Bacteriophage Engineering for Improved Copper Ion Binding.

In this study, fd viruses are genetically modified to display seven cropped versions (H, HG, HGF, HGFA, HGFAN, HGFANV and HGFANVA) of the previously identified Cu(II) specific peptide (HGFANVA). Atomic force microscopy (AFM) imaging reveals the typical filamentous structures of recombinant phages with thicknesses of ≈2-5 nm in dry state. Scanning electron microscopy (SEM) imaging shows that HGFANVA viruses form larger elongated assemblies than H viruses that are deposited with a mineral layer after Cu(II) treatment. C and N peaks are detected for virus samples through Energy dispersive X-ray spectroscopy (EDX) analyses confirming the presence of phage organic material. Cu peak is only detected for engineered viruses after Cu(II) exposure. Enzyme-linked immunosorbent assay (ELISA) analyses show the selective Cu(II) binding of engineered phages. Agarose gel electrophoresis (AGE) and zeta potential analyses reveal negative surface charges of engineered viral constructs. Positively charged Cytopore beads are coated with bacteriophages and used for Cu(II) ion sorption studies. ICP-MS analyses clearly show the improved Cu(II) binding of engineered viruses with respect to wild-type fd phages. Such bottom-up constructed, genetically engineered virus-based biomaterials may be applied in bioremediation studies targeting metal species from environmental samples.

Phage display selection of a Pb(II) specific peptide and its application as a biorecognition unit for colorimetric detection of Pb(II) ions.

Phage display technology employs a library of engineered filamentous M13 viruses infecting only bacteria. In this study, the genuine phage display selection technique was applied to identify a Pb(II) specific peptide. After three rounds of positive selection against Pb(II) coated agarose-based beads and the consecutive negative screenings against interfering metal ions (Al(III), Co(II), Fe(III), Ni(II), and Zn(II)), a final phage library with enhanced Pb(II) binding was obtained. Enzyme Linked Immunosorbent Assay (ELISA) analyses confirmed the selective Pb(II) binding of the enriched viruses. Twenty monoclonal phage plaques were randomly selected, single stranded DNAs (ssDNAs) were isolated and sequenced. Sequencing results revealed four different peptide sequences. Pb9 peptide (KASPYIT) showing the most specific Pb(II) binding was selected for detection studies. Pb9 was synthetically synthesized with additional three cysteine (3xCys) units at C-terminal. Twenty nanometers AuNPs were functionalized with Pb9-3xCys peptides through Au-thiol (Au-S) interaction. A colorimetric Pb(II) detection system was validated using the engineered peptide-AuNP complex at a calculated LOD of around 11 nM (3σ/k, n = 6) for the case study. The detection system was Pb(II) selective over various metal ions (Ag(II), Al(III), Au(III), Cd(II), Co(II), Cr (III), Cu(II), Fe(III), Hg(II), Mg(II), Mn(II), Ni(II), and Zn(II)). Such metal ion specific peptides can be further studied to develop simple, user friendly and cost-effective tools to design alternative detection and bioremediation systems for a circular economy.

Isolation, microscopic and magnetotactic characterization of Magnetospirillum moscoviense MS-24 from Banjosa Lake, Pakistan.

At currently, approximately 70 species of magnetotactic bacteria have been identified; thus, there is an urgent need to identify more magnetotactic bacteria from diverse environmental sources with potential applications in industry and biotechnology. To the best of our knowledge, this is the first magnetotactic bacterial strain discovered in Pakistan. The first magnetotactic bacteria, Magnetospirillum moscoviense MS-24, was isolated from Banjosa Lake (Rawalakot), Pakistan, in the current investigation. Magnetospirillum moscoviense MS-24 was screened using the Racetrack method. The Magnetospirillum moscoviense MS-24 were physically characterised using Atomic Force Microscopy, High-Resolution Scanning Electron Microscopy, and Transmission Electron Microscopy. The current study used microscopy to illustrate the shape of bacteria and to find a very obvious chain of magnetosomes within the bacterial cell. The Magnetospirillum moscoviense MS-24 measured about 4 ± 0.04 µm in length and 600 ± 0.02 nm in diameter. The microfluidic chip experiments were also used to detect magnetotaxis behaviour in bacteria.

An open-source automated magnetic optical density meter for analysis of suspensions of magnetic cells and particles.

We present a spectrophotometer (optical density meter) combined with electromagnets dedicated to the analysis of suspensions of magnetotactic bacteria. The instrument can also be applied to suspensions of other magnetic cells and magnetic particles. We have ensured that our system, called MagOD, can be easily reproduced by providing the source of the 3D prints for the housing, electronic designs, circuit board layouts, and microcontroller software. We compare the performance of our system to existing adapted commercial spectrophotometers. In addition, we demonstrate its use by analyzing the absorbance of magnetotactic bacteria as a function of their orientation with respect to the light path and their speed of reorientation after the field has been rotated by 90°. We continuously monitored the development of a culture of magnetotactic bacteria over a period of 5 days and measured the development of their velocity distribution over a period of one hour. Even though this dedicated spectrophotometer is relatively simple to construct and cost-effective, a range of magnetic field-dependent parameters can be extracted from suspensions of magnetotactic bacteria. Therefore, this instrument will help the magnetotactic research community to understand and apply this intriguing micro-organism.

