Addressing the Requirements of High-Sensitivity Single-Molecule Imaging of Low-Copy-Number Proteins in Bacteria

Plasmids play a major role in bacterial adaptation to environmental stress and often contribute to antibiotic resistance and disease virulence. Although the complete sequence of each plasmid is essential for studying plasmid biology, most antibiotic resistance and virulence plasmids in Salmonella are present only in a low copy number, making extraction and sequencing difficult. Long read sequencing technologies require higher concentrations of DNA to provide optimal results. To resolve this problem, we assessed the sufficiency of multiple displacement amplification (MDA) for replicating Salmonella plasmid DNA to a satisfactory concentration for accurate sequencing and multiplexing. Nine Salmonella enterica isolates, representing nine different serovars carrying plasmids for which sequence data are already available at NCBI, were cultured and their plasmids isolated using an alkaline lysis extraction protocol. We then used the Phi29 polymerase to perform MDA, thereby obtaining enough plasmid DNA for long read sequencing. These amplified plasmids were multiplexed and sequenced on one single molecule, real-time (SMRT) cell with the Pacific Biosciences (Pacbio) Sequel sequencer. We were able to close all Salmonella plasmids (sizes ranged from 38 to 166 Kb) with sequencing coverage from 24 to 2,582X. This protocol, consisting of plasmid isolation, MDA, and multiplex sequencing, is an effective and fast method for closing high-molecular weight and low-copy-number plasmids. This high throughput protocol reduces the time and cost of plasmid closure.

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High-throughput single-cell analysis of low copy number β-galactosidase by a laboratory-built high-sensitivity flow cytometer

Biosensors and Bioelectronics ◽

10.1016/j.bios.2013.03.078 ◽

2013 ◽

Vol 48 ◽

pp. 49-55 ◽

Cited By ~ 11

Author(s):

Lingling Yang ◽

Tianxun Huang ◽

Shaobin Zhu ◽

Yingxing Zhou ◽

Yunbin Jiang ◽

...

Keyword(s):

Single Cell ◽

High Throughput ◽

Copy Number ◽

Single Cell Analysis ◽

High Sensitivity ◽

Flow Cytometer ◽

Cell Analysis ◽

Low Copy Number

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Reconstitution of a prokaryotic minus end-tracking system using TubRC centromeric complexes and tubulin-like protein TubZ filaments

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1423746112 ◽

2015 ◽

Vol 112 (15) ◽

pp. E1845-E1850 ◽

Cited By ~ 23

Author(s):

Gero Fink ◽

Jan Löwe

Keyword(s):

Cell Division ◽

Bacillus Thuringiensis ◽

Single Molecule ◽

Copy Number ◽

Tracking System ◽

Adaptor Protein ◽

Dna Transport ◽

Dna Segregation ◽

Low Copy Number ◽

Fundamental Process

Segregation of DNA is a fundamental process during cell division. The mechanism of prokaryotic DNA segregation is largely unknown, but several low-copy-number plasmids encode cytomotive filament systems of the actin type and tubulin type important for plasmid inheritance. Of these cytomotive filaments, only actin-like systems are mechanistically well characterized. In contrast, the mechanism by which filaments of tubulin-like TubZ protein mediate DNA motility is unknown. To understand polymer-driven DNA transport, we reconstituted the filaments of TubZ protein (TubZ filaments) from Bacillus thuringiensis pBtoxis plasmid with their centromeric TubRC complexes containing adaptor protein TubR and tubC DNA. TubZ alone assembled into polar filaments, which annealed laterally and treadmilled. Using single-molecule imaging, we show that TubRC complexes were not pushed by filament polymerization; instead, they processively tracked shrinking, depolymerizing minus ends. Additionally, the TubRC complex nucleated TubZ filaments and allowed for treadmilling. Overall, our results indicate a pulling mechanism for DNA transport by the TubZRC system. The discovered minus end-tracking property of the TubRC complex expands the mechanistic diversity of the prokaryotic cytoskeleton.

