular evolution beginning with a defective fd phage carrying a random polypeptide of 139 amino acids in place of the g3p minor coat protein D2 domain, which is essential for phage infection. After 20 cycles of random substitution at sites 12-130 of the initial random polypeptide and selection for infectivity, the selected phage showed a 1.

Adaptive response of a gene network to environmental changes by fitness-induced attractor selection.

Cells switch between various stable genetic programs (attractors) to accommodate environmental conditions. Signal transduction machineries efficiently convey environmental changes to the gene regulation apparatus in order to express the appropriate genetic program. However, since the number of environmental conditions is much larger than that of available genetic programs so that the cell may utilize the same genetic program for a large set of conditions, it may not have evolved a signaling pathway for every environmental condition, notably those that are rarely encountered.

Nascent chain, mRNA, and ribosome complexes generated by a pure translation system.

Ribosome display is based on the concept that ternary complexes consisting of a nascent chain, ribosome, and mRNA can be generated, thereby establishing the linkage between genotype and phenotype that is essential for evolutionary experiments. With cell extract-based in vitro translation systems, it has been shown that ternary complexes can be generated by omitting the termination codon from the constructs, which can be stabilized at low temperature in the presence of high Mg2+ concentrations. Using an Escherichia coli-based reconstituted in vitro translation system (PURE system), in which all components necessary for the translation reaction were highly purified and reconstituted, ternary complexes could be generated equally well with a variety of sequences at the 3' end of the RNA, even those with a termination codon.

Inherent characteristics of gene expression for buffering environmental changes without the corresponding transcriptional regulations.

Gene expression patterning is crucial for environmental nutritional responses such as the nitrogen response in . The nitrogen response is primarily regulated by the expression of glutamine synthetase (GS), which catalyzes the sole reaction of glutamine formation, by cis-logic regulatory circuits. Here, by removing the entire corresponding operator and promoter regions required for the control of GS, we constructed an strain that enables the detection of the basal GS gene expression, which is expressed from a plain promoter unrelated to the nitrogen response, and measured by co-transcribed GFP expression, an indicator of GS expression.

Quantification of structural properties of cell-sized individual liposomes by flow cytometry.

We describe a new high-throughput method of quantifying the structural properties of individual cell-sized liposomes. An internal aqueous solution of liposomes was labeled with a green fluorescent marker and the membrane with a red marker. The double-labeled liposomes were analyzed using flow cytometry, and the internal aqueous volume and lipid membrane volume of each liposome were measured.

Femtoliter compartment in liposomes for in vitro selection of proteins.

The aqueous compartment in liposomes provides a reaction resembling the cell and therefore is used as a microcompartment in which to study enzymatic reactions. However, regardless of their method of preparation, the heterogeneity in size of cell-size liposomes limits their potential uses. We established a strategy to estimate the internal aqueous volume of cell-size liposomes using a fluorescence-activated cell sorter (FACS).

Functional Qbeta replicase genetically fusing essential subunits EF-Ts and EF-Tu with beta-subunit.

Qbeta replicase, an RNA-dependent RNA polymerase of RNA coliphage Qbeta, is a heterotetramer composed of a phage-encoded beta-subunit and three host-encoded proteins: the ribosomal protein S1 (alpha-subunit), EF-Tu, and EF-Ts. Several purification methods for Qbeta replicase were described previously. However, in our efforts to improve the production of Qbeta replicase, a substantial amount of the beta-subunit overproduced in Escherichia coli cells was found as insoluble aggregates.

Kinetic properties of Qbeta replicase, an RNA dependent RNA polymerase.

The kinetic properties of Qbeta replicase, an RNA-dependent RNA polymerase, were investigated experimentally. The reaction at the A-incorporation site was inhibited by UTP and CTP with inhibition constants of 3.2 and 2.

Synthesis of functional protein in liposome.

The liposome consisting of eggPC, cholesterol, and DSPE-PEG5000 with a molar ratio of 1.5:1:0.08 was used to entrap cell-free protein synthesis reaction mixture.

Effects of amino acid substitution on the physicochemical properties of artificial proteins with random sequences.

Physicochemical properties of four random proteins, each consisting of about 150 amino acid residues with different sequence identity, were compared to know the correlation between the physicochemical properties and its sequence. The results showed that the extent of the sequence alterations correlated well with the extent of differences in CD spectra, roughly with those in pH-solubility profiles and sedimentation velocity, and not with that in the binding of a hydrophobic fluorescent dye (ANS). Therefore, proteins with similar sequences can have different physicochemical properties, indicating that the extent of mutational effects varies in response to the sequence being altered.

Construction and characterization of phage libraries displaying artificial proteins with random sequences.

Three phage libraries, PL1, PL2, and PL3, displaying artificial proteins with random sequences were constructed. The artificial proteins, which are model of ancestral proteins, are derivatives of the 25 kinds of random proteins with about 140 amino acid residues produced via random mutagenesis and combinatorial recombination. The random proteins were displayed on the surface of filamentous bacteriophage as fusion protein with the pIII coat protein at an estimated average number on the phage particles in PL1, PL2, and PL3 of 0.

History dependent effects on phenotypic expression of a newly emerged gene.

In this study, we investigate the history dependence of the penetrance of a newly emerged gene. Penetrance is defined as the percentage of individuals with a given genotype who exhibit the phenotype associated with that particular genotype. Here, we used the glutamine synthetase gene and its mutants with lower fitness as model genes.

Expression of a cascading genetic network within liposomes.

Liposomes have long been used as possible compartments for artificial cells, and it has been shown that liposomes can sustain various types of biochemical reactions. To elevate the degree of molecular complexity of the system in liposomes, we have constructed a two-stage genetic network encapsulated in liposomes. This two-stage genetic network was constructed with the plasmid pTH, in which the protein product of the first stage (T7 RNA polymerase) is required to drive the protein synthesis of the second stage (GFP).

Evolution of an arbitrary sequence in solubility.

We have investigated the evolvability of an insoluble random polypeptide, RP3-34, to a soluble form through iterative mutation and selection with the aid of the green fluorescent protein (GFP) folding reporter. To assess the solubility of the polypeptides in the selected clones of each generation, the polypeptide genes were detached from the GFP fusions and expressed with a His(6) tag. The solubility of the variant random polypeptides increased in each generation within the scope of the evolutionary process, and the polypeptides assumed a soluble form from the fourth generation.