A novel copper (II) binding peptide for a colorimetric biosensor system design.

Filamentous bacteriophages are viruses infecting only bacteria. In this study, phage display technique was applied to identify highly selective Cu(II) binding peptides. After five rounds of positive screening against Cu(II) and various rounds of negative screenings against competitive metal ions (Al(III), Co(II), Fe(III), Ni(II) and Zn(II)), bacteriophages were enriched. Selective Cu(II) binding of final phages was confirmed by Enzyme Linked Immunosorbent Assay (ELISA), Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray spectroscopy (EDX) analyses. 15 phage plaques were randomly selected and sequenced. Cu-5 peptide (HGFANVA) with the highest frequency of occurrence and the strongest Cu(II) affinity was chosen for further Cu(II) detection and removal tests. Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) confirmed the strong Cu(II) binding potential of engineered viruses. Cu-5 peptides were synthetically synthesized with three Cysteine units at C-terminal and a AuNP-peptide biosensor system was developed based on aggregation behavior of AuNPs upon Cu(II) ion treatment. AuNP-based Cu(II) sensor was selective for Cu(II) and the LOD was 91.15 nM (ca. 5.8 × 10-3 mg/L; 3σ/k, n = 5, R2 = 0.992) for the case study which is considerably lower than the WHO's accepted guideline of 1.3 mg/L. This study provides an interdisciplinary approach to apply short peptides as recognition units for biosensor studies which are user friendly, not bulky and cost-effective.

Evaluation of biological activities of Barbarea integrifolia and isolation of a new glucosinolate derivated compound.

The aim of the present study is to determine the potent biological activities and carry out isolation studies on Barbarea integrifolia. The antioxidant capacity of the species was evaluated by total phenolic content, FRAP, CUPRAC, and DPPH radical scavenging activity. Anticancer activity studies were performed by MTT assay in MDA-MB-231, MCF-7, Hep3B, PC-3, A549, HCT116, L-929 cell lines. It was observed that the remaining aqueous fraction has higher total phenolic content while higher activity in the CUPRAC and FRAP assays was displayed for the methanolic extract and chloroform fraction. The extracts showed anticancer activity as compared with vincristine. It was observed that chloroform fraction has the highest anticancer activity on MCF-7 cell line, while ethyl acetate fraction has the highest anticancer activity on Hep-3B and A549 cell lines. Methanolic extract has the highest anticancer activity on HCT116 and MDA-MB-23 cell lines. The isolation studies have been performed using several chromatographic methods. The chemical structures of compounds have been identified by means of 1H NMR, 13C NMR, 2D-NMR, and MS. Five major compounds, one steroid (ß-Sitosterol), one phenolic acid (Rosmarinic acid), one flavonol heteroside (kaempferol 7-O-α-l-rhamnoside-3-O-ß-d-(2-O-ß- d -glucosyl)-ß-d-glucoside), and two glucosinolates (Gluconasturtiin, Gluconasturtiin choline salt) have been isolated.

Long-term observation of Magnetospirillum gryphiswaldense in a microfluidic channel.

We controlled and observed individual magneto-tactic bacteria (Magnetospirillum gryphiswaldense) inside a [Formula: see text]-high microfluidic channel for over 4 h. After a period of constant velocity, the duration of which varied between bacteria, all observed bacteria showed a gradual decrease in their velocity of about [Formula: see text]. After coming to a full stop, different behaviour was observed, ranging from rotation around the centre of mass synchronous with the direction of the external magnetic field, to being completely immobile. Our results suggest that the influence of the high-intensity illumination and the presence of the channel walls are important parameters to consider when performing observations of such long duration.

Recombinant bacteriophages as gold binding bio-templates.

Bacteriophages are nano-sized virion particles infecting bacteria. In this study, it is shown that metal binding properties of filamentous fd-bacteriophages can be enhanced by genetic engineering. Quartz crystal microbalance (QCM) analyses, UV-vis absorption spectra measurements and scanning electron microscopy (SEM) imaging revealed that expression of MMM short amino acid sequence on major coat protein p8 facilitates recombinant MMM-phage binding to Au surfaces and nanoparticles (NPs) via gold-sulfur (AuS) interaction. Electroless deposition of Au particles on phage assemblies was investigated upon chemical reduction reaction with NaBH4 at different HAuCl4 precursor concentrations. Energy dispersive X-ray spectroscopy (EDX) measurements confirmed the presence of Au on both AuNP decorated and chemically metallized phage structures. Further studies on patterning and controlled immobilization of recombinant bacteriophages on specific surfaces may contribute to bio-templated nanowire development field and biosensor application studies.

