Improved CUT&RUN chromatin profiling and analysis tools

AbstractWe previously described a novel alternative to Chromatin Immunoprecipitation, Cleavage Under Targets & Release Using Nuclease (CUT&RUN), in which unfixed permeabilized cells are incubated with antibody, followed by binding of a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein (1). Upon activation of tethered MNase, the bound complex is excised and released into the supernatant for DNA extraction and sequencing. Here we introduce four enhancements to CUT&RUN: 1) a hybrid Protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification; 2) a modified digestion protocol that inhibits premature release of the nuclease-bound complex; 3) a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein; and 4) a novel peak-calling strategy customized for the low-background profiles obtained using CUT&RUN. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.

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Improved CUT&RUN chromatin profiling tools

eLife ◽

10.7554/elife.46314 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 35

Author(s):

Michael P Meers ◽

Terri D Bryson ◽

Jorja G Henikoff ◽

Steven Henikoff

Keyword(s):

Fusion Protein ◽

High Efficiency ◽

Low Cost ◽

Protein A ◽

Micrococcal Nuclease ◽

Permeabilized Cells ◽

E Coli ◽

Carry Over ◽

Chromatin Profiling ◽

Premature Release

Previously, we described a novel alternative to chromatin immunoprecipitation, CUT&RUN, in which unfixed permeabilized cells are incubated with antibody, followed by binding of a protein A-Micrococcal Nuclease (pA/MNase) fusion protein (Skene and Henikoff, 2017). Here we introduce three enhancements to CUT&RUN: A hybrid protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification, a modified digestion protocol that inhibits premature release of the nuclease-bound complex, and a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.

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Assessment of Signal Peptides to Optimize Interleukin 2 (IL-2) Folding and Expression

Current Proteomics ◽

10.2174/1570164615666181024113612 ◽

2019 ◽

Vol 16 (3) ◽

pp. 188-198 ◽

Cited By ~ 3

Author(s):

Narges Nazifi ◽

Mojtaba Tahmoorespur ◽

Mohammad Hadi Sekhavati ◽

Alireza Haghparast ◽

Mohammad Ali Behroozikhah

Keyword(s):

Cellular Localization ◽

High Efficiency ◽

Low Cost ◽

Expression System ◽

Interleukin 2 ◽

Signal Peptides ◽

Gram Negative Bacteria ◽

E Coli ◽

Secretion Pathway ◽

Physico Chemical

Background:Using a bacterial expression system such as Escherichia coli (E. coli) is very common for protein expression because of its simplicity, low cost and high efficiency.Objective:In order to express proteins that contain di-sulfide bands, an oxidative space such as the periplasmic environment of the bacteria is required. Therefore, a leader sequence which named Signal Peptide (SP) is needed to direct recombinant protein to fold in periplasmic space. Interleukin-2 (IL-2) is a prominent cytokine which known as growth factor for T-cells and typically produced by a variety of immune cells that stimulate and regulate inflammatory and immune responses.Methods:This study was designed to predict the best signal peptides to express IL-2 in E. coli. To predict the best signal peptides for the expression of IL-2 in Gram-negative bacteria (E. coli), forty-five sequences of SPs were extracted from data base. Some most important details such as n, h and c regions of signal peptides and their probability were studied through the signalP software. Afterwards, physico–chemical features of SPs were analyzed by Portparam and Solpro tools. Finally, secretion-pathway and sub-cellular localization sites were evaluated by PRED-TAT and ProtcompB softwares.Results:At the end of the in-silico analyzes, it was determined that ccmH, PelB, traU, yohN, lolA, yhcN are the most reliable SPs, respectively, with highest score and best performing to express the IL-2 protein in E. coli.

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Copper ions binding regulation for the high-efficiency biodegradation of ciprofloxacin and tetracycline-HCl by low-cost permeabilized-cells

Bioresource Technology ◽

10.1016/j.biortech.2021.126297 ◽

2021 ◽

pp. 126297

Author(s):

Yu Zhou ◽

Shengping You ◽

Jiaxing Zhang ◽

Min Wu ◽

Xiaohui Yan ◽

...

Keyword(s):

High Efficiency ◽

Copper Ions ◽

Low Cost ◽

Permeabilized Cells ◽

Tetracycline Hcl

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Positive Selection of Specific Antibodies Produced against Fusion Proteins

Methods and Protocols ◽

10.3390/mps3020037 ◽

2020 ◽

Vol 3 (2) ◽

pp. 37

Author(s):

Lukas Kramberger-Kaplan ◽

Tina Austerlitz ◽

Holger Bohlmann

Keyword(s):

