Short-time movement of E. coli chromosomal loci depends on coordinate and subcellular localization

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis

Bioresources and Bioprocessing ◽

10.1186/s40643-021-00413-2 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Liyuan Zhang ◽

Xiaomei Lin ◽

Ting Wang ◽

Wei Guo ◽

Yuan Lu

Keyword(s):

Protein Synthesis ◽

Protein Production ◽

Influential Factors ◽

Competent Cell ◽

Free Protein ◽

Application Range ◽

E Coli ◽

Short Time ◽

Cell Free Protein Synthesis

AbstractCell-free protein synthesis (CFPS) systems have become an ideal choice for pathway prototyping, protein production, and biosensing, due to their high controllability, tolerance, stability, and ability to produce proteins in a short time. At present, the widely used CFPS systems are mainly based on Escherichia coli strain. Bacillus subtilis, Corynebacterium glutamate, and Vibrio natriegens are potential chassis cells for many biotechnological applications with their respective characteristics. Therefore, to expand the platform of the CFPS systems and options for protein production, four prokaryotes, E. coli, B. subtilis, C. glutamate, and V. natriegens were selected as host organisms to construct the CFPS systems and be compared. Moreover, the process parameters of the CFPS system were optimized, including the codon usage, plasmid synthesis competent cell selection, plasmid concentration, ribosomal binding site (RBS), and CFPS system reagent components. By optimizing and comparing the main influencing factors of different CFPS systems, the systems can be optimized directly for the most influential factors to further improve the protein yield of the systems. In addition, to demonstrate the applicability of the CFPS systems, it was proved that the four CFPS systems all had the potential to produce therapeutic proteins, and they could produce the receptor-binding domain (RBD) protein of SARS-CoV-2 with functional activity. They not only could expand the potential options for in vitro protein production, but also could increase the application range of the system by expanding the cell-free protein synthesis platform.

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Efficacy of compressed sodium chloride (CSC) against E. coli and Candida auris in minutes and methods improvement for testing

Scientific Reports ◽

10.1038/s41598-020-79212-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lan N. Truong ◽

Brayden D. Whitlock

Keyword(s):

Sodium Chloride ◽

Improved Method ◽

Candida Auris ◽

Antimicrobial Surfaces ◽

E Coli ◽

Route Of Transmission ◽

The World ◽

Short Time ◽

First Time ◽

Time Point

AbstractControlling infections has become one of the biggest problems in the world, whether measured in lives lost or money spent. This is worsening as pathogens continue becoming resistant to therapeutics. Antimicrobial surfaces are one strategy being investigated in an attempt to decrease the spread of infections through the most common route of transmission: surfaces, including hands. Regulators have chosen two hours as the time point at which efficacy should be measured. The objectives of this study were to characterize the new antimicrobial surface compressed sodium chloride (CSC) so that its action may be understood at timepoints more relevant to real-time infection control, under two minutes; to develop a sensitive method to test efficacy at short time points; and to investigate antifungal properties for the first time. E. coli and Candida auris are added to surfaces, and the surfaces are monitored by contact plate, or by washing into collection vats. An improved method of testing antimicrobial efficacy is reported. Antimicrobial CSC achieves at least 99.9% reduction of E. coli in the first two minutes of contact, and at least 99% reduction of C. auris in one minute.

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Evaluation of biofilm performance as a protective barrier against biocorrosion using an enzyme electrode

Water Science & Technology ◽

10.2166/wst.2011.091 ◽

2011 ◽

Vol 64 (8) ◽

pp. 1736-1742 ◽

Cited By ~ 1

Author(s):

S. Soleimani ◽

B. Ormeci ◽

O. B. Isgor ◽

S. Papavinasam

Keyword(s):

Escherichia Coli ◽

Growth Conditions ◽

Microbiologically Influenced Corrosion ◽

Simple Method ◽

E Coli ◽

Concrete Deterioration ◽

Protective Barrier ◽

Microbial Biofilms ◽

Short Time ◽

Selection Of

Sulfide is known to be an important factor in microbiologically influenced corrosion (MIC) of metals and concrete deterioration in wastewater treatment structures and sewer pipelines. A sulfide biosensor was used to determine the effectiveness of Escherichia coli DH5α biofilm as a protective barrier against MIC. The biofilm was shown to be effective in protecting surfaces from sulfide and helping to reduce MIC using amperometric measurements. The results also indicated that the growth conditions of E. coli DH5α may have an impact on the performance of the biofilm as a sulfide barrier. The simple method provided in this work enables the comparison of several microbial biofilms and selection of the ones with potential to prevent MIC in a relatively short time.