Bacteriophages as templates for manufacturing supramolecular structures.

SS phages are genetically enginnered by replacing 2 N-terminal amino acids of the p8 coat protein of the fd phage. AGE and zeta potential measurements show that SS phages carry at least 1/4 less net negative surface charge than fd phages. Morphology and thickness of phages are studied at different counterion concentrations (10(-3) , 10(-2) and 10(-1) M) by AFM, SEM and immunofluorescence analyses. Bundles induced by CoCl2 and CaCl2 are either metallized by chemical reduction or biomineralized for apatite-like material formation. EDX spectroscopy confirms the presence of Co, P and Ca peaks in mineralized samples. Such bottom-up manufactured phage scaffolds might be applied in bioengineering studies.

Formation of tubes during self-assembly of bacterial surface layers.

Based on experimental studies on tube formation during self-assembly of bacterial surface (S)-layers, a mechanistic model for describing the underlying basic mechanisms is proposed and the effect of process parameters on growth velocity and tube radius is investigated. The S-layer is modeled as a curved sheet with discrete binding sites for the association of monomers distributed along the S-layer edges. Reported changes of the tube radius owing to genetic protein modifications are explained within the framework of continuum mechanics. S-layer growth velocity and shape development are analyzed by Monte Carlo simulation in their dependence on the attachment and detachment frequencies of monomers at the S-layer. For curved S-layer patches, a criterion for the formation of S-layer tubes is derived. Accordingly, tubes can form only within a certain range of the initial monomer concentration. Furthermore, the effect of calcium ion concentration on tube formation is discussed, including recent experimental findings on the calcium effect.

Metallization and investigation of electrical properties of in vitro recrystallized mSbsC-eGFP assemblies.

Surface layer (SL) proteins are self-assembling nanosized arrays which can be recrystallized in solution or on surfaces. In this paper, we investigate the metallization, contact potential difference and conductivity of in vitro recrystallized mSbsC-eGFP tube-like assemblies for possible applications in nanobiotechnology. Treatment of mSbsC-eGFP tube-like structures with 150 mM Pt salt solution resulted in the formation of metallized SL assemblies decorated with Pt nanoparticles (∅ > 3 nm) which were closely packed and aggregated into metal clusters. Kelvin probe force microscopy (KPFM) measurements revealed that metallized and unmetallized SL templates showed different surface potential behaviours, demonstrating that the metal coating changes the electrostatic surface characteristics of SL assemblies. In situ conductivity measurements showed that unmetallized SL assemblies were not conductive. Metallized samples showed linear I-V dependence between - 1 and + 1 V with a conductivity of ∼ 10(3) S m( - 1).

Calcium dependent formation of tubular assemblies by recombinant S-layer proteins in vivo and in vitro.

Surface layer proteins have the appealing property to self-assemble in nanosized arrays in solution and on solid substrates. In this work, we characterize the formation of assembly structures of the recombinant surface layer protein SbsC of Geobacillus stearothermophilus ATTC 12980, which was tagged with enhanced green fluorescent protein and expressed in the yeast Saccharomyces cerevisiae. The tubular structures formed by the protein in vivo are retained upon bursting the cells by osmotic shock; however, their average length is decreased. During dialysis, monomers obtained by treatment with chaotropic chemicals recrystallize again to form tube-like structures. This process is strictly dependent on calcium (Ca(2+)) ions, with an optimal concentration of 10 mM. Further increase of the Ca(2+) concentration results in multiple non-productive nucleation points. We further show that the lengths of the S-layer assemblies increase with time and can be controlled by pH. After 48 h, the average length at pH 9.0 is 4.13 µm compared to 2.69 µm at pH 5.5. Successful chemical deposition of platinum indicates the potential of recrystallized mSbsC-eGFP structures for nanobiotechnological applications.

Expression and assembly of recombinant surface layer proteins in Saccharomyces cerevisiae.

Most bacterial surface layers (SLs) are formed by self-assembly of a single type of protein. Native and recombinant surface layer monomers are capable to self-assemble on solid substrates and in solution to highly regular nanosized arrays which make them attractive for nanobiotechnological applications. In this study, we expressed the surface layer protein SbsC of Bacillus stearothermophilus ATTC 12980, tagged with Enhanced Green Fluorescent Protein, in the yeast Saccharomyces cerevisiae. We observed a network of tubular structures in the cytosol of the transformed yeast cells that did not colocalize with microtubules or the actin cytoskeleton. Time-resolved analysis of the formation of these structures during vegetative growth and sporulation was investigated by live fluorescence microscopy. While in meiosis ascospores seemed to receive assembled structures from the diploid cells, during mitosis, SL structures were formed de novo in the buds. SL assembly always started with the appearance of a dot-like structure in the cytoplasm, suggesting a single nucleation point.