When recombineering bacterial artificial chromosomes (BACs), it is common practice to design the ends of the donor molecule with 50 bp of homology specifying its insertion site. We demonstrate that desired recombinants can be produced using intermolecular homologies as short as 15 bp. Although the use of shorter donor end regions decreases total recombinants by several fold, the frequency of recombinants with correctly inserted donor molecules was high enough for easy detection by simple polymerase chain reaction (PCR) screening. This observation may have important implications for the design of oligonucleotides for recombineering, including significant cost savings, especially for high-throughput projects that use large quantities of primers.
Sample preparation for scanning electron microscope analysis involves reagents and equipment that are expensive and often hazardous. Here we demonstrate a circumvention of Osmium tetroxide and critical point drying, greatly reducing the duration, complexity and cost of the process. We captured early stage interactions of invasive-bacteria and HeLa cells during the process of bacteria-mediated gene delivery and illustrate sufficient clarity can be obtained using this procedure to preserve and clearly visualize relevant cellular structures. This protocol is significantly cheaper and easier to adapt compared to conventional methods, and will allow routine preparation/viewing of eukaryotic or bacterial samples for basic morphological studies.
Rapid detection of foodborne pathogens is crucial as ingestion of contaminated food products may endanger human health. Thus, the objective of this study was to develop a biosensor using reduced graphene oxide-carbon nanotubes (rGO-CNT) nanocomposite via the hydrothermal method for accurate and rapid label-free electrochemical detection of pathogenic bacteria such as Salmonella enterica. The rGO-CNT nanocomposite was characterized using Fourier transform infrared spectroscopy, Raman spectroscopy, X-ray diffraction and transmission electron microscopy. The nanocomposite was dropped cast on the glassy carbon electrode and further modified with amino-modified DNA aptamer. The resultant ssDNA/rGO-CNT/GCE aptasensor was then used to detect bacteria by using differential pulse voltammetry (DPV) technique. Synergistic effects of aptasensor was evident through the combination of enhanced electrical properties and facile chemical functionality of both rGO and CNT for the stable interface. Under optimal experimental conditions, the aptasensor could detect S. Typhimurium in a wide linear dynamic range from 101 until 108 cfu mL-1 with a 101 cfu mL-1 of the limit of detection. This aptasensor also showed good sensitivity, selectivity and specificity for the detection of microorganisms. Furthermore, we have successfully applied the aptasensor for S. Typhimurium detection in real food samples.
Quantification of bacterial invasion into eukaryotic cells is a prerequisite to unfold the molecular mechanisms of this vector's function to obtain insights for improving its efficiency. Invasion is traditionally quantified by antibiotic protection assays that require dilution plating and counting of colony-forming units rescued from infected cells. However, to differentiate between attached and internalized bacteria vector, this assay requires supplementation by a time-consuming and tedious immunofluorescence staining, making it laborious and reduces its reliability and reproducibility. Here we describe a new red fluorescent protein (RFP)-based high-throughput and inexpensive method for tracking bacterial adherence and internalization through flow cytometry to provide a convenient and real-time quantification of bacterial invasiveness in a heterogeneous population of cells. We invaded MCF-7, A549, and HEK-293 cells with the E. coli vector and measured RFP using imaging flow cytometry. We found high cellular infection of up to 70.47% in MCF-7 compared to 27.4% and 26.2% in A549 and HEK-293 cells, respectively. The quantitative evaluation of internalized E. coli is rapid and cell-dependent, and it distinctively differentiates between attached and cytosolic bacteria while showing the degree of cellular invasiveness. This imaging flow cytometry approach can be applied broadly to study host-bacteria interaction.
Green fluorescent protein (GFP) is a versatile reporter protein and has been widely used in biological research. However, its quantitation requires expensive equipment such as a spectrofluorometer. In the current study, a gel documentation imaging system using a native polyacrylamide gel for the quantitation of GFP was developed. The assay was evaluated for its precision, linearity, reproducibility, and sensitivity in the presence of Escherichia coli cells and was compared with the spectrofluorometric method. Using this newly established, gel-based imaging technique; the amount of GFP can be quantified accurately.
