scholarly journals PTO-QuickStep: A Fast and Efficient Method for Cloning Random Mutagenesis Libraries

2019 ◽  
Vol 20 (16) ◽  
pp. 3908 ◽  
Author(s):  
Pawel Jajesniak ◽  
Kang Lan Tee ◽  
Tuck Seng Wong
Keyword(s):  
Directed Evolution ◽  
Random Mutagenesis ◽  
Cost Effective ◽  
Conventional Pcr ◽  
Cost Effective Method ◽  
Cloning Method ◽  
Point Integration ◽  
First Time ◽  
Plasmid Amplification ◽  
Target Plasmid

QuickStep is a cloning method that allows seamless point integration of a DNA sequence at any position within a target plasmid using only Q5 High-Fidelity DNA Polymerase and DpnI endonuclease. This efficient and cost-effective method consists of two steps: two parallel asymmetric PCRs, followed by a megaprimer-based whole-plasmid amplification. To further simplify the workflow, enhance the efficiency, and increase the uptake of QuickStep, we replaced the asymmetric PCRs with a conventional PCR that uses phosphorothioate (PTO) oligos to generate megaprimers with 3′ overhangs. The ease and speed of PTO-QuickStep were demonstrated through (1) right-first-time cloning of a 1.8 kb gene fragment into a pET vector and (2) creating a random mutagenesis library for directed evolution. Unlike most ligation-free random mutagenesis library creation methods (e.g., megaprimer PCR of whole plasmid [MEGAWHOP]), PTO-QuickStep does not require the gene of interest to be precloned into an expression vector to prepare a random mutagenesis library. Therefore, PTO-QuickStep is a simple, reliable, and robust technique, adding to the ever-expanding molecular toolbox of synthetic biology and expediting protein engineering via directed evolution.

scholarly journals Analysis of CMA-DAPI bands and preparation of fluorescent karyotypes in thirty Indian cultivars of Lens culinaris

Caryologia ◽  
2021 ◽  
Author(s):  
Timir baran Jha ◽  
Biplab Kumar Bhowmick ◽  
Partha Roy
Keyword(s):  
Cost Effective ◽  
Fluorochrome Banding ◽  
Banding Patterns ◽  
Plant Chromosomes ◽  
Cost Effective Method ◽  
Production And Consumption ◽  
Breeding Programmes ◽  
Cultivated Species ◽  
Protein Rich ◽  
First Time

India holds a significant rank in production and consumption of the age old protein rich crop Lentil with only one cultivated species and a large number of phenotypically similar cultivars. The need for a reliable and cost effective method of genetic characterization to unravel differences within the Lentil cultivars was felt. The present paper adopted EMA based chromosome preparation followed by staining with two contrasting fluorochromes dyes CMA and DAPI that bind directly to GC and AT rich heterochromatic segments on chromosomes. Analysis of fluorochrome banding pattern furnished a comparative account of genetic diversity within the cultivars that could not be achieved by traditional karyotyping. The marker pair of nucleolar chromosomes (4th and 3rd, majorly) occupied a pivotal position to intensify differences between cultivars in terms of banding patterns around secondary constrictions, suggestive of  yet unknown variation in heterochromatin composition. Our study has strengthened genetic background and relationships of Lentil cultivars. We observed certain types of unusual fluorochrome bands that put forward the exclusivity of Indian germplasm and have questioned the mainstream heterochromatin elements of plant chromosomes captured by CMA-DAPI stains. The comprehensive fluorescent karyotypes of 30 L. culinaris cultivars prepared for the first time, serve as an archetype for the benefit of future breeding programmes in any Indian crop. 

scholarly journals Natural iron ore as a novel substrate for the biosynthesis of bioactive-stable ZnO@CuO@iron ore NCs: a magnetically recyclable and reusable superior nanocatalyst for the degradation of organic dyes, reduction of Cr(vi) and adsorption of crude oil aromatic compounds, including PAHs

RSC Advances ◽  
2018 ◽  
Vol 8 (62) ◽  
pp. 35557-35570 ◽  
Author(s):  
S. Mohammad Sajadi ◽  
Kamal Kolo ◽  
Mohammad Pirouei ◽  
Sarbast A. Mahmud ◽  
Jagar. A. Ali ◽  
...  
Keyword(s):  
Crude Oil ◽  
Aromatic Compounds ◽  
Iron Ore ◽  
Organic Dyes ◽  
Cost Effective ◽  
Natural Substrate ◽  
Cost Effective Method ◽  
Natural Iron ◽  
First Time

For the first time, stable ZnO@CuO@iron ore nanocomposites (NCs) were green synthesized using magnetic iron ore as a natural substrate through an eco-friendly, simple and cost-effective method.

One-pot controllable synthesis of CoFe2O4solid, hollow and multi-shell hollow nanospheres as superior anode materials for lithium ion batteries

2017 ◽  
Vol 5 (41) ◽  
pp. 21994-22003 ◽  
Author(s):  
Yun Qi ◽  
Bingqiu Liu ◽  
Lingyu Zhang ◽  
Yuqiu Huo ◽  
Lu Li ◽  
...  
Keyword(s):  
Lithium Ion Batteries ◽  
Anode Materials ◽  
Large Scale ◽  
Lithium Ion ◽  
Cost Effective ◽  
Hollow Nanospheres ◽  
Controllable Synthesis ◽  
One Pot ◽  
Cost Effective Method ◽  
First Time

CFO-SNSs, CFO-HNSs and MS-CFO-HNSs are controllably fabricated for the first time by a straightforward and cost-effective method on a large scale as superior anode materials for lithium ion batteries.

