Genetic Approaches to Study the Interplay Between Transcription and Nucleoid-Associated Proteins in Escherichia coli.

Bacterial nucleoid-associated proteins are important factors in regulation of transcription, in nucleoid structuring, and in homeostasis of DNA supercoiling. Vice versa, transcription influences DNA supercoiling and can affect DNA binding of nucleoid-associated proteins (NAPs) such as H-NS in Escherichia coli. Here we describe genetic tools to study the interplay between transcription and nucleoid-associated proteins in E. coli. These methods include construction of genomic and plasmidic transcriptional and translational lacZ reporter gene fusions to study regulation of promoters; insertion of promoter cassettes to drive transcription into a locus of interest in the genome, for example, an H-NS-bound locus; and construction of isogenic hns and stpA mutants and precautions in doing so.

High Abundance of Transcription Regulators Compacts the Nucleoid in Escherichia coli.

In enteric bacteria organization of the circular chromosomal DNA into a highly dynamic and toroidal-shaped nucleoid involves various factors, such as DNA supercoiling, nucleoid-associated proteins (NAPs), the structural maintenance of chromatin (SMC) complex, and macrodomain organizing proteins. Here, we show that ectopic expression of transcription regulators at high levels leads to nucleoid compaction. This serendipitous result was obtained by fluorescence microscopy upon ectopic expression of the transcription regulator and phosphodiesterase PdeL of Escherichia coli. Nucleoid compaction by PdeL depends on DNA-binding, but not on its enzymatic phosphodiesterase activity. Nucleoid compaction was also observed upon high-level ectopic expression of the transcription regulators LacI, RutR, RcsB, LeuO, and Cra, which range from single-target gene regulators to global regulators. In the case of LacI, its high-level expression in the presence of the gratuitous inducer IPTG (isopropyl-ß-d-thiogalactopyranoside) also led to nucleoid compaction, indicating that compaction is caused by unspecific DNA-binding. In all cases nucleoid compaction correlated with misplacement of the FtsZ ring and loss of MukB foci, a subunit of the SMC complex. Thus, high levels of several transcription regulators cause nucleoid compaction with consequences for replication and cell division. IMPORTANCE The bacterial nucleoid is a highly organized and dynamic structure for simultaneous transcription, replication, and segregation of the bacterial genome. Compaction of the nucleoid and disturbance of DNA segregation and cell division by artificially high levels of transcription regulators, as described here, reveals that an excess of DNA-binding protein disturbs nucleoid structuring. The results suggest that ectopic expression levels of DNA-binding proteins for genetic studies of their function but also for their purification should be carefully controlled and adjusted.

Deletion of FRT-sites by no-SCAR recombineering in Escherichia coli.

Lambda-Red recombineering is the most commonly used method to create point mutations, insertions or deletions in Escherichia coli and other bacteria, but usually an Flp recognition target (FRT) scar-site is retained in the genome. Alternative scarless recombineering methods, including CRISPR/Cas9-assisted methods, generally require cloning steps and/or complex PCR schemes for specific targeting of the genome. Here we describe the deletion of FRT scar-sites by the scarless Cas9-assisted recombineering method no-SCAR using an FRT-specific guide RNA, sgRNAFRT, and locus-specific ssDNA oligonucleotides. We applied this method to construct a scarless E. coli strain suitable for gradual induction by l-arabinose. Genome sequencing of the resulting strain and its parent strains demonstrated that no additional mutations were introduced along with the simultaneous deletion of two FRT scar-sites. The FRT-specific no-SCAR selection by sgRNAFRT/Cas9 may be generally applicable to cure FRT scar-sites of E. coli strains constructed by classical λ-Red recombineering.

The transcription regulator and c-di-GMP phosphodiesterase PdeL represses motility in Escherichia coli.

PdeL is a transcription regulator and catalytically active c-di-GMP phosphodiesterases (PDE) in Escherichia coli PdeL has been shown to be a transcription autoregulator, while no other target genes have been identified so far. Here, we show that PdeL represses transcription of the flagella class II operon, fliFGHIJK, and activates sslE encoding an extracellular anchored metalloprotease, among additional loci. DNA-binding studies and expression analyses using plasmidic reporters suggest that regulation of the fliF and sslE promoters by PdeL is direct. Transcription repression of the fliFGHIJK operon, encoding protein required for assembly of the flagellar basal body, results in inhibition of motility on soft agar plates and reduction of flagella assembly, as shown by fluorescence staining of the flagella hook protein FlgE. PdeL-mediated repression of motility is independent of its phosphodiesterase activity. Thus, in motility control the transcription regulator function of PdeL reducing the number of assembled flagella is apparently epistatic to its phosphodiesterase function, which can indirectly promote the activity of the flagellar motor by lowering the c-di-GMP concentration.Bacteria adopt different lifestyles depending on their environment and physiological condition. In Escherichia coli and other enteric bacteria the transition between the motile and the sessile state is controlled at multiple levels from the regulation of gene expression to the modulation of various processes by the second messenger c-di-GMP as signaling molecule. The significance of our research is in identifying PdeL, a protein of dual function that hydrolyzes c-di-GMP and that regulates transcription of genes, as a repressor of Flagella gene expression and an inhibitor of motility, which adds an additional regulatory switch to the control of motility.