Fusion Protein ◽

Affinity Chromatography ◽

Positive Selection ◽

Fusion Proteins ◽

Protein A ◽

High Specificity ◽

Western Blots ◽

Specific Antibodies ◽

E Coli ◽

Selection Of

A method for the positive selection of specific antibodies for target proteins expressed as fusion proteins for the production of antiserum is presented. As proof of concept, the fusion protein FLAG::His::GFP::His::FLAG was expressed in Escherichia coli, purified, and used for the immunization of rabbits. The obtained serum was precleared via protein A affinity. A CusF::FLAG fusion protein was expressed in the periplasm of E. coli and purified. GFP without tags was also expressed in E. coli and purified via organic extraction. These proteins were then coupled to NHS-activated sepharose and used for the positive selection of Anti-GFP and Anti-FLAG antibodies. The obtained sera were tested for their specificity against different protein samples and fusion proteins in Western blots. A high specificity of the antibodies could be achieved by a single affinity chromatography step. In general, we advise to express the target protein with different tags and in different E. coli compartments for antibody production and affinity chromatography.

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Cu 2+ Binding Regulation for the High-Efficiency Biodegradation of Ciprofloxacin and Tetracycline-HCl by Low-Cost Permeabilized-Cells

SSRN Electronic Journal ◽

10.2139/ssrn.3924283 ◽

2021 ◽

Author(s):

Yu Zhou ◽

Shengping You ◽

Min Wu ◽

Yuxuan Liu ◽

Jiaxing Zhang ◽

...

Keyword(s):

High Efficiency ◽

Low Cost ◽

Permeabilized Cells ◽

Tetracycline Hcl

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Influence of MBE Growth Conditions on Dislocation Densities of GaAs/Ge Epilayers Grown on Silicon Substrates

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100118692 ◽

1985 ◽

Vol 43 ◽

pp. 364-365

Author(s):

K.M. Hones ◽

P. Sheldon ◽

B.G. Yacobi ◽

A. Mason

Keyword(s):

High Efficiency ◽

Lattice Mismatch ◽

Low Cost ◽

Growth Conditions ◽

Nonradiative Recombination ◽

Device Structure ◽

Si Substrates ◽

Transmission Electron ◽

Dislocation Densities ◽

Dislocation Type

There is increasing interest in growing epitaxial GaAs on Si substrates. Such a device structure would allow low-cost substrates to be used for high-efficiency cascade- junction solar cells. However, high-defect densities may result from the large lattice mismatch (∼4%) between the GaAs epilayer and the silicon substrate. These defects can act as nonradiative recombination centers that can degrade the optical and electrical properties of the epitaxially grown GaAs. For this reason, it is important to optimize epilayer growth conditions in order to minimize resulting dislocation densities. The purpose of this paper is to provide an indication of the quality of the epitaxially grown GaAs layers by using transmission electron microscopy (TEM) to examine dislocation type and density as a function of various growth conditions. In this study an intermediate Ge layer was used to avoid nucleation difficulties observed for GaAs growth directly on Si substrates. GaAs/Ge epilayers were grown by molecular beam epitaxy (MBE) on Si substrates in a manner similar to that described previously.

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Improvement of Ti/RuO2–IrO2 Anode Lifetime and Electrocatalytical Properties by Using an Eco-Friendly Thermal Decomposition Method Using Polyvinyl Alcohol as Solvent

10.26434/chemrxiv.7991129 ◽

2019 ◽

Author(s):

Charlys Bezerra ◽

Géssica Santos ◽

Marilia Pupo ◽

Maria Gomes ◽

Ronaldo Silva ◽

...

Keyword(s):

Thermal Decomposition ◽

Polyvinyl Alcohol ◽

High Efficiency ◽

Electrochemical Impedance ◽

Pechini Method ◽

Low Cost ◽

Charge Transfer Resistance ◽

Transfer Resistance ◽

Calcination Temperatures ◽

Service Lifetime

Electrochemical oxidation processes are promising solutions for wastewater treatment due to their high efficiency, easy control and versatility. Mixed metal oxides (MMO) anodes are particularly attractive due to their low cost and specific catalytic properties. Here, we propose an innovative thermal decomposition methodology using <a>polyvinyl alcohol (PVA)</a> as a solvent to prepare Ti/RuO2–IrO2 anodes. Comparative anodes were prepared by conventional method employing a polymeric precursor solvent (Pechini method). The calcination temperatures studied were 300, 400 and 500 °C. The physical characterisation of all materials was performed by X-ray diffraction and scanning electron microscopy coupled with energy dispersive spectroscopy, while electrochemical characterisation was done by cyclic voltammetry, accelerated service lifetime and electrochemical impedance spectroscopy. Both RuO2 and IrO2 have rutile-type structures for all anodes. Rougher and more compact surfaces are formed for the anodes prepared using PVA. Amongst temperatures studied, 300 °C using PVA as solvent is the most suitable one to produce anodes with expressive increase in voltammetric charge (250%) and accelerated service lifetime (4.3 times longer) besides reducing charge-transfer resistance (8 times lower). Moreover, the electrocatalytic activity of the anodes synthesised with PVA toward the Reactive Blue 21 dye removal in chloride medium (100 % in 30 min) is higher than that prepared by Pechini method (60 min). Additionally, the removal total organic carbon point out improved mineralisation potential of PVA anodes. Finally, this study reports a novel methodology using PVA as solvent to synthesise Ti/RuO2–IrO2 anodes with improved properties that can be further extended to synthesise other MMO compositions.

