Rapid, Affordable, and Uncomplicated Production of Bacterial Cell-free Lysate.

Cell-free gene expression offers the power of biology without the complications of a living organism. Although many such gene expression systems exist, most are quite expensive to buy and/or require special equipment and finely honed expertise to produce effectively. This protocol describes a method to produce bacterial cell-free lysate that supports high levels of gene expression, using only standard laboratory equipment and requiring minimal processing. The method uses an Escherichia coli strain producing an endolysin that does not affect growth, but which efficiently lyses a harvested cell pellet following a simple freeze-thaw cycle. The only further processing required is a brief incubation followed by centrifugation to clear the autolysate of cellular debris. Dynamic gene circuits can be achieved through heterologous expression of the ClpX protease in the cells before harvesting. An E. coli strain lacking the lacZ gene can be used for high-sensitivity, cell-free biosensing applications using a colorimetric or fluorescent readout. The entire protocol requires as few as 8-9 hours, with only 1-2 hours of hands-on labor from inoculation to completion. By reducing the cost and time to obtain cell-free lysate, this method should increase the affordability of cell-free gene expression for various applications.

The Inhibition of Icing and Frosting on Glass Surfaces by the Coating of Polyethylene Glycol and Polypeptide Mimicking Antifreeze Protein.

The development of anti-icing, anti-frosting transparent plates is important for many reasons, such as poor visibility through the ice-covered windshields of vehicles. We have fabricated new glass surfaces coated with polypeptides which mimic a part of winter flounder antifreeze protein. We adopted glutaraldehyde and polyethylene glycol as linkers between these polypeptides and silane coupling agents applied to the glass surfaces. We have measured the contact angle, the temperature of water droplets on the cooling surfaces, and the frost weight. In addition, we have conducted surface roughness observation and surface elemental analysis. It was found that peaks in the height profile, obtained with the atomic force microscope for the polypeptide-coated surface with polyethylene glycol, were much higher than those for the surface without the polypeptide. This shows the adhesion of many polypeptide aggregates to the polyethylene glycol locally. The average supercooling temperature of the droplet for the polypeptide-coated surface with the polyethylene glycol was lower than for the polypeptide-coated surface with glutaraldehyde and the polyethylene-glycol-coated surface without the polypeptide. In addition, the average weight of frost cover on the specimen was lowest for the polypeptide-coated surface with the polyethylene glycol. These results argue for the effects of combined polyethylene glycol and polypeptide aggregates on the locations of ice nuclei and condensation droplets. Thus, this polypeptide-coating with the polyethylene glycol is a potential contender to improve the anti-icing and anti-frosting of glasses.

Microfluidic Device with an Integrated Freeze-Dried Cell-Free Protein Synthesis System for Small-Volume Biosensing.

Microfluidic devices enable the precise operation of liquid samples in small volumes. This motivates why microfluidic devices have been applied to point-of-care (PoC) liquid biopsy. Among PoC liquid biopsy studies, some report diagnostic reagents being freeze-dried in such microfluidic devices. This type of PoC microfluidic device has distinct advantages, such as simplicity of the procedures, compared with other PoC devices using liquid-type diagnostic reagents. Despite the attractive characteristic, only diagnostic reagents based on the cloned enzyme donor immunoassay (CEDIA) have been freeze-dried in the microfluidic device. However, development of the PoC device based on the CEDIA method is time-consuming and labor-intensive. Here, we employed a molecule-responsive protein synthesis system as the diagnostic reagent to be freeze-dried in the microfluidic device. Such molecule-responsive protein synthesis has been well investigated in the field of molecular biology. Therefore, using the accumulated information, PoC devices can be efficiently developed. Thus, we developed a microfluidic device with an integrated freeze-dried molecule-responsive protein synthesis system. Using the developed device, we detected two types of bio-functional molecules (i.e., bacterial quorum sensing molecules and mercury ions) by injecting 1 µL of sample solution containing these molecules. We showed that the developed device is applicable for small-volume biosensing.

Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression.

Cell-free gene expression systems are emerging as an important platform for a diverse range of synthetic biology and biotechnology applications, including production of robust field-ready biosensors. Here, we combine programmed cellular autolysis with a freeze-thaw or freeze-dry cycle to create a practical, reproducible, and a labor- and cost-effective approach for rapid production of bacterial lysates for cell-free gene expression. Using this method, robust and highly active bacterial cell lysates can be produced without specialized equipment at a wide range of scales, making cell-free gene expression easily and broadly accessible. Moreover, live autolysis strain can be freeze-dried directly and subsequently lysed upon rehydration to produce active lysate. We demonstrate the utility of autolysates for synthetic biology by regulating protein production and degradation, implementing quorum sensing, and showing quantitative protection of linear DNA templates by GamS protein. To allow versatile and sensitive ß-galactosidase (LacZ) based readout we produce autolysates with no detectable background LacZ activity and use them to produce sensitive mercury(II) biosensors with LacZ-mediated colorimetric and fluorescent outputs. The autolysis approach can facilitate wider adoption of cell-free technology for cell-free gene expression as well as other synthetic biology and biotechnology applications, such as metabolic engineering, natural product biosynthesis, or proteomics.

Repetitive formation of optically-observable planar lipid bilayers by rotating chambers on a microaperture.