Many proteins derived from the latex of Hevea brasiliensis that remain soluble in trichloroacetic acid (TCA) can be precipitated by phosphotungstic acid (PTA). A combination of 5% TCA and 0.2% PTA precipitates a wide range of proteins effectively even when they are present in low concentrations (below 1 microgram ml-1). In addition to its protein purification function, acid precipitation also increases the sensitivity of the subsequent protein assay by allowing the test sample to be concentrated. Another advantage of protein precipitation by TCA and PTA is that very small amounts of protein (of the order of 10 micrograms) can be repeatably recovered without the use of precipitate-bulking agents such as sodium deoxycholate. This general procedure of protein purification and concentration is simple and rapid, but the use of PTA may not be fully compatible with the Bradford protein assay. A modified Lowry microassay is described which enables about 3 micrograms ml-1 to be quantitated at the photometric absorbance of 0.05. When used in conjunction with protein concentration by precipitating with TCA/PTA, approximately 0.4 microgram ml-1 protein present in 6 ml of solution can be assayed.
Phage N15 protelomerase (TelN) cleaves double-stranded circular DNA containing a telomerase-occupancy-site (tos) and rejoins the resulting linear-ends to form closed-hairpin-telomeres in Escherichia coli (E. coli). Continued TelN expression is essential to support resolution of the linear structure. In mammalian cells, no enzyme with TelN-like activities has been found. In this work, we show that phage TelN, expressed transiently and stably in human and mouse cells, recapitulates its native activities in these exogenous environments. We found TelN to accurately resolve tos-DNA in vitro and in vivo within human and mouse cells into linear DNA-containing terminal telomeres that are resistant to RecBCD degradation, a hallmark of protelomerase processing. In stable cells, TelN activity was detectable for at least 60 days, which suggests the possibility of limited silencing of its expression. Correspondingly, linear plasmid containing a 100 kb human β-globin gene expressed for at least 120 h in non-β-globin-expressing mouse cells with TelN presence. Our results demonstrate TelN is able to cut and heal DNA as hairpin-telomeres within mammalian cells, providing a tool for creating novel structures by DNA resolution in these hosts. The TelN protelomerase may be useful for exploring novel technologies for genome interrogation and chromosome engineering.
The variation in the extracellular metabolites of RAW 264.7 cells obtained from different passage numbers (passage 9, 12 and 14) was examined. The impact of different harvesting protocols (trypsinization and scraping) on recovery of intracellular metabolites was then assessed. The similarity and variation in the cell metabolome was investigated using 1H NMR metabolic profiling modeled using multivariate data analysis. The characterization and quantification of metabolites was performed to determine the passage-related and harvesting-dependent effects on impacted metabolic networks. The trypsinized RAW cells from lower passages gave higher intensities of most identified metabolites, including asparagine, serine and tryptophan. Principal component analysis revealed variation between cells from different passages and harvesting methods, as indicated by the formation of clusters in score plot. Analysis of S-plots revealed metabolites that acted as biomarkers in discriminating cells from different passages including acetate, serine, lactate and choline. Meanwhile lactate, glutamine and pyruvate served as biomarkers for differentiating trypsinized and scraped cells. In passage-dependent effects, glycolysis and TCA cycle were influential, whereas glycerophospholipid metabolism was affected by the harvesting method. Overall, it is proposed that typsinized RAW cells from lower passage numbers are more appropriate when conducting experiments related to NMR metabolomics.
In multiple biological processes, molecular recognition performs an integral role in detecting bio analytes. Molecular imprinted polymers (MIPs) are tailored sensing materials that can biomimic the biologic ligands and can detect specific target molecules selectively and sensitively. The formulation of molecularly imprinted polymers is followed by the formulation of a control termed as non-imprinted polymer (NIP), which, in the absence of a template, is commonly formulated to evaluate whether distinctive imprints have been produced for the template. Given the difficulties confronting bioanalytical researchers, it is inevitable that this strategy would come out as a central route of multidisciplinary studies to create extremely promising stable artificial receptors as a replacement or accelerate biological matrices. The ease of synthesis, low cost, capability to 'tailor' recognition element for analyte molecules, and stability under harsh environments make MIPs promising candidates as a recognition tool for biosensing. Compared to biological systems, molecular imprinting techniques have several advantages, including high recognition ability, long-term durability, low cost, and robustness, allowing molecularly imprinted polymers to be employed in drug delivery, biosensor technology, and nanotechnology. Molecular imprinted polymer-based sensors still have certain shortcomings in determining biomacromolecules (nucleic acid, protein, lipids, and carbohydrates), considering the vast volume of the latest literature on biomicromolecules. These potential materials are still required to address a few weaknesses until gaining their position in recognition of biomacromolecules. This review aims to highlight the current progress in molecularly imprinted polymers (MIPs)-based sensors for the determination of deoxyribonucleic acid (DNA) or nucleobases.
