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

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

Unfolded states of ribonuclease A were used to investigate the effects macromolecular crowding on compactness and protein folding. The extent folding measured by circular dichroism spectroscopy, fluorescence correlation NMR spectroscopy in presence polyethylene glycol (PEG) or Ficoll as agent. unfolded state RNase a 2.4 M urea solution at pH 3.0 became native conformation addition 35% PEG 20000 70. In addition, inert macromolecule investigated using Fluorescence-labeled test macromolecule....

The discovery of two fundamental laws concerning cellular dynamics with recursive growth is reported. Firstly, the chemical abundances measured over many cells were found to obey a log-normal distribution and secondly, relationship between average standard deviation was be linear. ubiquity these explored both theoretically experimentally. By means model catalytic reaction network, shown exist near critical state efficient self-reproduction. Additionally, by measuring distributions...

Mechanisms that enabled primitive cell membranes to self-reproduce have been discussed based on the physicochemical properties of fatty acids; however, there must be a transition modern composed phospholipids [Budin I, Szostak JW (2011) Proc Natl Acad Sci USA 108:5249-5254]. Thus, growth-division mechanism does not depend chemical nature amphiphilic molecules existed. Here, we show giant unilamellar vesicles can undergo coupled process fusion and budding transformation, which mimics growth...

In all living systems, the genome is replicated by proteins that are encoded within itself. This universal reaction essential to allow system evolve. Here, we have constructed a simplified involving encapsulated macromolecules termed "self-encoding system", in which genetic information self-encoded replicase liposomes. That is, was reconstituted microcompartment bound lipid bilayer. The assembled using one template RNA sequence as molecule and an vitro translation from purified factors...

Abstract Background Understanding ethanol tolerance in microorganisms is important for the improvement of bioethanol production. Hence, we performed parallel-evolution experiments using Escherichia coli cells under stress to determine phenotypic changes necessary tolerance. Results After cultivation 1,000 generations 5% stress, obtained 6 ethanol-tolerant strains that showed an approximately 2-fold increase their specific growth rate comparison with ancestor. Expression analysis microarrays...

Lipid vesicles have been used as model cell systems, in which an in-vitro transcription–translation system (IVTT) is encapsulated to carry out intravesicular protein synthesis. Despite a large number of previous studies, quantitative understanding how synthesis inside the affected by lipid membrane remains elusive. This mainly because heterogeneity structural properties experiments. We investigated effects phospholipid on green fluorescent (GFP) occurring cell-sized giant unilamellar (GUV),...

Significance The directed evolution of proteins in vitro has generated highly functional and contributed to elucidating the sequence–function relationship proteins. However, available methods consider globular proteins, not membrane despite biological pharmaceutical importance latter. We report development a method called liposome display, which enables applied evolve pore-forming activity α-hemolysin from Staphylococcus aureus obtained mutant with 30-fold higher than WT. Given its high...

Significance We reconstituted a simple in vitro molecular ecosystem using two self-replicating RNA molecules. The host species produces an RNA-dependent polymerase for their replication, and the parasitic relies on same enzyme its replication as well. found that only when compartmentalized into microscale droplets did show typical host–parasite oscillatory dynamics. Furthermore, after long cultivation, evolved to allow coexistence of species. These results demonstrate cell-size...

Highly parallel experimental biology is offering opportunities to not just accomplish work more easily, but explore for underlying governing principles. Recent analysis of the large-scale organization gene expression has revealed its complex and dynamic nature. However, dynamics that generate cellular are yet understood. To comprehensively quantitatively elucidate these dynamics, we have analyzed genome-wide in many conditions Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana,...

A general relationship between fluctuation and response in a biological system is presented. The given by the variance of some quantity, whereas as average change that quantity for parameter change. We propose where two are proportional, similar way to fluctuation–dissipation theorem physics. By studying an evolution experiment fluorescence protein bacteria increases, we confirm our relation observing positive correlation speed phenotypic over clone bacteria. generality well its relevance discussed.

It remains to be determined experimentally whether increasing fitness is related positive selection, while stationary neutral evolution. Long-term laboratory evolution in Escherichia coli was performed under conditions of thermal stress defined conditions. The complete cell growth data showed common continuous recovery every 2°C or 4°C stepwise temperature upshift, finally resulting an evolved E. strain with improved upper limit as high 45.9°C after 523 days serial transfer, equivalent 7,560...

Next-generation sequencing technologies enable the rapid cost-effective production of sequence data. To evaluate performance these technologies, investigation quality reads obtained from methods is important. In this study, we analyzed and SNP detection using three commercially available next-generation sequencers, i.e., Roche Genome Sequencer FLX System (FLX), Illumina Analyzer (GA), Applied Biosystems SOLiD system (SOLiD). A common genomic DNA sample Escherichia coli strain DH1 was applied...

Giant unilamellar vesicles (GUVs) are vesicles>1 μm in diameter that provide an environment which the effect of a confined reaction volume on intravesicular reactions can be investigated. By synthesizing EmrE, multidrug transporter from Escherichia coli, as model membrane protein using reconstituted vitro transcription-translation system inside GUVs, we investigated synthesis and integration EmrE. Flow cytometry was used to analyze multiple properties quantify EmrE GUVs composed only...

A major challenge in constructing artificial cells is the establishment of a recursive genome replication system coupled with gene expression from itself. One simplest schemes DNA rolling-circle circular recombination. In this study, we attempted to develop based on scheme using self-encoded phi29 polymerase and externally supplied Cre recombinase. We first identified that polymerization significantly inhibited by To overcome problem, performed vitro evolution obtained an evolved can...

Diphtheria toxin (DT) receptor associates with a 27-kD membrane protein (DRAP27) in monkey Vero cells. A cDNA encoding DRAP27 was isolated, and its nucleotide sequence determined. The deduced amino acid revealed that is the homologue of human CD9 antigen. recognized by antibodies. human-mouse hybrid cell line (3279-10) possessing chromosome 5, sensitive to DT, but not expressing antigen, used for transfection experiments DRAP27. When cloned transiently expressed 3279-10 cells, total DT...

The fitness landscape in sequence space determines the process of biomolecular evolution. To plot protein function, we carried out vitro molecular evolution beginning with a defective fd phage carrying random polypeptide 139 amino acids place g3p minor coat D2 domain, which is essential for infection. After 20 cycles substitution at sites 12–130 initial and selection infectivity, selected showed 1.7×104-fold increase defined as number infected cells per ml suspension. Fitness was logarithm...