Blue-white screening as a new readout for deoxyribozyme activity in bacterial cells

Sadeghi, Samira; Ahmadi, Najmeh; Esmaeili, Abolghasem; Javadi‐Zarnaghi, Fatemeh

doi:10.1039/c7ra09679h

Cited by 10 publications

(5 citation statements)

References 31 publications

(32 reference statements)

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“…Figure 4 showed single colonies that grew after the transformation product, with different volumes, on Luria Bertani (LB) media supplemented with carbenicillin antibiotics and X-gal for blue-white screening. As a result, the colour of the colonies will serve as a marker for the existence of the GOI [50]. All of the plates showed many white colonies and only a few blue colonies appeared after being incubated overnight at 37 o C. As white colony indicated that the colony contained construct with insert (recombinant) while blue colony contained construct without insert [50].…”

Section: Vector Constructionmentioning

confidence: 99%

“…As a result, the colour of the colonies will serve as a marker for the existence of the GOI [50]. All of the plates showed many white colonies and only a few blue colonies appeared after being incubated overnight at 37 o C. As white colony indicated that the colony contained construct with insert (recombinant) while blue colony contained construct without insert [50]. The single white colonies were picked and proceeded with plasmid purification and PCRanalysed using the same forward and reverse primers used in PCR of CRISPR plasmid Level 0.…”

Section: Vector Constructionmentioning

confidence: 99%

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Development of CRISPR/Cas9 Construct in Rice (Oryza sativa subsp. indica) Using Golden Gate Cloning Method Towards Drought Tolerance

Yusof,

Tamizi,

Zim

et al. 2023

J.Trop.Life.Science

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Rice (Oryza sativa) is a staple food consumed by the majority of the world’s population. Climate change, however, has created a significant threat to our food security as it posed severe effects on rice production. The emergence of genome editing technology has opened a new era in crop improvement. Hence, this study aims to develop the CRISPR/Cas9 construct of drought tolerance for O. sativa subsp. indica cv. IR64 using Golden Gate cloning method. For this purpose, the generation of CRISPR/Cas9 constructs involved several stages, i.e., characterization of SUMO E2-Conjugating Enzyme (OsSCE1) gene, single-guide RNA (sgRNA) design and vector construction. FGENESH, GeneMarkS, InterProScan, and Blast2GO programmes – were used for the OsSCE1 gene characterisation. The putative OsSCE1 gene isolated from IR64 was then verified by sequencing, and the gene was 585 bp long and showed 99% identity with O. sativa on chromosome 10. In silico analysis concluded the gene is involved in abiotic stress regulation. The 20 bp sgRNA was designed manually with the aid of gRNA prediction programmes including CCTop, and Benchling. The virtual vector was validated using the Golden Gate Cloning approach and later confirmed through sequencing. The assembly involved separate vectors containing the OsSCE1 sgRNA sequence, plant selectable marker, and Cas9 cassette to construct standardised elements for hierarchical modular cloning (MoClo). This study demonstrated that our format, as the gene insertion are achievable, resulting in a speedier and more efficient assembly process which may contribute to improve drought tolerance in indica rice. Further study on the Agrobacterium-mediated transformation using the developed construct may be conducted to determine the efficacy of knocking out candidate genes in improving drought tolerance ability O. sativa

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Section: Vector Constructionmentioning

confidence: 99%

Section: Vector Constructionmentioning

confidence: 99%

Development of CRISPR/Cas9 Construct in Rice (Oryza sativa subsp. indica) Using Golden Gate Cloning Method Towards Drought Tolerance

Yusof,

Tamizi,

Zim

et al. 2023

J.Trop.Life.Science

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“…The LAMP reaction products are produced with high efficiency and can provide colorimetric signals for visual detection with bare eyes. Visual detection methods are the method of choice in many detection systems (Sadeghi et al 2017 ; Javani et al 2017 ; Anantharaj et al 2020 ). The signal could be generated with either pH indicators or magnesium pyrophosphate indicators (Scott et al 2019 ; Gachugia et al 2020 ; Goto et al 2009 ).…”

