Effect of Liposome Size on Internal RNA Replication Coupled with Replicase Translation.

Cell membranes inhibit the diffusion of intracellular materials, and compartment size can strongly affect the intracellular biochemical reactions. To assess the effect of the size of microcompartments on intracellular reactions, we constructed a primitive cell model consisting of giant liposomes and a translation-coupled RNA replication (TcRR) system. The RNA was replicated by Qß replicase, which was translated from the RNA in giant liposomes encapsulating the cell-free translation system. A reporter RNA encoding the antisense strand of ß-glucuronidase was introduced into the system to yield a TcRR read-out (green fluorescence). We demonstrate that TcRR was hardly detectable in larger liposomes (230 fL) but was more effective in smaller (7.7 fL) liposomes. Our experimental and theoretical results show that smaller microcompartments considerably enhance TcRR because the synthesized molecules, such as RNA and replicases, are more concentrated in smaller liposomes.

In vitro evolution of α-hemolysin using a liposome display.

In vitro methods have enabled the rapid and efficient evolution of proteins and successful generation of novel and highly functional proteins. However, the available methods consider only globular proteins (e.g., antibodies, enzymes) and not membrane proteins despite the biological and pharmaceutical importance of the latter. In this study, we report the development of a method called liposome display that can evolve the properties of membrane proteins entirely in vitro. This method, which involves in vitro protein synthesis inside liposomes, which are cell-sized phospholipid vesicles, was applied to the pore-forming activity of α-hemolysin, a membrane protein derived from Staphylococcus aureus. The obtained α-hemolysin mutant possessed only two point mutations but exhibited a 30-fold increase in its pore-forming activity compared with the WT. Given the ability to synthesize various membrane proteins and modify protein synthesis and functional screening conditions, this method will allow for the rapid and efficient evolution of a wide range of membrane proteins.

The evolutionary enhancement of genotype-phenotype linkages in the presence of multiple copies of genetic material.

Genetic evolutionary mechanisms employed by protolife developed without accompanying regulatory mechanisms for the amounts of genetic material in protocells. When many copies of genetic material are present, inactive copies generated by mutations are not effectively excluded through phenotypic selection. We demonstrate a model of gene evolution initiated with different amounts of DNA inside artificial protocells. We adopted transcription- and translation-coupled RNA replication and liposome-based in vitro compartmentalization. Despite the fact that the average number of DNA copies in each liposome was 6.4, DNA encoding active genes was maintained until the 16th selection round. Our experimental and theoretical results indicated that gene evolution can occur in the presence of multiple DNA copies. Most genetic material became junk code through gene mutations, and consequently the linkage between genotype and phenotype was enhanced through the associated decreases in active genetic material.

Kinetic analysis of aptazyme-regulated gene expression in a cell-free translation system: modeling of ligand-dependent and -independent expression.

Aptazymes are useful as RNA-based switches of gene expression responsive to several types of compounds. One of the most important properties of the switching ability is the signal/noise (S/N) ratio, i.e., the ratio of gene expression in the presence of ligand to that in the absence of ligand. The present study was performed to gain a quantitative understanding of how the aptazyme S/N ratio is determined by factors involved in gene expression, such as transcription, RNA self-cleavage, RNA degradation, protein translation, and their ligand dependencies. We performed switching of gene expression using two on-switch aptazymes with different properties in a cell-free translation system, and constructed a kinetic model that quantitatively describes the dynamics of RNA and protein species involved in switching. Both theoretical and experimental analyses consistently demonstrated that factors determining both the absolute value and the dynamics of the S/N ratio are highly dependent on the routes of translation in the absence of ligand: translation from the ligand-independently cleaved RNA or leaky translation from the noncleaved RNA. The model obtained here is useful to assess the factors that restrict the S/N ratio and to improve aptazymes more efficiently.

Kinetic analysis of ß-galactosidase and ß-glucuronidase tetramerization coupled with protein translation.

Both ß-galactosidase (GAL) and ß-glucuronidase (GUS) are tetrameric enzymes used widely as reporter proteins. However, little is known about the folding and assembly of these enzymes. Although the refolding kinetics of GAL from a denatured enzyme have been reported, it is not known how the kinetics differ when coupled with a protein translation reaction. Elucidating the assembly kinetics of GAL and GUS when coupled with protein translation will illustrate the differences between these two reporter proteins and also the assembly process under conditions more relevant to those in vivo. In this study, we used an in vitro translation/transcription system to synthesize GAL and GUS, measured the time development of the activity and oligomerization state of these enzymes, and determined the rate constants of the monomer to tetramer assembly process. We found that at similar concentrations, GAL assembles into tetramers faster than GUS. The rate constant of monomer to dimer assembly of GAL was 50-fold faster when coupled with protein translation than that of refolding from the denatured state. Furthermore, GAL synthesis was found to lack the rate-limiting step in the assembly process, whereas GUS has two rate-limiting steps: monomer to dimer assembly and dimer to tetramer assembly. The consequence of these differences when used as reporter proteins is discussed.