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Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy

Essays in Biochemistry ◽

10.1042/ebc20200023 ◽

2020 ◽

Author(s):

Nikolas Hundt

Keyword(s):

Single Molecule ◽

Cellular Systems ◽

Single Molecule Imaging ◽

Label Free ◽

Measurement Sensitivity ◽

Mass Distributions ◽

Quantitative Measurements ◽

Contrast Mechanism ◽

Cellular Components ◽

Scattering Signal

Abstract Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.

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Low Copy Number of the Salivary Amylase Gene (AMY1) Is Associated with Obesity, Dyslipidemia, and Chronic Low-Grade Inflammation but Not Insulin Sensitivity and Secretion

Diabetes ◽

10.2337/db18-58-lb ◽

2018 ◽

Vol 67 (Supplement 1) ◽

pp. 58-LB

Author(s):

BARBORA DE COURTEN ◽

AYA MOUSA ◽

NEGAR NADERPOOR

Keyword(s):

Insulin Sensitivity ◽

Copy Number ◽

Low Grade ◽

Amylase Gene ◽

Salivary Amylase ◽

Low Copy Number ◽

Low Grade Inflammation

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Direct Read and Quantify Damage Nucleotide from an Oligonucleotide Using a Single Molecule Interface

10.26434/chemrxiv.10080638.v2 ◽

2019 ◽

Author(s):

Jiajun Wang ◽

Meng-Yin Li ◽

Jie Yang ◽

Ya-Qian Wang ◽

Xue-Yuan Wu ◽

...

Keyword(s):

Dna Sequencing ◽

Single Molecule ◽

Genetic Diseases ◽

High Sensitivity ◽

Dna Lesions ◽

Lesion Detection ◽

The Novel ◽

Structural Variations ◽

Dna Lesion ◽

Base Lesion

DNA lesion such as metholcytosine(mC), 8-OXO-guanine(OG), inosine(I) etc could cause the genetic diseases. Identification of the varieties of lesion bases are usually beyond the capability of conventional DNA sequencing which is mainly designed to discriminate four bases only. Therefore, lesion detection remain challenge due to the massive varieties and less distinguishable readouts for minor structural variations. Moreover, standard amplification and labelling hardly works in DNA lesions detection. Herein, we designed a single molecule interface from the mutant K238Q Aerolysin, whose confined sensing region shows the high compatible to capture and then directly convert each base lesion into distinguishable current readouts. Compared with previous single molecule sensing interface, the resolution of the K238Q Aerolysin nanopore is enhanced by 2-order. The novel K238Q could direct discriminate at least 3 types (mC, OG, I) lesions without lableing and quantify modification sites under mixed hetero-composition condition of oligonucleotide. Such nanopore could be further applied to diagnose genetic diseases at high sensitivity.

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A Molecular Logic Gate Enables Super-Resolved Imaging of Intracellular Lipid Droplets

10.26434/chemrxiv.9784799.v1 ◽

2019 ◽

Author(s):

Adam Eördögh ◽

Carolina Paganini ◽

Dorothea Pinotsi ◽

Paolo Arosio ◽

Pablo Rivera-Fuentes

Keyword(s):

Single Molecule ◽

Lipid Droplets ◽

Neutral Lipids ◽

Logic Gate ◽

Living Cells ◽

Three Dimensions ◽

Single Molecule Imaging ◽

Molecular Logic Gate ◽

Molecular Logic ◽

Cellular Compartments

<div>Photoactivatable dyes enable single-molecule imaging in biology. Despite progress in the development of new fluorophores and labeling strategies, many cellular compartments remain difficult to image beyond the limit of diffraction in living cells. For example, lipid droplets, which are organelles that contain mostly neutral lipids, have eluded single-molecule imaging. To visualize these challenging subcellular targets, it is necessary to develop new fluorescent molecular devices beyond simple on/off switches. Here, we report a fluorogenic molecular logic gate that can be used to image single molecules associated with lipid droplets with excellent specificity. This probe requires the subsequent action of light, a lipophilic environment and a competent nucleophile to produce a fluorescent product. The combination of these requirements results in a probe that can be used to image the boundary of lipid droplets in three dimensions with resolutions beyond the limit of diffraction. Moreover, this probe enables single-molecule tracking of lipids within and between droplets in living cells.</div>

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