Section: Introductionmentioning

confidence: 99%

Malachite Green-Based Detection of SARS-CoV-2 by One-Step Reverse Transcription Loop-Mediated Isothermal Amplification

et al. 2023

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The pandemic of severe acute respiratory syndrome 2 (SARS-CoV-2) revealed the necessity of diagnosis of the infected people to prevent the prevalence infection cycle. Many commercial pathogen diagnosis methods are based on the detection of genomic materials. Isothermal amplification methods such as loop-mediated-isothermal amplification (LAMP) are the method of choice in these cases. Reverse transcription steps are efficiently coupled to LAMP for the detection of pathogens with genomic RNAs such as SARS-CoV-2. Many detection systems for LAMP include fluorescent readout systems. Although such systems result in desirable limits of detection, the need for special instrumentation is the main dispute of such systems to become real point of care assays. In contrast, colorimetric detection methods would reduce costs and improve the applicability of the system. In this study one-step reverse transcription-LAMP reaction was established that enables visual detection of the SARS-CoV-2 genome. Nasopharyngeal RNA samples were first validated by reverse transcription quantitative polymerase chain reaction and then subjected to RT-LAMP. To lower the cost associated with the readout system equipment, malachite green (MG) was used. The color change of MG to blue allowed visual detection of the virus. Firstly, experiments were set up as two-step RT-LAMP reaction to identify the best primer sets. In addition, MG concentration was optimized with the significant colorimetric signal for the positive samples. Next, a one-step colorimetric method was developed for the detection of SARS-CoV-2 based on MG color shift in 2 h.

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“…Since the report of the first DNAzyme ( 5 ), DNAzymes catalyzing a growing variety of chemical reactions have been reported ( 7–19 ). Among them, RNA-cleaving DNAzymes have been vastly pursued due to their important applications in molecular ( 20 ) and synthetic biology ( 21 , 22 ), as therapeutic and diagnostic tools ( 23–26 ), as biosensors ( 27–29 ) and as molecular logic gates ( 30 , 31 ).…”

Section: Introductionmentioning

confidence: 99%

DNAzymeBuilder, a web application for in situ generation of RNA/DNA-cleaving deoxyribozymes

Mohammadi-Arani

Javadi‐Zarnaghi

Boccaletto

et al. 2022

Nucleic Acids Research

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Nucleic acid cleaving DNAzymes are versatile and robust catalysts that outcompete ribozymes and protein enzymes in terms of chemical stability, affordability and ease to synthesize. In spite of their attractiveness, the choice of which DNAzyme should be used to cleave a given substrate is far from obvious, and requires expert knowledge as well as in-depth literature scrutiny. DNAzymeBuilder enables fast and automatic assembly of DNAzymes for the first time, superseding the manual design of DNAzymes. DNAzymeBuilder relies on an internal database with information on RNA and DNA cleaving DNAzymes, including the reaction conditions under which they best operate, their kinetic parameters, the type of cleavage reaction that is catalyzed, the specific sequence that is recognized by the DNAzyme, the cleavage site within this sequence, and special design features that might be necessary for optimal activity of the DNAzyme. Based on this information and the input sequence provided by the user, DNAzymeBuilder provides a list of DNAzymes to carry out the cleavage reaction and detailed information for each of them, including the expected yield, reaction products and optimal reaction conditions. DNAzymeBuilder is a resource to help researchers introduce DNAzymes in their day-to-day research, and is publicly available at https://iimcb.genesilico.pl/DNAzymeBuilder.

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Blue-white screening as a new readout for deoxyribozyme activity in bacterial cells

Abstract: Demonstration of 10–23 deoxyribozyme activity in viable E. coli using blue-white screening as the readout system.

Cited by 10 publications

References 31 publications

Development of CRISPR/Cas9 Construct in Rice (Oryza sativa subsp. indica) Using Golden Gate Cloning Method Towards Drought Tolerance

Development of CRISPR/Cas9 Construct in Rice (Oryza sativa subsp. indica) Using Golden Gate Cloning Method Towards Drought Tolerance

Malachite Green-Based Detection of SARS-CoV-2 by One-Step Reverse Transcription Loop-Mediated Isothermal Amplification

DNAzymeBuilder, a web application for in situ generation of RNA/DNA-cleaving deoxyribozymes

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