α-Complementation in an artificial genome replication system in liposomes.

Genome size is considered one of the limiting factors for the replication of primitive life forms. However, the relationship between genome size and replication efficiency has not been tested experimentally. In this study, we examined the effect of genome size on genome replication by using an artificial cell model: a self-replicating RNA genome encapsulated in a liposome. For the reduced genome size we used α-complementation of the lacZ gene. We first characterized α-complementation in the purified translation system and then applied α-complementation to the genome replication system. The reduction in the genome size together with the addition of ω-fragment increased the replication efficiency approximately eightfold. This result provides experimental evidence that genome size can be a limiting factor for primitive self-replication systems; it also implies that this artificial cell model could be a useful experimental model to identify possible mechanisms of genome enlargement.

Bayesian-based decipherment of in-depth information in bacterial chemical sensing beyond pleasant/unpleasant responses.

Chemical sensing is vital to the survival of all organisms. Bacterial chemotaxis is conducted by multiple receptors that sense chemicals to regulate a single signalling system controlling the transition between the direction (clockwise vs. counterclockwise) of flagellar rotation. Such an integrated system seems better suited to judge chemicals as either favourable or unfavourable, but not for identification purposes though differences in their affinities to the receptors may cause difference in response strength. Here, an experimental setup was developed to monitor behaviours of multiple cells stimulated simultaneously as well as a statistical framework based on Bayesian inferences. Although responses of individual cells varied substantially, ensemble averaging of the time courses seemed characteristic to attractant species, indicating we can extract information of input chemical species from responses of the bacterium. Furthermore, two similar, but distinct, beverages elicited attractant responses of cells with profiles distinguishable with the Bayesian procedure. These results provide a basis for novel bio-inspired sensors that could be used with other cell types to sense wider ranges of chemicals.

In vitro selection of proteins that undergo covalent labeling with small molecules by thiol-disulfide exchange by using ribosome display.

There is a great deal of interest in proteins that can bind covalently to target molecules, as they allow unambiguous experiments by tight binding to molecules of interest. Here, we report the generation of proteins that undergo covalent labeling with small molecules through in vitro selection by using ribosome display. Selection was performed from a mutant library of the WW domain with a biotinylated peptide as its binding target, in which the biotin and the peptide are connected by a disulfide bond. After five rounds of selection, we identified mutants carrying a particular cysteine mutation. The binding target reacted specifically with the selected mutant, even in the presence of other proteins, and resulted in the generation of biotin- or peptide-labeled WW domains by thiol-disulfide exchange. When the mutant was fused to a protein of interest, the fusion protein was also labeled with biotin. Thus, the characteristics of the selected mutant should be suitable as a tag sequence that can be covalently labeled with small synthetic molecules. These results indicate that the rapid and efficient generation of such proteins is possible by ribosome display.

Compartmentalization in a water-in-oil emulsion repressed the spontaneous amplification of RNA by Q beta replicase.

During RNA replication mediated by Qbeta replicase, self-replicating RNAs (RQ RNAs) are amplified without the addition of template RNA. This undesired amplification makes the study of target RNA replication difficult, especially for long RNA such as genomic RNA of Qbeta phage. This perhaps is one of the reasons why the precise rate of genomic RNA replication in the presence of host factor Hfq has not been reported in vitro. Here, we report a method to repress RQ RNA amplification by compartmentalization of the reaction using a water-in-oil emulsion but maintaining the activity of Qbeta replicase. This method allowed us to amplify the phage Qbeta genome RNA exponentially without detectable amplification of RQ RNA. Furthermore, we found that the rate constant of genome RNA replication in the exponential phase at the optimum Hfq concentration was approximately 4.6 times larger than that of a previous report, close to in vivo data. This result indicates that the replication rate in vivo is largely explained by the presence of Hfq. This easy method paves the way for the study of genomic RNA replication without special care for the undesired RQ RNA amplification.

Quantifying epistatic interactions among the components constituting the protein translation system.