Following hematopoietic stem cell transplantation (HSCT), it is important to determine whether engraftment is successful and to track the dynamic changes of the graft. Tandem repeats such as minisatellites and microsatellites are currently the most established markers for chimerism application. We have developed a reliable method to quantitatively evaluate engraftment status in post-allogeneic HSCT patients using variable number of tandem repeat (VNTR) markers and "lab-on-a-chip" microfluidic electrophoresis technology. Following identification of an informative marker by conventional polymerase chain reaction (PCR), donor chimerism percentage was calculated based on a standard curve generated from artificially mixed patient-donor DNA-specific alleles in serial dilutions. All PCR products were mixed with commercial gel dye and loaded into Agilent DNA 1000 microfluidic LabChips for DNA sizing and quantitation. In 44 patients, separation of pretransplant and donor DNA fragments was resolved clearly and accomplished rapidly within 30min. Chimerism analysis using this platform is able to detect an amount as low as 6.3% donor DNA with acceptable coefficient of variation values. We also demonstrated concordant chimerism analysis findings using both microchip tandem repeats and real-time PCR quantitation of insertion-deletion polymorphisms. This microchip platform obviates the need for fluorescently labeled primers or any post-PCR sample manipulation. Quantitative monitoring of post-HSCT chimerism status using microfluidic electrophoresis is a useful tool for both large- and small-scale post-HSCT chimerism centers.
A method using solid phase extraction and liquid chromatography-tandem mass spectrometry to quantitatively detect mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine in human urine samples was developed and validated. The relevant metabolites were identified using multiple reaction monitoring in positive ionization mode using nalorphine as an internal standard. The method was validated for accuracy, precision, recovery, linearity, and lower limit of quantitation. The intra- and inter-day accuracy and precision were found in the range of 83.6-117.5% with coefficient of variation less than 13%. The percentage of recovery for mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine was within the range of 80.1-118.9%. The lower limit of quantification was 1 ng/mL for mitragynine, 2 ng/mL for 16-carboxy mitragynine, and 50 ng/mL for 9-O-demethyl mitragynine. The developed method was reproducible, high precision and accuracy with good linearity and recovery for mitragynine, 16-carboxy mitragynine, and 9-O-demethyl mitragynine in human urine.
The performance of aptamers as versatile tools in numerous analytical applications is critically dependent on their high target binding specificity and selectivity. However, only the technical or methodological aspects of measuring aptamer-target binding affinities are focused, ignoring the equally important mathematical components that play pivotal roles in affinity measurements. In this study, we aim to provide a comprehensive review regarding the utilization of different mathematical models and equations, along with a detailed description of the computational steps involved in mathematically deriving the binding affinity of aptamers against their specific target molecules. Mathematical models ranging from one-site binding to multiple aptameric binding site-based models are explained in detail. Models applied in several different approaches of affinity measurements such as thermodynamics and kinetic analysis, including cooperativity and competitive-assay based mathematical models have been elaborately discussed. Mathematical models incorporating factors that could potentially affect affinity measurements are also further scrutinized.
The effect of DNA topology on transfection efficiency of mammalian cells has been widely tested on plasmids smaller than 10 kb, but little is known for larger DNA vectors carrying intact genomic DNA containing introns, exons, and regulatory regions. Here, we demonstrate that circular BACs transfect more efficiently than covalently closed linear BACs. We found up to 3.1- and 8.9- fold higher eGFP expression from circular 11 kb and 100 kb BACs, respectively, compared to linear BACs. These findings provide insights for improved vector development for gene delivery and expression studies of large intact transgenes in mammalian cells.
Anti-salbutamol antibodies remain as important tools for the detection of salbutamol abuse in athletic doping. This study evaluated the feasibility and efficiency of the chicken (Gallus gallus domesticus) as an immunization host to generate anti-salbutamol scFv antibodies by phage display. A phage display antibody library was constructed from a single chicken immunized against salbutamol-KLH conjugate. After a stringent biopanning strategy, a novel scFv clone which was inhibited by free salbutamol recorded the highest affinity. This scFv was expressed as soluble and functional protein in Escherichia coli T7 SHuffle Express B (DE3) strain. Cross-reactivity studies of the scFv towards other relevant β2-agonists revealed that the scFv cross-reacted significantly towards clenbuterol. The determined IC50 of the scFv towards the two β2-agonists were; IC50 salbutamol = ∼0.310 μg/ml, IC50 clenbuterol = ∼0.076 μg/ml. The generated scFv demonstrated poor stability based on accelerated stability studies. The scFv was used to develop an competitive indirect ELISA (LOD = 0.125 μg/ml) for detection of parent salbutamol in spiked human urine (n = 18) with ∼83.4% reliability at the cut-off of 1 μg/ml currently implemented by WADA and may be of potential use in human doping urinalysis.
