scholarly journals PlasmidMaker: a Versatile, Automated, and High Throughput End-to-End Platform for Plasmid Construction

2022 ◽  
Author(s):  
Behnam Enghiad ◽  
Pu Xue ◽  
Nilmani Singh ◽  
Aashutosh Girish Boob ◽  
Chengyou Shi ◽  
...  
Keyword(s):  
High Throughput ◽  
Pyrococcus Furiosus ◽  
Restriction Enzymes ◽  
Dna Assembly ◽  
Assembly Method ◽  
Free Construction ◽  
Artificial Restriction ◽  
End To End ◽  
Plasmid Design ◽  
User Friendly

Plasmids are used extensively in basic and applied biology. However, design and construction of plasmids, specifically the ones carrying complex genetic information, remains one of the most time-consuming, labor-intensive, and rate-limiting steps in performing sophisticated biological experiments. Here, we report the development of a versatile, robust, automated end-to-end platform named PlasmidMaker that allows error-free construction of plasmids with virtually any sequences in a high-throughput manner. This platform consists of a most versatile DNA assembly method using Pyrococcus furiosus Argonaute (PfAgo)-based artificial restriction enzymes, a user-friendly frontend for plasmid design, and a backend that streamlines the workflow and integration with a robotic system. As a proof of concept, we used this platform to generate 101 plasmids from six different species ranging from 5 to 18 kb in size from up to 11 DNA fragments within 3 days. PlasmidMaker should greatly expand the potential of synthetic biology.

scholarly journals Direct preparation of Cas9 ribonucleoprotein from E. coli for PCR-free seamless DNA assembly

2018 ◽  
Author(s):  
Wenqiang Li ◽  
Shuntang Li ◽  
Jie Qiao ◽  
Fei Wang ◽  
Yang Liu ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Restriction Enzymes ◽  
General Strategy ◽  
Dna Assembly ◽  
E Coli ◽  
Assembly Method ◽  
Direct Preparation

AbstractCRISPR-Cas9 is a versatile and powerful genome engineering tool. Recently, Cas9 ribonucleoprotein (RNP) complexes have been used as promising biological tools with plenty of in vivo and in vitro applications, but there are by far no efficient methods to produce Cas9 RNP at large scale and low cost. Here, we describe a simple and effective approach for direct preparation of Cas9 RNP from E. coli by co-expressing Cas9 and target specific single guided RNAs. The purified RNP showed in vivo genome editing ability, as well as in vitro endonuclease activity that combines with an unexpected superior stability to enable routine uses in molecular cloning instead of restriction enzymes. We further develop a RNP-based PCR-free method termed Cas-Brick in a one-step or cyclic way for seamless assembly of multiple DNA fragments with high fidelity up to 99%. Altogether, our findings provide a general strategy to prepare Cas9 RNP and supply a convenient and cost-effective DNA assembly method as an invaluable addition to synthetic biological toolboxes.

Comparison of Small- and Large-scale Expression of Selected Pyrococcus furiosus Genes as an Aid to High-throughput Protein Production

2005 ◽  
Vol 6 (2-3) ◽  
pp. 149-158 ◽  
Author(s):  
Frank J. Sugar ◽  
Francis E. Jenney ◽  
Farris L. Poole ◽  
Phillip S. Brereton ◽  
Michi Izumi ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Protein Production ◽  
Pyrococcus Furiosus

scholarly journals High-Throughput Analysis of Selected Urinary Hydroxy Polycyclic Aromatic Hydrocarbons by an Innovative Automated Solid-Phase Microextraction

Molecules ◽  
2018 ◽  
Vol 23 (8) ◽  
pp. 1869 ◽  
Author(s):  
Stefano Dugheri ◽  
Alessandro Bonari ◽  
Matteo Gentili ◽  
Giovanni Cappelli ◽  
Ilenia Pompilio ◽  
...  
Keyword(s):  
Polycyclic Aromatic Hydrocarbons ◽  
High Throughput ◽  
Aromatic Hydrocarbons ◽  
High Throughput Screening ◽  
Solid Phase Microextraction ◽  
Solid Phase ◽  
User Friendliness ◽  
High Throughput Analysis ◽  
Polycyclic Aromatic ◽  
User Friendly

High-throughput screening of samples is the strategy of choice to detect occupational exposure biomarkers, yet it requires a user-friendly apparatus that gives relatively prompt results while ensuring high degrees of selectivity, precision, accuracy and automation, particularly in the preparation process. Miniaturization has attracted much attention in analytical chemistry and has driven solvent and sample savings as easier automation, the latter thanks to the introduction on the market of the three axis autosampler. In light of the above, this contribution describes a novel user-friendly solid-phase microextraction (SPME) off- and on-line platform coupled with gas chromatography and triple quadrupole-mass spectrometry to determine urinary metabolites of polycyclic aromatic hydrocarbons 1- and 2-hydroxy-naphthalene, 9-hydroxy-phenanthrene, 1-hydroxy-pyrene, 3- and 9-hydroxy-benzoantracene, and 3-hydroxy-benzo[a]pyrene. In this new procedure, chromatography’s sensitivity is combined with the user-friendliness of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide on-fiber SPME derivatization using direct immersion sampling; moreover, specific isotope-labelled internal standards provide quantitative accuracy. The detection limits for the seven OH-PAHs ranged from 0.25 to 4.52 ng/L. Intra-(from 2.5 to 3.0%) and inter-session (from 2.4 to 3.9%) repeatability was also evaluated. This method serves to identify suitable risk-control strategies for occupational hygiene conservation programs.

scholarly journals SSFinder: High Throughput CRISPR-Cas Target Sites Prediction Tool