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A human homologue of Escherichia coli ClpP caseinolytic protease: recombinant expression, intracellular processing and subcellular localization

Biochemical Journal ◽

10.1042/bj3310309 ◽

1998 ◽

Vol 331 (1) ◽

pp. 309-316 ◽

Cited By ~ 43

Author(s):

Thomas J. CORYDON ◽

Peter BROSS ◽

Henrik U. HOLST ◽

Søren NEVE ◽

Karsten KRISTIANSEN ◽

...

Keyword(s):

Escherichia Coli ◽

Subcellular Localization ◽

Laser Scanning Microscopy ◽

Rat Liver Mitochondria ◽

Pulse Labelling ◽

Intact Cells ◽

Free System ◽

E Coli ◽

Intracellular Processing ◽

Chang Cells

We have recently cloned a human cDNA (hClpP) with significant sequence similarity to the ATP-dependent Escherichia coli ClpP protease [Bross, Andresen, Knudsen, Kruse and Gregersen (1995) FEBS Lett. 377, 249–252]. In the present study, synthesis, intracellular processing and subcellular localization of hClpP have been analysed in intact cells and in a cell-free system. Using pulse-labelling/immunoprecipitation of Chang cells transfected with the hClpP cDNA, we observed two major bands with apparent molecular masses of approx. 39 and 37 kDa. A pulse–chase experiment showed that these bands were converted into one mature-enzyme band with a molecular mass of approx. 32 kDa that was stable for at least 24 h. The 37 kDa band co-migrated with a band produced upon expression of full-length hClpP in E. coli, and the 32 kDa band co-migrated with the product of E. coli-expressed hClpP in which the 56 N-terminal residues had been deleted, indicating that the 37 kDa moiety represents the precursor and that approx. 56 residues are cleaved off during maturation. The processing of hClpP in intact cells was dependent on mitochondrial membrane potential. These results were confirmed in an import assay system using in vitro transcription and translation directed by the hClpP cDNA and isolated rat liver mitochondria. No protease activity towards a series of fluorogenic peptides could be observed in extracts of Chang cells overexpressing hClpP, indicating that the protease may not be active without co-factors. Immunofluorescence studies using confocal-laser-scanning microscopy showed co-localization of the hClpP and the mitochondrially located Hsp60 (heat-shock protein 60). Taken together, the results reported here show that hClpP is localized inside mitochondria and that the trafficking and processing of hClpP resembles the typical biogenesis pathway for nuclear-encoded mitochondrial proteins.

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LONG DISTANCE AND SHORT TIME MOVEMENT OF A SMALL NEOTROPICAL MARSUPIAL

Oecologia Australis ◽

10.4257/oeco.2016.2003.09 ◽

2016 ◽

Vol 20 (03) ◽

pp. 396-400 ◽

Cited By ~ 1

Author(s):

Camila S. Barros ◽

Thaís Kubik Martins ◽

Thomas Puettker ◽

Renata Pardini

Keyword(s):

Long Distance ◽

Short Time ◽

Time Movement

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Enhanced Photocatalytic and Antibacterial Performance of ZnO Nanoparticles Prepared by an Efficient Thermolysis Method

Catalysts ◽

10.3390/catal9070608 ◽

2019 ◽

Vol 9 (7) ◽

pp. 608 ◽

Cited By ~ 11

Author(s):

Md. Hanif ◽

Insup Lee ◽

Jeasmin Akter ◽

Md. Islam ◽

Ali Zahid ◽

...

Keyword(s):

Photocatalytic Activity ◽

Zno Nanoparticles ◽

Zinc Acetate Dihydrate ◽

Hexagonal Wurtzite Structure ◽

Thermal Method ◽

X Ray Diffraction ◽

Zno Nps ◽

E Coli ◽

Antibacterial Performance ◽

Short Time

ZnO nanoparticles (ZnO-NPs) were synthesized by a straightforward modified thermal method using only one chemical: zinc acetate dihydrate. The process is environmentally safer than other methods because it does not involve other chemicals or a catalyst, acid, or base source. X-ray diffraction analysis indicated that the ZnO-NPs crystallize in the hexagonal wurtzite structure. The UV–vis absorption spectra revealed a marked redshift, which is critical for enhanced photocatalytic activity. We used methylene blue for photocatalytic activity tests and found an excellent degradation percentage (99.7%) within a short time (80 min). The antibacterial activity of the synthesized ZnO-NPs was tested against Escherichia coli at different concentrations of ZnO-NPs. The analysis revealed that the minimum inhibitory concentration (MIC) of the ZnO-NPs against E. coli was 30–50 μg/mL. Our ZnO-NPs were found to be more effective than previously reported ZnO-NPs synthesized via other methods.

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