Optical observation of a planar lipid bilayer is an effective method of lipid bilayer characterization. However, previous methods for optically observable lipid bilayer formation are unsuitable for repetitive formation of lipid bilayers. In this paper, we propose a system that facilitates repetitive formation of horizontal lipid bilayers via mechanical rotation of the rotating part. We show that multiple bilayers can be observed within a short period, and that the electrical and optical characteristics of a bilayer can be analyzed simultaneously.

An inhalation anesthetic device for stereotaxic operation on mouse pups.

BACKGROUND: Mouse pups are invaluable model animals for understanding the molecular and neural basis underlying behavioral development. Stereotaxic operations with anesthetic control are useful tools in systems neuroscience. However, there are no commercially available anesthetic or stereotaxic devices for mouse pups. Current devices have several problems such as invasive approach for stabilization, poor sanitary control, and less flexibility to combine other surgical apparatuses. NEW METHOD: Here, we developed an inhalation anesthetic device equipped with stereotaxic function for mouse pups, by using polydimethylsiloxane (PDMS). PDMS is tolerant to heat and water exposure, and soft enough to cut or make a hole. The anesthetic and the stereotaxic parts were fabricated from the three-dimensional computer-aided design (3D CAD) data obtained from the head of a real mouse pup. RESULTS: To confirm its utility, a tracer was injected into the brain. We were able to anesthetize and stabilize pups at once in a non-invasive manner using the PDMS device. The histological staining revealed that tracer injection was successful. Our device was compatible with various types of commercial stereotaxic and anesthetic apparatuses via trimming and tube insertion, respectively. COMPARISON WITH EXISTING METHOD(S): To our knowledge, this is the first report of a device that can stabilize the mouse pup's head with the non-invasive manner and functions as an inhalation anesthetic device that can be sterilized. CONCLUSIONS: The present fabrication method will provide a handy and functional instrument for stereotaxic operations in animal models at various developmental stages.

pH-induced motion control of self-propelled oil droplets using a hydrolyzable gemini cationic surfactant.

Self-propelled motion of micrometer-sized substances has drawn much attention as an autonomous transportation system. One candidate vehicle is a chemically driven micrometer-sized oil droplet. However, to the best of our knowledge, there has been no report of a chemical reaction system controlling the three-dimensional motion of oil droplets underwater. In this study, we developed a molecular system that controlled the self-propelled motion of 4-heptyloxybenzaldehyde oil droplets by using novel gemini cationic surfactants containing carbonate linkages (2G12C). We found that, in emulsions containing sodium hydroxide, the motion time of the self-propelled oil droplets was longer in the presence of 2G12C than in the presence of gemini cationic surfactants without carbonate linkages. Moreover, in 2G12C solution, oil droplets at rest underwent unidirectional, self-propelled motion in a gradient field toward a higher concentration of sodium hydroxide. Even though they stopped within several seconds, they restarted in the same direction. 2G12C was gradually hydrolyzed under basic conditions to produce a pair of the corresponding monomeric surfactants, which exhibit different interfacial properties from 2G12C. The prolonged and restart motion of the oil droplets were explained by the increase in the heterogeneity of the interfacial tension of the oil droplets.

Lipid bilayers on a picoliter microdroplet array for rapid fluorescence detection of membrane transport.

This paper describes picoliter-sized lipid bilayer chambers and their theoretical model for the rapid detection of membrane transport. To prepare the chambers, semispherical aqueous droplets are patterned on a hydrophilic/hydrophobic substrate and then brought into contact with another aqueous droplet in lipid-dispersed organic solvent, resulting in the formation of the lipid bilayers on the semispherical droplets. The proposed method implements the lipid bilayer chambers with 25-fold higher ratio of lipid membrane area (S) to chamber volume (V) compared to the previous spherical droplet chambers. Using these chambers, we are able to trace the time-course of Ca(2+) influx through α-hemolysin pores by a fluorescent indicator. Moreover, we confirm that the detection time of the substrate transport is inversely proportional to the S/V ratio of the developed chambers, which is consistent with the simulation results based on the developed model. Our chambers and model might be useful for rapid functional analyses of membrane transport phenomena.

Magnetically responsive microflaps reveal cell membrane boundaries from multiple angles.

A microflap system to incline adherent cells in the desired orientation is described. Inclination angles of cell-laden microflaps are precisely controlled by the applied magnetic field, enabling us to observe cell-membrane boundaries from multiple angles. This system is equipped with conventional microscopes, allowing clear focused images of cell-membrane boundaries to be obtained with high magnification.

Induced chirality through electromagnetic coupling between chiral molecular layers and plasmonic nanostructures.

We report a new approach for creating chiral plasmonic nanomaterials. A previously unconsidered, far-field mechanism is utilized which enables chirality to be conveyed from a surrounding chiral molecular material to a plasmonic resonance of an achiral metallic nanostructure. Our observations break a currently held preconception that optical properties of plasmonic particles can most effectively be manipulated by molecular materials through near-field effects. We show that far-field electromagnetic coupling between a localized plasmon of a nonchiral nanostructure and a surrounding chiral molecular layer can induce plasmonic chirality much more effectively (by a factor of 10(3)) than previously reported near-field phenomena. We gain insight into the mechanism by comparing our experimental results to a simple electromagnetic model which incorporates a plasmonic object coupled with a chiral molecular medium. Our work offers a new direction for the creation of hybrid molecular plasmonic nanomaterials that display significant chiroptical properties in the visible spectral region.