2014 ◽  
Vol 2014 ◽  
pp. 1-4 ◽  
Author(s):  
Santosh Kumar Upadhyay ◽  
Shailesh Sharma
Keyword(s):  
Operating Systems ◽  
High Throughput ◽  
Genomic Data ◽  
Prediction Tool ◽  
High Demand ◽  
Specific Target ◽  
Target Sites ◽  
High Throughput Detection ◽  
User Friendly

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) system facilitates targeted genome editing in organisms. Despite high demand of this system, finding a reliable tool for the determination of specific target sites in large genomic data remained challenging. Here, we report SSFinder, a python script to perform high throughput detection of specific target sites in large nucleotide datasets. The SSFinder is a user-friendly tool, compatible with Windows, Mac OS, and Linux operating systems, and freely available online.

scholarly journals Automata for DNA Splicing Languages with Palindromic and Non-Palindromic Restriction Enzymes using Grammars

MATEMATIKA ◽  
2019 ◽  
Vol 35 (4) ◽  
pp. 1-14
Author(s):  
Wan Heng Fong ◽  
Nurul Izzaty Ismail ◽  
Nor Haniza Sarmin
Keyword(s):  
Dna Sequences ◽  
Formal Language ◽  
Restriction Enzymes ◽  
Language Theory ◽  
Dna Assembly ◽  
Dna Molecules ◽  
Splicing Systems ◽  
Transition Graphs ◽  
Palindromic Sequences ◽  
Dna Splicing

In DNA splicing system, DNA molecules are cut and recombined with the presence of restriction enzymes and a ligase. The splicing system is analyzed via formal language theory where the molecules resulting from the splicing system generate a language which is called a splicing language. In nature, DNA molecules can be read in two ways; forward and backward. A sequence of string that reads the same forward and backward is known as a palindrome. Palindromic and non-palindromic sequences can also be recognized in restriction enzymes. Research on splicing languages from DNA splicing systems with palindromic and non-palindromic restriction enzymes have been done previously. This research is motivated by the problem of DNA assembly to read millions of long DNA sequences where the concepts of automata and grammars are applied in DNA splicing systems to simplify the assembly in short-read sequences. The splicing languages generated from DNA splicing systems with palindromic and nonpalindromic restriction enzymes are deduced from the grammars which are visualised as automata diagrams, and presented by transition graphs where transition labels represent the language of DNA molecules resulting from the respective DNA splicing systems.

GEView (Gene Expression View) Tool for Intuitive and High Accessible Visualization of Expression Data for Non-Programmer Biologists

2020 ◽  
pp. 580-592
Author(s):  
Libi Hertzberg ◽  
Assif Yitzhaky ◽  
Metsada Pasmanik-Chor
Keyword(s):  
Gene Expression ◽  
Quality Control ◽  
User Interface ◽  
High Throughput ◽  
Graphical User Interface ◽  
Differential Expression Analysis ◽  
Biological Data ◽  
Expression Data ◽  
Batch Correction ◽  
User Friendly

This article describes how the last decade has been characterized by the production of huge amounts of different types of biological data. Following that, a flood of bioinformatics tools have been published. However, many of these tools are commercial, or require computational skills. In addition, not all tools provide intuitive and highly accessible visualization of the results. The authors have developed GEView (Gene Expression View), which is a free, user-friendly tool harboring several existing algorithms and statistical methods for the analysis of high-throughput gene, microRNA or protein expression data. It can be used to perform basic analysis such as quality control, outlier detection, batch correction and differential expression analysis, through a single intuitive graphical user interface. GEView is unique in its simplicity and highly accessible visualization it provides. Together with its basic and intuitive functionality it allows Bio-Medical scientists with no computational skills to independently analyze and visualize high-throughput data produced in their own labs.

M6A2Target: a comprehensive database for targets of m6A writers, erasers and readers

2020 ◽  
Author(s):  
Shuang Deng ◽  
Hongwan Zhang ◽  
Kaiyu Zhu ◽  
Xingyang Li ◽  
Ying Ye ◽  
...  
Keyword(s):  
High Throughput ◽  
Target Gene ◽  
Target Genes ◽  
Biological Processes ◽  
Public Repository ◽  
Reversible Process ◽  
Comprehensive Database ◽  
Dynamic Modification ◽  
Gene Database ◽  
User Friendly

Abstract N6-methyladenosine (m6A) is the most abundant posttranscriptional modification in mammalian mRNA molecules and has a crucial function in the regulation of many fundamental biological processes. The m6A modification is a dynamic and reversible process regulated by a series of writers, erasers and readers (WERs). Different WERs might have different functions, and even the same WER might function differently in different conditions, which are mostly due to different downstream genes being targeted by the WERs. Therefore, identification of the targets of WERs is particularly important for elucidating this dynamic modification. However, there is still no public repository to host the known targets of WERs. Therefore, we developed the m6A WER target gene database (m6A2Target) to provide a comprehensive resource of the targets of m6A WERs. M6A2Target provides a user-friendly interface to present WER targets in two different modules: ‘Validated Targets’, referred to as WER targets identified from low-throughput studies, and ‘Potential Targets’, including WER targets analyzed from high-throughput studies. Compared to other existing m6A-associated databases, m6A2Target is the first specific resource for m6A WER target genes. M6A2Target is freely accessible at http://m6a2target.canceromics.org.

GoldenGateway: A DNA Assembly Method for Plant Biotechnology

2021 ◽  
Vol 26 (1) ◽  
pp. 95-96
Author(s):  
Mansour Karimi ◽  
Thomas B. Jacobs
Keyword(s):  
Plant Biotechnology ◽  
Dna Assembly ◽  
Assembly Method
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