Endosomal escape is considered a crucial barrier that needs to be overcome by integrin-mediated E. coli for gene delivery into mammalian cells. Bafilomycin, a potent inhibitor of the H+ proton pump commonly employed to lower endosomal pH, was evaluated as part of the E. coli protocol during delivery. We found an increase in green fluorescent protein expression up 6.9, 3.2, 5.0, 2.8, and 4.5 fold in HeLa, HEK-293, A549, HT1080, and MCF-7 respectively, compared to untreated cells. Our result showed for the first time that Inhibition of lysosomal V-ATPase enhances E. coli efficiency.
Distinctive bioactivities possessed by luteolin (3', 4', 5, 7-tetrahydroxy-flavone) are advantageous for sundry practical applications. This paper reports the in vitro selection and characterization of single stranded-DNA (ssDNA) aptamers, specific for luteolin (LUT). 76-mer library containing 1015 randomized ssDNA were screened via systematic evolution of ligands by exponential enrichment (SELEX). The recovered ssDNA pool from the 8th round was amplified with unlabeled primers and cloned into PSTBlue-1 vector prior to sequencing. 22 of LUT-binding aptamer variants were further classified into one of the seven groups based on their N40 random sequence regions, wherein one representative from each group was characterized. The dissociation constant of aptamers designated as LUT#28, LUT#20 and LUT#3 was discerned to be 107, 214 and 109 nM, respectively with high binding affinity towards LUT. Prediction analysis of the secondary structure suggested discrete features with typical loop and stem motifs. Furthermore, LUT#3 displayed higher specificity with insignificant binding toward kaempferol and quercetin despite its structural and functional similarity compared to LUT#28 and LUT#20. Further LUT#3 can detect free luteolin within 0.2-1 mM in solution. It was suggested that LUT#3 aptamer were the most suitable for LUT recognition tool at laboratory scale based on the condition tested.
In eukaryotes, serine proteases are cellular localized hydrolases reported to regulate essential biological reactions. Improved industrial applications of proteins are aided by prediction and analysis of their 3-dimensional structures (3D). A serine protease was identified from CTG-clade yeast Meyerozyma guilliermondii strain SO and its 3D structure as well as its catalytic attributes have not been fully understood yet, thus we seek to report on the catalytic mechanism of M. guilliermondii strain SO MgPRB1 using substrate PMSF via in silico docking as well as its stability by way of disulfide bonds formation. Herein, bioinformatics tools and techniques were used to predict, validate and analyze the possible changes of CUG ambiguity (if any) in strain SO using template PDB ID: 3F7O. Structural assessments confirmed the classic catalytic triad Asp305, His337, and Ser499. Superimposition of MgPRB1 and template 3F7O structures revealed the unlinked cysteine residues between Cys341, Cys440, Cys471 and Cys506 of MgPRB1 compared to template 3F7O with two disulfide bonds formation, which confers structural stability. In conclusion, serine protease structure from strain SO was successfully predicted and studies towards understanding at the molecular level may be undertaken for its potential applications in the degradation of peptide bonds.
The low presentation efficiency of Fab (fragment antigen binding) fragments during phage display is largely due to the complexity of disulphide bond formation. This can result in the presentation of Fab fragments devoid of a light chain during phage display. Here we propose the use of a coplasmid system encoding several molecular chaperones (DsbA, DsbC, FkpA, and SurA) to improve Fab packaging. A comparison was done using the Fab fragment from IgG and IgD. We found that the use of the coplasmid during phage packaging was able to improve the presentation efficiency of the Fab fragment on phage surfaces. A modified version of panning using the coplasmid system was evaluated and was successful at enriching Fab binders. Therefore, the coplasmid system would be an attractive alternative for improved Fab presentation for phage display.
Immunoassays are often coupled to peroxidase activity for antigen detection. Sensitivity and speed of detection has been increased by the advent of hybrid methods such as immuno-PCR (polymerase chain reaction). However, a more simplified immunoassay that retains both colorimetric peroxidase detection and effective DNA amplification in a setting closer to field application conditions has been nonexistent. Here we describe a method that successfully combines a competitive immunoassay with the new isothermal quadruplex-primed amplification (QPA) to generate excess quadruplex reporter molecules with intrinsic peroxidase DNAzyme activity.