CAD Of Anticorrosive Protection Of Steel Structures

2019 ◽  
pp. 18-23
Keyword(s):  
Protective Coating ◽  
Computer Aided Design ◽  
Steel Structures ◽  
Cost Effective ◽  
Operating Conditions ◽  
Computational Tool ◽  
Protection Method ◽  
Cost Effective Method ◽  
The Cost ◽  
Aided Design

The choice of cost-effective method of anticorrosive protection of steel structures is an urgent and time consuming task, considering the significant number of protection ways, differing from each other in the complex of technological, physical, chemical and economic characteristics. To reduce the complexity of solving this problem, the author proposes a computational tool that can be considered as a subsystem of computer-aided design and used at the stage of variant and detailed design of steel structures. As a criterion of the effectiveness of the anti-corrosion protection method, the cost of the protective coating during the service life is accepted. The analysis of existing methods of steel protection against corrosion is performed, the possibility of their use for the protection of the most common steel structures is established, as well as the estimated period of effective operation of the coating. The developed computational tool makes it possible to choose the best method of protection of steel structures against corrosion, taking into account the operating conditions of the protected structure and the possibility of using a protective coating.

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

Monitoring effluent quality in hypertrophic wastewater reservoirs using remote sensing

1996 ◽  
Vol 33 (8) ◽  
pp. 23-29 ◽  
Author(s):  
I. Dor ◽  
N. Ben-Yosef
Keyword(s):  
Water Quality ◽  
Theoretical Model ◽  
Principal Component ◽  
Cost Effective ◽  
Operational Parameters ◽  
Effluent Quality ◽  
Optical Images ◽  
Routine Monitoring ◽  
Cost Effective Method ◽  
Wastewater Quality

About one hundred and fifty wastewater reservoirs store effluents for irrigation in Israel. Effluent qualities differ according to the inflowing wastewater quality, the degree of pretreatment and the operational parameters. Certain aspects of water quality like concentration of organic matter, suspended solids and chlorophyll are significantly correlated with the water column transparency and colour. Accordingly optical images of the reservoirs obtained from the SPOT satellite demonstrate pronounced differences correlated with the water quality. The analysis of satellite multispectral images is based on a theoretical model. The model calculates, using the radiation transfer equation, the volume reflectance of the water body. Satellite images of 99 reservoirs were analyzed in the chromacity space in order to classify them according to water quality. Principal Component Analysis backed by the theoretical model increases the method sensitivity. Further elaboration of this approach will lead to the establishment of a time and cost effective method for the routine monitoring of these hypertrophic wastewater reservoirs.

A Cost-Effective Method to Prepare Pure Vildagliptin

2013 ◽  
Vol 10 (3) ◽  
pp. 159-163 ◽  
Author(s):  
Jun Peng ◽  
Yue Feng ◽  
Zhu Tao ◽  
Yingjie Chen ◽  
Xiangnan Hu
Keyword(s):  
Cost Effective ◽  
Cost Effective Method

Green Synthesis and Spectroscopic Studies of Ag-rGO Nanocomposites for Highly Selective Mercury (II) Sensing

2018 ◽  
Vol 9 (1) ◽  
pp. 101-108 ◽  
Author(s):  
Shubhangi J. Mane-Gavade ◽  
Sandip R. Sabale ◽  
Xiao-Ying Yu ◽  
Gurunath H. Nikam ◽  
Bhaskar V. Tamhankar
Keyword(s):  
Green Synthesis ◽  
Color Change ◽  
Limit Of Detection ◽  
Aqueous Media ◽  
Suitable Condition ◽  
Cost Effective ◽  
Spectroscopic Studies ◽  
Calibration Plot ◽  
First Time

Introduction: Herein we report the green synthesis and characterization of silverreduced graphene oxide nanocomposites (Ag-rGO) using Acacia nilotica gum for the first time. Experimental: We demonstrate the Hg2+ ions sensing ability of the Ag-rGO nanocomposites form aqueous medium. The developed colorimetric sensor method is simple, fast and selective for the detection of Hg2+ ions in aqueous media in presence of other associated ions. A significant color change was noticed with naked eye upon Hg2+ addition. The color change was not observed for cations including Sr2+, Ni2+, Cd2+, Pb2+, Mg2+, Ca2+, Fe2+, Ba2+ and Mn2+indicating that only Hg2+ shows a strong interaction with Ag-rGO nanocomposites. Under the most suitable condition, the calibration plot (A0-A) against concentration of Hg2+ was linear in the range of 0.1-1.0 ppm with a correlation coefficient (R2) value 0.9998. Results & Conclusion The concentration of Hg2+ was quantitatively determined with the Limit of Detection (LOD) of 0.85 ppm. Also, this method shows excellent selectivity towards Hg2+ over nine other cations tested. Moreover, the method offers a new cost effective, rapid and simple approach for the detection of Hg2+ in water samples.

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