In principle, the accumulation of knowledge regarding the molecular basis of biological systems should allow the development of large-scale kinetic models of their functions. However, the development of such models requires vast numbers of parameters, which are difficult to obtain in practice. Here, we used an in vitro translation system, consisting of 69 defined components, to quantify the epistatic interactions among changes in component concentrations through Bahadur expansion, thereby obtaining a coarse-grained model of protein synthesis activity. Analyses of the data measured using various combinations of component concentrations indicated that the contributions of larger than 2-body inter-component epistatic interactions are negligible, despite the presence of larger than 2-body physical interactions. These findings allowed the prediction of protein synthesis activity at various combinations of component concentrations from a small number of samples, the principle of which is applicable to analysis and optimization of other biological systems. Moreover, the average ratio of 2- to 1-body terms was estimated to be as small as 0.1, implying high adaptability and evolvability of the protein translation system.

Comprehensive analysis of the effects of Escherichia coli ORFs on protein translation reaction.

Protein synthesis is one of the most important reactions in the cell. Recent experimental studies indicated that this complex reaction can be achieved with a minimum complement of 36 proteins and ribosomes by reconstituting an Escherichia coli-based in vitro translation system with these protein components highly purified on an individual basis. From the protein-protein interaction (PPI) network of E. coli proteins, these minimal protein components are known to interact physically with large numbers of proteins. However, it is unclear what fraction of E. coli proteins are linked functionally with the minimal protein synthesis system. We investigated the effects of each of the 4194 E. coli ORF products on the minimal protein synthesis system; at least 12% of the entire ORF products, a significant fraction of the gene product of E. coli, affect the activity of this system. Furthermore 34% of these functional modifiers present in the PPI network were shown by mapping to be directly linked (i.e. to interact physically) with the minimal components of the PPI network. Topological analysis of the relationships between modifiers and the minimal components in the PPI network indicated clustering of the minimal components. The modifiers showed no such clustering, indicating that the location of functional modifiers is spread across the PPI network rather than clustering close to the minimal protein components. These observations may reflect the evolutionary process of the protein synthesis system.

A transcription and translation-coupled DNA replication system using rolling-circle replication.

All living organisms have a genome replication system in which genomic DNA is replicated by a DNA polymerase translated from mRNA transcribed from the genome. The artificial reconstitution of this genome replication system is a great challenge in in vitro synthetic biology. In this study, we attempted to construct a transcription- and translation-coupled DNA replication (TTcDR) system using circular genomic DNA encoding phi29 DNA polymerase and a reconstituted transcription and translation system. In this system, phi29 DNA polymerase was translated from the genome and replicated the genome in a rolling-circle manner. When using a traditional translation system composition, almost no DNA replication was observed, because the tRNA and nucleoside triphosphates included in the translation system significantly inhibited DNA replication. To minimize these inhibitory effects, we optimized the composition of the TTcDR system and improved replication by approximately 100-fold. Using our system, genomic DNA was replicated up to 10 times in 12 hours at 30 °C. This system provides a step toward the in vitro construction of an artificial genome replication system, which is a prerequisite for the construction of an artificial cell.

Development of a reporter peptide that catalytically produces a fluorescent signal through α-complementation.

In α-complementation, inactive N-terminal (α-domain) and C-terminal (ω-domain) fragments of ß-galactosidase associate to reconstitute the active protein. To date, the effect of α-domain size on α-complementation activity has not been systematically investigated. In this study, we compared the complementation activities of α-domains of various sizes using an in vitro system. We found that the complementation activities are similar for α-domains comprising between 45 and 229 N-terminal residues but are significantly decreased for those containing less than 37 residues. However, these smaller α-domains (15 and 25 residues) exhibited sufficient α-complementation activity for application as reporters.

Adaptive evolution of an artificial RNA genome to a reduced ribosome environment.

The reconstitution of an artificial system that has the same evolutionary ability as a living thing is a major challenge in the in vitro synthetic biology. In this study, we tested the adaptive evolutionary ability of an artificial RNA genome replication system, termed the translation-coupled RNA replication (TcRR) system. In a previous work, we performed a study of the long-term evolution of the genome with an excess amount of ribosome. In this study, we continued the evolution experiment in a reduced-ribosome environment and observed that the mutant genome compensated for the reduced ribosome concentration. This result demonstrated the ability of the TcRR system to adapt and may be a step toward generating living things with evolutionary ability.

Evolutionary Consequence of a Trade-Off between Growth and Maintenance along with Ribosomal Damages.