High-Rate Printing of Micro/Nanoscale Patterns Using Interfacial Convective Assembly.

Printing of electronics has been receiving increasing attention from academia and industry over the recent years. However, commonly used printing techniques have limited resolution of micro- or sub-microscale. Here, a directed-assembly-based printing technique, interfacial convective assembly, is reported, which utilizes a substrate-heating-induced solutal Marangoni convective flow to drive particles toward patterned substrates and then uses van der Waals interactions as well as geometrical confinement to trap the particles in the pattern areas. The influence of various assembly parameters including type of mixing solvent, substrate temperature, particle concentration, and assembly time is investigated. The results show successful assembly of various nanoparticles in patterns of different shapes with a high resolution down to 25 nm. In addition, the assembly only takes a few minutes, which is two orders of magnitude faster than conventional convective assembly. Small-sized (diameter below 5 nm) nanoparticles tend to coalesce during the assembly process and form sintered structures. The fabricated silver nanorods show single-crystal structure with a low resistivity of 8.58 × 10-5 Ω cm. With high versatility, high resolution, and high throughput, the interfacial convective assembly opens remarkable opportunities for printing next generation nanoelectronics and sensors.

Directed assembly-based printing of homogeneous and hybrid nanorods using dielectrophoresis.

Printing nano and microscale three-dimensional (3D) structures using directed assembly of nanoparticles has many potential applications in electronics, photonics and biotechnology. This paper presents a reproducible and scalable 3D dielectrophoresis assembly process for printing homogeneous silica and hybrid silica/gold nanorods from silica and gold nanoparticles. The nanoparticles are assembled into patterned vias under a dielectrophoretic force generated by an alternating current (AC) field, and then completely fused in situ to form nanorods. The assembly process is governed by the applied AC voltage amplitude and frequency, pattern geometry, and assembly time. Here, we find out that complete assembly of nanorods is not possible without applying both dielectrophoresis and electrophoresis. Therefore, a direct current offset voltage is used to add an additional electrophoretic force to the assembly process. The assembly can be precisely controlled to print silica nanorods with diameters from 20-200 nm and spacing from 500 nm to 2 µm. The assembled nanorods have good uniformity in diameter and height over a millimeter scale. Besides homogeneous silica nanorods, hybrid silica/gold nanorods are also assembled by sequentially assembling silica and gold nanoparticles. The precision of the assembly process is further demonstrated by assembling a single particle on top of each nanorod to demonstrate an additional level of functionalization. The assembled hybrid silica/gold nanorods have potential to be used for metamaterial applications that require nanoscale structures as well as for plasmonic sensors for biosensing applications.

High-Rate Assembly of Nanomaterials on Insulating Surfaces Using Electro-Fluidic Directed Assembly.

Conductive or semiconducting nanomaterials-based applications such as electronics and sensors often require direct placement of such nanomaterials on insulating surfaces. Most fluidic-based directed assembly techniques on insulating surfaces utilize capillary force and evaporation but are diffusion limited and slow. Electrophoretic-based assembly, on the other hand, is fast but can only be utilized for assembly on a conductive surface. Here, we present a directed assembly technique that enables rapid assembly of nanomaterials on insulating surfaces. The approach leverages and combines fluidic and electrophoretic assembly by applying the electric field through an insulating surface via a conductive film underneath. The approach (called electro-fluidic) yields an assembly process that is 2 orders of magnitude faster compared to fluidic assembly. By understanding the forces on the assembly process, we have demonstrated the controlled assembly of various types of nanomaterials that are conducting, semiconducting, and insulating including nanoparticles and single-walled carbon nanotubes on insulating rigid and flexible substrates. The presented approach shows great promise for making practical devices in miniaturized sensors and flexible electronics.

Novel Nanoprinting for Oral Delivery of Poorly Soluble Drugs.

Many of the newly developed drugs for cancer, and some of those for cardiovascular disease, are poorly soluble in water and cannot be taken orally. This can be overcome by employing a new and effective delivery system utilizing nanotechnology. We present a new method for oral preparation of poorly soluble drugs that entails assembling (printing) drug-loaded polymeric micelles into sub-100 nm orally acceptable nanorods (NRs). Due to their small size, these NRs will have a high permeability through cells and thus should transport through the intestine to allow for drug delivery in the blood. These NRs drugs are expected to penetrate tumors more efficiently and much faster than individual nanoparticles and may also be useful for drug delivery to atherosclerotic plaque. This should lead to better bioavailability of the drug with reduced toxicity and side effects. Currently used micellar formulations are administered intravenously, which is invasive and could be toxic due to high doses and interaction with normal healthy tissues. Oral drug administration is the easiest and most desirable way to deliver most drugs, including those that are poorly soluble.