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Light-Weight, Low Cost, High-Efficiency Solar Cells Space Planar Arrays.

10.21236/adb209013 ◽

1996 ◽

Author(s):

Michael L. Timmons

Keyword(s):

Solar Cells ◽

High Efficiency ◽

Low Cost ◽

Light Weight ◽

Planar Arrays ◽

High Efficiency Solar Cells

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Contradiction Analysis and Solution in the R&D of High-Power Solar LED Street Lamp Controller

Recent Advances in Electrical & Electronic Engineering (Formerly Recent Patents on Electrical & Electronic Engineering) ◽

10.2174/2352096511666180314141920 ◽

2019 ◽

Vol 12 (1) ◽

pp. 12-19

Author(s):

Zhengwang Xu ◽

Wei Mei ◽

Jiaqi Yu ◽

Jiarui Zhang ◽

Yuchun Yi ◽

...

Keyword(s):

Power Supply ◽

High Efficiency ◽

Low Cost ◽

Solar Panel ◽

Battery Voltage ◽

Supply Circuit ◽

Led Light ◽

Power Supply Circuit ◽

Side Mode ◽

Dual Power

As being restricted by factors such as cost, efficiency and size, the development of high-power solar LED street light controller is faced with plenty of difficulties. In case that a structure of two independent DC/DC is applied as the main circuit, it has to face problems such as large size and high cost; in case of applying the bidirectional BUCK/BOOST circuit, it requires change-over switches to control the solar panel and LED light. As being restricted by withstanding voltage, on-resistance and cost, a PMOS device cannot be used as the change-over switch of solar panel and LED light. However, when being used as a change-over switch, an NMOS device must apply the low-side mode under which the negative ends of the mentioned three parts are cut off. In the condition of applying the low-side mode, a differential circuit must be used to detect the voltage of the solar panel. Furthermore, in order to make sure batteries can still be regularly charged after wearing out in daylight, the controller must be supplied with power through a dual power supply circuit that can obtain power from both the solar panel and the battery. The demander has a requirement on extremely low standby power consumption of the product, and thus it is necessary to minimize the circuit that is live while working in standby mode. Methods: The bidirectional BUCK/BOOST circuit structure is applied to the main circuit to realize a higher change-over efficiency while giving considerations to both cost and size. The NMOS device, model IRFB4410ZPBF, with a price of about three yuan, is used as the switching device, and the low-side mode is applied, that is the switches inserted in between negative end of the solar panel or LED light and that of the DC/DC circuit. The low-cost rail-to-rail operational amplifier LM358 is used to form a differential amplification circuit for detecting the voltage of the solar panel. A XL1509-12E1 chip that only costs 0.88 yuan/pc is selected as the main change-over chip for the power supply, which has realized the highly-efficient and low-cost change-over of the power supply. A dual power supply circuit and a step-down protective circuit are designed for the XL1509-12E1 change-over chip. By comparing solar panel voltage with battery voltage, the solar panel booting circuit is realized. Only when solar panel voltage is higher than battery voltage, does the system program start to power it up for running, so that the outage of most of the circuits of the system under standby mode does not consume energy. Furthermore, the solar panel voltage detecting circuit, the solar panel booting circuit and several return difference functions are corrected during system debugging. Results: The circuit board of the entire controller features small size, low cost and high efficiency. It measures about 100*62*18mm in size, costs about 60 yuan, and the charge/discharge change-over efficiency reaches up to over 95%. The controller has many functions: it is capable of operating within a large scope, in which, solar panel voltage is subject to 15~50V, LED light voltage is subject to 15~60V, battery voltage is subject to 10~35V and battery-end charge/discharge current is 10A; it is capable of adapting to monocrystalline silicon/multicrystalline silicon/thin-film and many other kinds of solar panels, as well as lithium/lead-acid and many other kinds of batteries; it is capable of detecting the conversion of day and night, automatically controlling charging and discharging and automatically making adaptive adjustment according to seasonal variations; the current to be consumed during standby will be maintained below 3mA, and thus the power consumption is extremely low. Conclusion: By selecting the bidirectional BUCK/BOOST circuit structure, applying low-side mode for switching of solar panel and LED light, using a differential circuit to detect solar panel voltage, using a low-cost DC/DC chip to realize power supply change-over, designing a dual power supply circuit, introducing solar panel booting circuit and other hardware design, as well as MPPT algorithm, state recognition and control, return difference control and other software design, a solar LED street light control product featuring small size, low cost, high efficiency and multiple functions is successfully developed.

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