Microorganisms in nature are constantly subjected to a limited availability of resources and experience repeated starvation and nutrition. Therefore, microbial life may evolve for both growth fitness and sustainability. By contrast, experimental evolution, as a powerful approach to investigate microbial evolutionary strategies, often targets the increased growth fitness in controlled, steady-state conditions. Here, we address evolutionary changes balanced between growth and maintenance while taking nutritional fluctuations into account. We performed a 290-day-long evolution experiment with a histidine-requiring Escherichia coli strain that encountered repeated histidine-rich and histidine-starved conditions. The cells that experienced seven rounds of starvation and re-feed grew more sustainably under prolonged starvation but dramatically lost growth fitness under rich conditions. The improved sustainability arose from the evolved capability to use a trace amount of histidine for cell propagation. The reduced growth rate was attributed to mutations genetically disturbing the translation machinery, that is, the ribosome, ultimately slowing protein translation. This study provides the experimental demonstration of slow growth accompanied by an enhanced affinity to resources as an evolutionary adaptation to oscillated environments and verifies that it is possible to evolve for reduced growth fitness. Growth economics favored for population increase under extreme resource limitations is most likely a common survival strategy adopted by natural microbes.

Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system.

In this study, the amount of protein synthesized using an in vitro protein synthesis system composed of only highly purified components (the PURE system) was optimized. By varying the concentrations of each system component, we determined the component concentrations that result in the synthesis of 0.38 mg/mL green fluorescent protein (GFP) in batch mode and 3.8 mg/mL GFP in dialysis mode. In dialysis mode, protein concentrations of 4.3 and 4.4 mg/mL were synthesized for dihydrofolate reductase and ß-galactosidase, respectively. Using the optimized system, the synthesized protein represented 30% (w/w) of the total protein, which is comparable to the level of overexpressed protein in Escherichia coli cells. This optimized reconstituted in vitro protein synthesis system may potentially be useful for various applications, including in vitro directed evolution of proteins, artificial cell assembly, and protein structural studies.

Effects of ribosomes on the kinetics of Qß replication.

Bacteriophage Qß utilizes some host cell translation factors during replication. Previously, we constructed a kinetic model that explains replication of long RNA molecules by Qß replicase. Here, we expanded the previous kinetic model to include the effects of ribosome concentration on RNA replication. The expanded model quantitatively explained single- and double-strand formation kinetics during replication with various ribosome concentrations for two artificial long RNAs. This expanded model and the knowledge obtained in this study provide useful frameworks to understand the precise replication mechanism of Qß replicase with ribosomes and to design amplifiable RNA genomes in translation-coupling systems.

Liposome display for in vitro selection and evolution of membrane proteins.

Liposome display is a novel method for in vitro selection and directed evolution of membrane proteins. In this approach, membrane proteins of interest are displayed on liposome membranes through translation from a single DNA molecule by using an encapsulated cell-free translation system. The liposomes are probed with a fluorescence indicator that senses membrane protein activity and selected using a fluorescence-activated cell sorting (FACS) instrument. Consequently, DNA encoding a protein with a desired function can be obtained. By implementing this protocol, researchers can process a DNA library of 10(7) different mutants. A single round of the selection procedure requires 24 h for completion, and multiple iterations of this technique, which take 1-5 weeks, enable the isolation of a desired gene. As this protocol is conducted entirely in vitro, it enables the engineering of various proteins, including pore-forming proteins, transporters and receptors. As a useful example of the approach, here we detail a procedure for the in vitro evolution of α-hemolysin from Staphylococcus aureus for its pore-forming activity.

A controllable gene expression system in liposomes that includes a positive feedback loop.

We introduced a positive feedback loop into a LacI-dependent gene expression system in lipid vesicles, producing a cell-like system that senses and responds to an external signal with a high signal-to-noise ratio. This fully reconstituted system will be a useful tool in future applications in in vitro synthetic biology.

Kinetic model of double-stranded RNA formation during long RNA replication by Qß replicase.

Qß replicase is an RNA-dependent RNA polymerase, which synthesizes the complementary RNA using a single-stranded RNA as a template. The formation of non-replicable double-stranded RNA (dsRNA) by hybridization between newly synthesized RNA and the template RNA hinders the broader application of Qß replicase. Here, we developed a kinetic model of Qß RNA replication consisting of two reaction pathways of dsRNA formation, which quantitatively explains the dynamics of dsRNA formation of three template RNAs. We also found that part of the Qß phage genomic RNA sequence including the central hairpin loop significantly decreases the rate of dsRNA formation.