3-D perpendicular assembly of single walled carbon nanotubes for complimentary metal oxide semiconductor interconnects.

Due to their superior electrical properties such as high current density and ballistic transport, carbon nanotubes (CNT) are considered as a potential candidate for future Very Large Scale Integration (VLSI) interconnects. However, direct incorporation of CNTs into Complimentary Metal Oxide Semiconductor (CMOS) architecture by conventional chemical vapor deposition (CVD) growth method is problematic since it requires high temperatures that might damage insulators and doped semiconductors in the underlying CMOS circuits. In this paper, we present a directed assembly method to assemble aligned CNTs into pre-patterned vias and perpendicular to the substrate. A dynamic electric field with a static offset is applied to provide the force needed for directing the SWNT assembly. It is also shown that by adjusting assembly parameters the density of the assembled CNTs can be significantly enhanced. This highly scalable directed assembly method is conducted at room temperature and pressure and is accomplished in a few minutes. I-V characterization of the assembled CNTs was conducted using a Zyvex nanomanipulator in a scanning electron microscope (SEM) and the measured value of the resistance is found to be 270 komega s.

Three-dimensional crystalline and homogeneous metallic nanostructures using directed assembly of nanoparticles.

Directed assembly of nano building blocks offers a versatile route to the creation of complex nanostructures with unique properties. Bottom-up directed assembly of nanoparticles have been considered as one of the best approaches to fabricate such functional and novel nanostructures. However, there is a dearth of studies on making crystalline, solid, and homogeneous nanostructures. This requires a fundamental understanding of the forces driving the assembly of nanoparticles and precise control of these forces to enable the formation of desired nanostructures. Here, we demonstrate that colloidal nanoparticles can be assembled and simultaneously fused into 3-D solid nanostructures in a single step using externally applied electric field. By understanding the influence of various assembly parameters, we showed the fabrication of 3-D metallic materials with complex geometries such as nanopillars, nanoboxes, and nanorings with feature sizes as small as 25 nm in less than a minute. The fabricated gold nanopillars have a polycrystalline nature, have an electrical resistivity that is lower than or equivalent to electroplated gold, and support strong plasmonic resonances. We also demonstrate that the fabrication process is versatile, as fast as electroplating, and scalable to the millimeter scale. These results indicate that the presented approach will facilitate fabrication of novel 3-D nanomaterials (homogeneous or hybrid) in an aqueous solution at room temperature and pressure, while addressing many of the manufacturing challenges in semiconductor nanoelectronics and nanophotonics.

Highly sensitive microscale in vivo sensor enabled by electrophoretic assembly of nanoparticles for multiple biomarker detection.

This paper describes a microscale in vivo sensor platform device for the simultaneous detection of multiple biomarkers. We designed the polymer-based biosensors incorporating multiple active isolated areas, as small as 70 µm × 70 µm, for antigen detection. The fabrication approach involved conventional micro- and nano-fabrication processes followed by site-specific electrophoretic directed assembly of antibody-functionalized nanoparticles. To ensure precise and large-scale manufacturing of these biosensors, we developed a semi-automated system for the attachment of the 250-µm biosensor to a 300-µm catheter probe. Our fabrication and post-processing procedures should enable large-scale production of such biosensor devices at lower manufacturing cost. The principle of detection with these biosensors involved a simple fluorescence-based enzyme-linked immunosorbent assay. These biosensors exhibit high selectivity (ability to selectively detect multiple biomarkers of different diseases), specificity (ability to target generic to specific disease biomarkers), rapid antigen uptake, and low detection limits (for carcinoembryonic antigen, 31.25 pg mL(-1); for nucleosomes, 62.5 pg mL(-1)), laying the foundation for potential early detection of various diseases.

Size-selective template-assisted electrophoretic assembly of nanoparticles for biosensing applications.

The precise, size-selective assembly of nanoparticles gives rise to many applications where the assembly of nano building blocks with different biological or chemical functionalizations is necessary. We introduce a simple, fast, reproducible-directed assembly technique that enables a complete sorting of nanoparticles with single-particle resolution. Nanoparticles are size-selectively assembled into prefabricated via arrays using a sequential template-directed electrophoretic assembly method. Polystyrene latex (PSL) nanoparticles with diameters ranging from 200 to 50 nm are selectively assembled into vias comparable to nanoparticle diameter. We investigate the effects of particle size and via size on the sorting efficiency. We show that complete sorting can be achieved when the size of the vias is close to the diameter of the nanoparticles and the size distribution of the chosen nanoparticles does not overlap. The results also show that it is necessary to keep the electric field on during the insertion and removal of the template. To elucidate the versatility and nil effects that the electrophoresis assembly technique has on the assembled nanoparticle characteristics, we have assembled cancer-specific monoclonal antibody-2C5-coated nanoparticles and have also shown that they can successfully measure low concentrations of the nucleosome (NS) antigen.